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Purification and Characterization of Vegetative Storage Proteins from Alfalfa (Medicago sativa L.) Taproots
Abstract
Alfalfa (Medicago sativa L.) accumulates C and N reserves in taproots and utilizes these reserves for shoot growth in spring and for shoot regrowth after defoliation. Three proteins are very abundant in taproots and undergo a cyclic pattern of utilization during early shoot growth followed by reaccumulation during late shoot development. Our objectives were to purify and characterize these putative vegetative storage proteins from alfalfa taproots. The proteins were purified using organic-solvent and ionic-precipitation techniques, gel filtration, and affinity chromatography. Polyclonal antibodies were raised against the purified proteins, and electrophoresis and immunoblotting were utilized to determine protein distribution and relative abundance. These proteins are present in high concentrations in alfalfa taproots, but were not found in seeds, nodules, leaves, or stems of alfalfa. Taproots of all perennial Medicago species examined contained these proteins, whereas roots of annual Medicago species had very low to undetectable amounts of these proteins. Taproots of other forage legume species (Lotus, Melilotus, and Trifolium) did not contain proteins that cross-reacted with antibodies raised against the three alfalfa taproot proteins. The three proteins have molecular masses of 15, 19, and 32 ku, are glycosylated, and have epitopes in common. The amino acids asparagine and aspartate make up 15 mole percent of the three alfalfa taproot proteins. These proteins possess features consistent with their role being vegetative storage proteins.
... Nitrogen-containing compounds in alfalfa roots (such as amino acids and proteins) have been shown to be positively associated with the rate of herbage regrowth after defoliation (Kim et al., 1991;Hendershot and Volenec, 1993b;Ourry et al., 1994;Barber et al., 1996;Volenec et al., 1996) and in the spring when shoot growth resumes (Volenec et al., 1991;Hendershot and Volenec, 1993a;Li et al., 1996). Three polypeptides constituting approximately 40% of the root's soluble protein pool have been isolated and characterized (Cunningham and Volenec, 1996). We believe that these polypeptides are VSPs because they accumulate in alfalfa taproots in early autumn and disappear in the spring and after plants are defoliated, in a manner that is consistent with functions assigned to VSPs (Cyr and Bewley, 1990). ...
... Defoliation reduces nitrogenase activity and nitrogen fixation (Vance et al., 1979). Alfalfa root-amylase shows patterns of activity similar to three abundant VSPs we have characterized from alfalfa roots (Cunningham and Volenec, 1996). These four polypeptides (including-amylase) accumulate in alfalfa taproots in early autumn and disappear in spring, when shoot growth resumes, and during shoot regrowth when plants are defoliated. ...
... Further work will concentrate on enhancing our understanding of the function of-amylase using antisense DNA transformation studies. Work is also under way to clone and characterize the cDNAs for the three alfalfa taproot VSPs that we described previously (Cunningham and Volenec, 1996). We hope that understanding the structural and biological features of these VSPs will reveal the reasons that-amylase serves a similar role in the roots of this species. ...
Alfalfa (Medicago sativa L.) roots contain large quantities of β-amylase, but little is known about its role in vivo. We studied this by isolating a β-amylase cDNA and by examining signals that affect its expression. The β-amylase cDNA encoded a 55.95-kD polypeptide with a deduced amino acid sequence showing high similarity to other plant β-amylases. Starch concentrations, β-amylase activities, and β-amylase mRNA levels were measured in roots of alfalfa after defoliation, in suspension-cultured cells incubated in sucrose-rich or -deprived media, and in roots of cold-acclimated germ plasms. Starch levels, β-amylase activities, and β-amylase transcripts were reduced significantly in roots of defoliated plants and in sucrose-deprived cell cultures. β-Amylase transcript was high in roots of intact plants but could not be detected 2 to 8 d after defoliation. β-Amylase transcript levels increased in roots between September and October and then declined 10-fold in November and December after shoots were killed by frost. Alfalfa roots contain greater β-amylase transcript levels compared with roots of sweetclover (Melilotus officinalis L.), red clover (Trifolium pratense L.), and birdsfoot trefoil (Lotus corniculatus L.). Southern analysis indicated that β-amylase is present as a multigene family in alfalfa. Our results show no clear association between β-amylase activity or transcript abundance and starch hydrolysis in alfalfa roots. The great abundance of β-amylase and its unexpected patterns of gene expression and protein accumulation support our current belief that this protein serves a storage function in roots of this perennial species.
... Based on criteria given by Cyr and Bewley (1990), initial results obtained in alfalfa by Hendershot and Volenec (1993 a , b ) were confirmed by subsequent studies (Avice et al. 1996 a ;Cunningham and Volenec 1996), identifying three polypeptides (32, 19 and 15 kDa) in taproots. These Abbreviations used: DW, dry weight; JA, jasmonic acid; LD, long-day; MeJA, methyl jasmonate; PBS, phosphate buffered saline; pI, isoelectric point; PVDF, polyvinylidene difluoride; PVPP, polyvinylpolypyrrolidone; SD, short-day; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; SSC, saline sodium citrate; VSP, vegetative storage protein. ...
... After defoliation, the abundance of these proteins is substantially reduced (up to 84% for the 32-kDa VSP) during the first 6 d of shoot regrowth, while total soluble proteins in taproots declined only by 33% (Avice et al. 1996 a ). Cunningham and Volenec (1996) have clearly demonstrated using immunoblot analysis that these VSPs accumulate specifically in taproot tissue of perennial Medicago . Deposition of VSPs in roots begins about 50 d after planting in Medicago sativa cv. ...
... The method for protein analysis was adapted from Cunningham and Volenec (1996). Proteins were extracted by suspending 200 mg of ground, freeze-dried taproot at 4°C with 200 mg polyvinyl polypyrrolidone (PVPP) in 5 mL of 50 m M imidazole-HCl buffer (pH 6.5) containing 2 mM phenylmethyl-sulfonyl fluoride, 10 mM 2-mercaptoethanol and 100 µL L -1 Triton X-100. ...
Our objectives were to study the regulation of N partitioning within tissues
of non-nodulated alfalfa (Medicago sativa L.) and N
storage in taproots as vegetative storage proteins (VSP) of 15, 19, and 32 kDa
and β-amylase (57 kDa) by environmental (photoperiod, temperature, N
availability) and endogenous factors (methyl jasmonate). When compared to
long-day conditions (LD, 16 h day/8 h night), short-day (SD, 8 h
day/16 h night), exposure to low temperature (5˚C) or application of
methyl jasmonate (MeJA, 100 M ) for 35 d reduced the biomass shoot/ root
ratio and modified the source–sink relationships for N. SD and MeJA
treatments resulted in partitioning of N to taproots and a concomitant
accumulation of VSPs. In comparison with LD, SD treatment also stimulated
β-amylase gene expression 2.5-fold. Although low temperature increased the
N partitioning to root tissues and the accumulation of soluble proteins in
taproot, VSP concentration and β-amylase mRNA levels remained low.
Increasing N concentration from 1 to 5 mM KNO3 doubled
the total dry matter but did not affect the N partitioning within the plant,
VSP accumulation, or ‚ β-amylase expression. These results
suggested that short photoperiod can result in preferential N allocation
toward taproots with a concomitant induction of VSP accumulation.
... of N, total soluble proteins (TSP) and VSP in taproots N content in taproot was measured as described by Meuriot et al. [38] using a C/N analyzer linked to an isotope ratio mass spectrometer (IRMS, Roboprep CN and mass spectrometer, PDZ Europa Scientific Ltd., Crewe, UK). The method used for protein analyses was adapted from Cunningham and Volenec [39] and Noquet et al. [33]. Proteins were extracted by suspending 150 mg of ground, freeze-dried taproot at 4 8C with 150 mg polyvinyl polypyrrolidone (PVPP) in 5 mL of 100 mM sodium phosphate buffer (pH 7) containing 10 mM 2-mercaptoethanol. ...
... SDS-PAGE was performed using 150 g L À1 acrylamide separation gel and stained with Coomassie brilliant blue R-250 [43]. Separated proteins were blotted onto PVDF membrane for Western blotting analysis using the anti-32, -19, and -15 kDa VSP antibodies [33,39] to confirm SDS-PAGE results. All parameters were measured in plants at the end of the vegetative normal growth harvest (60-day-old plants) and after 30 days of regrowth (90-day-old plants). ...
The increasing atmospheric CO2 concentration resulting as a consequence of economic development generally leads to increased plant biomass production. However, little attention has been paid to the effects of combined factors, such as CO2, temperature or water availability, on plant regrowth after cutting or grazing, which represent the usual methods of managing forage legumes like alfalfa. It has been demonstrated that nitrogen pools in alfalfa taproot, especially vegetative storage proteins (VSP), condition new regrowing shoots. The aim of our study was to determine the effect of CO2 (ambient, around 350 μmol mol−1versus 700 μmol mol−1), temperature (ambient versus ambient + 4 °C) and water availability (well-irrigated versus partially irrigated) on taproot N accumulation, especially VSP, in nodulated alfalfa before defoliation and after 1 month of regrowth. At the end of vegetative normal growth, elevated CO2 enhanced dry matter production only in plants grown under high temperature and irrigated at field capacity. The taproot VSP content was increased by drought during this period, and this increase may explain the reduction in production differences between well-watered and drought plants at the end of cutting/regrowth cycle. After 1 month of regrowth, drought increased the VSP level again and it could be expected a greater forage production in drought treatments under equal regrowth conditions during the next cutting-regrowth cycle.
... It is widely recognized that alfalfa regrowth is not only affected by the availability of C reserves but also depends on the mobilization of organic N from storage organs and their translocation to new growing leaves (Ourry et al., 1994;Volenec et al., 1996;Avice et al., 1997). Amino acids such as asparagine and aspartic acid, as well as specific soluble proteins of 15, 19, 32 and 57 kDa, harbouring characteristics typical of vegetative storage proteins (VSPs), are thought to be involved in alfalfa regrowth (Hendershot and Volenec, 1993a;Avice et al., 1996;Cunningham and Volenec, 1996). Using plants acclimated to winter conditions in an unheated greenhouse, Dhont et al. (2003) recently reported positive correlations between both concentrations and pools of root N reserves and alfalfa spring regrowth. ...
... This field study confirms that the three major soluble proteins of 32, 19 and 15 kDa observed on SDS-PAGE correspond to the alfalfa VSPs identified by Cunningham and Volenec (1996), which are preferentially mobilized during regrowth of alfalfa Li et al., 1996;Avice et al., 1997). As previously observed under unheated greenhouse conditions ( Dhont et al., 2003), the abundance of these specific proteins was reduced in 2-or 3-year-old alfalfa stands by an early autumn defoliation at 400 or 500 GDD. ...
The objective of the study was to characterize variations in proline, arginine, histidine, vegetative storage proteins, and cold-inducible gene expression in overwintering roots of field-grown alfalfa, in response to autumn defoliation, and in relation to spring regrowth and winter survival.
Field trials, established in 1996 in eastern Canada, consisted of two alfalfa cultivars ('AC Caribou' and 'WL 225') defoliated in 1997 and 1998 either only twice during the summer or three times with the third defoliation taken 400, 500 or 600 growing degree days (basis 5 degrees C) after the second summer defoliation.
The root accumulation of proline, arginine, histidine and soluble proteins of 32, 19 and 15 kDa, characterized as alfalfa vegetative storage proteins, was reduced the following spring by an early autumn defoliation at 400 or 500 growing degree days in both cultivars; the 600-growing-degree-days defoliation treatment had less or no effect. Transcript levels of the cold-inducible gene msaCIA, encoding a glycine-rich protein, were markedly reduced by autumn defoliation in 'WL 225', but remained unaffected in the more winter-hardy cultivar 'AC Caribou'. The expression of another cold-inducible gene, the dehydrin homologue msaCIG, was not consistently affected by autumn defoliation. Principal component analyses, including components of root organic reserves at the onset of winter, along with yield and plant density in the following spring, revealed that (a) amino acids and soluble proteins are positively related to the vigour of spring regrowth but poorly related to winter survival and (b) winter survival, as indicated by plant density in the spring, is associated with higher concentrations of cryoprotective sugars in alfalfa roots the previous autumn.
An untimely autumn defoliation of alfalfa reduces root accumulation of specific N reserves such as proline, arginine, histidine and vegetative storage proteins that are positively related to the vigour of spring regrowth but poorly related to winter survival.
... It has been well documented that alfalfa regrowth is not only affected by the availability of C reserves but strongly depends on the translocation of organic N stored in vegetative organs to new growing leaves when N 2 fixation and mineral N uptake are limited (Kim et al., 1993a, b; Hendershot and Volenec, 1993a; Ourry et al., 1994). Compelling evidence indicates that root soluble proteins of 15, 19, 32, and 57 kDa, which exhibit characteristics typical of vegetative storage proteins (VSPs), are preferentially mobilized during alfalfa shoot growth in the spring or its regrowth after defoliation in the summer (Cunningham and Volenec, 1996; Avice et al., 1997; Justes et al., 2002). Furthermore, Dhont et al. (2003 recently concluded that the total amounts of N reserve components in roots are a better determinant of alfalfa shoot regrowth than their concentrations. ...
... However, the alfalfa VSP of 57 kDa was found to be the translational product of a b-amylase gene with no demonstrable starch hydrolytic activity, suggesting a loss of original function (Gana et al., 1998). In addition, the progressive increase in total soluble protein (TSP) concentrations in roots of alfalfa from October to December (Hendershot and Volenec, 1993b; Li et al., 1996), as well as a higher abundance of VSPs in a winter hardy perennial than in the annual species of Medicago (Cunningham and Volenec, 1996), also suggest the potential involvement of soluble proteins in the cold-hardening process and long-term persistence of alfalfa. This link is further supported by the positive relationship observed between reduced winter injury and high TSP concentrations in roots of alfalfa selected for higher autumn dormancy (Cunningham et al., 1998Cunningham et al., , 2001). ...
This study describes the time-course of the accumulation of total soluble proteins (TSPs) and vegetative storage proteins (VSPs) and of the transcripts of cold-inducible (CI) and VSP-encoding genes in taproots of two alfalfa cultivars (AC Caribou and Europe) during their acclimation to natural autumn hardening and overwintering conditions in eastern Canada. The impact of a defoliation in September on these winter hardening-related changes was also assessed. Both concentrations and pools of VSPs increased significantly between early September and mid-October and remained unchanged thereafter, concomitantly with the disappearance of VSP-encoding transcripts. Other soluble protein constituents continued to increase later in the autumn and early winter and accounted for nearly 60% of taproot TSP pools in winter. As a result, VSP abundance relative to TSPs decreased markedly during the winter. The increase in the levels of CI transcripts was induced by lowering temperatures, and distinct patterns suggest differences in the regulation of their accumulation. RNA analyses revealed that the accumulation of VSP transcripts during the autumn precedes the accumulation of CI transcripts. Autumn defoliation interrupted the accumulation of both TSPs and VSPs during autumn hardening and repressed the transcript levels of two CI genes differentially between cultivars. The well-documented impact of autumn defoliation on the vigour of spring regrowth and long-term persistence of alfalfa could be related to its negative impact on the accumulation of VSPs and TSPs and on the expression of genes encoding CI proteins potentially involved in cold tolerance and pathogen resistance.
... This risk might be mitigated somewhat by the prolonged dry-down period that would be expected of the clay soils where bottomland red oaks are typically found. Furthermore, given that most bottomland red oaks are moderately shade intolerant (Collins and Battaglia 2008;Johnson 1975), the low light conditions often found in the understory of bottomland forests (Cunningham and Volenec 1996;Jenkins and Chambers 1989) might impede the recovery of Nuttall oak seedlings that are flooded in the early spring, especially if flooding delays development of full photosynthetic capacity until after overstory leaf expansion. Hence, our results may represent a best-case scenario for recovery from flooding prior to the next winter, perhaps limiting Nuttall oak to locations where flood subsides early in spring. ...
Key message
Whereas Shumard oak seedlings are intolerant of dormant season flood, Nuttall oak seedlings are tolerant. Flooding more than 1–2 months beyond budbreak may have persistent negative impacts on Nuttall oaks.
Abstract
Since flooding in winter and spring is an integral part of bottomland hardwood ecosystems in the southeastern United States, moderately flood-tolerant oaks, like Nuttall oak (Quercus texana), should be well adapted to flooding during these seasons. To quantify the potential for injury from different lengths of winter flooding, we flooded seedlings of Nuttall oak and moderately flood intolerant Shumard oak (Q. shumardii) for 0, 1, 2, and 3 months, with the first month of flooding occurring during the dormant season. Flooding during dormancy had no effect on Nuttall oak, but Shumard oak seedlings had reduced growth in the spring. Flooding that extended beyond budbreak resulted in reduced leaf area and root biomass accumulation in spring for both species, while Shumard oaks also experienced high mortality. At the end of the growing season, Nuttall oaks that had been flooded accumulated tissue biomasses similar to non-flooded seedlings, except taproot biomass, which was reduced 40% by 3 months of flooding. It appears that Nuttall oak delayed fully investing in spring growth until after flooding subsided, and then was largely able to compensate following flooding that extended one month beyond budbreak. However, flooded Shumard oaks did not show similar signs of recovery. Thus, sites that flood at any time of year would not be suitable for Shumard oak. Our results suggest that natural or human-imposed flooding can extend several weeks beyond budbreak without harming Nuttall oaks, but inundation prolonged several months beyond budbreak could weaken the ability to respond to subsequent stresses.
... This is consistent with Avice et al. (1996), who suggested root reserves show intense N depletion in the first ~10 days post-harvest. Furthermore, Cunningham and Volenec (1996) demonstrated cell division was highly sensitive to N supply in the early regrowth period. This lack of N post-defoliation might also explain the slow LAER for treatments (SS) with diminished root reserves . ...
Lucerne (Medicago sativa L.) canopy expansion, as quantified by leaf area index (LAI), is the crop process that determines the amount of intercepted total radiation during each regrowth cycle. A challenge is to capture seasonal changes of canopy expansion rate in response to the environment. This research integrates parameters and functions of lucerne canopy expansion into the Agricultural Production Systems sIMulator (APSIM) next generation (APSIM NextGen) model (LeafArea module) to simulate canopy expansion and light interception. Over 20 years of detailed field experimental datasets, with multiple treatments, from Lincoln University were used for model development. Functions derived from a fall dormancy (FD) 5 rated genotype were grown under an industry standard defoliation treatment to parametrize the model. These functions were tested further using genotypes with an FD2 or FD10 rating under longer and shorter defoliation regimes, all under irrigated conditions. The APSIM NextGen lucerne model predicted the LAI expansion pattern in each growth cycle as a double sigmoid curve requiring functions that define the lag phase, basal bud initiation, the linear leaf area expansion rate (LAER; m² m⁻² °Cd), and canopy senescence which represents the loss of LAI over time. LAI was well predicted for experiments under the standard (42-day) and long (84 day) defoliation treatments for FD5, with Nash-Sutcliffe efficiency (NSE) of 0.61 and 0.55. However, the derived parameters and functions overestimated LAI under an extreme short defoliation treatment (28-day), NSE values ranged from 0.38 to 0.78. LAER was lower for the short-defoliation intervals (28-day), probably due to a depletion of carbon and nitrogen reverses in perennial organs. For FD2 and FD10, different LAER functions were generated from field observed data and used to improve simulation agreement. There was fair agreement for the 84-day treatment (NSE of 0.32) and the 42-day treatment (NSE of 0.38), but poor agreement for the 28-day treatment for FD10 (NSE = −0.88). The estimated extinction coefficient (k) was the same for seedling and regrowth crops, and consistent across defoliation treatments and FD classes. With the LeafArea module and k value, the APSIM NextGen lucerne model can now estimate daily LAI and intercepted radiation. Future model development includes validating the LeafArea module in different environments. However, a more mechanistic model approach is required to link canopy expansion to carbon and nitrogen reserves in lucerne plants that experience intense defoliation.
... Total nonstructural carbohydrates (sugar and starch) have long been positively associated with improved winter survival of alfalfa (Graber et al., 1927;Bula et al., 1956). More recently, the underlying physiological basis for improved winter survival and rapid shoot growth of alfalfa after harvest has been expanded to include taproot N reserves, including protein and amino acid pools (Hendershot and Volenec, 1993;Avice et al., 1996;Barber et al., 1996;Cunningham and Volenec, 1996;Volenec et al., 1996;Berg et al., 2009). Adequate P and K nutrition also has a positive impact on alfalfa yield and persistence (Berg et al., 2005(Berg et al., , 2007Lissbrant et al., 2010). ...
Phosphorus (P) and potassium (K) impact alfalfa (Medicago sativa L.) performance, but how these nutrients alter taproot physiology during fall acclimation and subsequent growth in spring is unclear. Our objectives were to: (1) determine seasonal patterns for taproot P and K concentrations during fall acclimation and during initial shoot growth in spring; (2) determine how P and K nutrition impacts accumulation of taproot C and N reserves during fall and their subsequent use when shoot growth resumes in spring; and (3) assess how addition of P and K fertilizer impacts survival and shoot growth in spring. Two P (0 and 75 kg ha⁻¹) and two K (0 and 400 kg ha⁻¹) treatments were applied and taproots were sampled between September and December, and again from March to May over 2 years. Concentrations of taproot sugar, starch, buffer-soluble protein, amino-N, and RNA pools were determined. While P and K fertilizer application increased taproot P and K concentrations two- to three-fold, concentrations of P and K in taproots over time did not change markedly during cold acclimation in fall, however, taproot P declined in spring as plant growth resumed. Compared to the 0K-0P treatment, taproots of plants fertilized with 400K-75P had higher starch, protein, amino-N, and RNA, but reduced sugar concentrations in fall. Concentrations of all these pools, except starch, declined during the initial 2 weeks of sampling beginning in late March as shoot growth resumed in spring. Herbage yield in May was highest for the 400K-75P treatment and least for the 0K-0P treatment, differences that were associated with variation in mass shoot⁻¹ and not shoots m⁻². High yield of the 400K-75P plants in May was consistently associated with greater concentrations and use of amino-N, soluble protein, and RNA pools in taproots, and not with accumulation and use of starch and sugar pools. Understanding factors leading to the accumulation of taproot N reserves and RNA during cold acclimation in fall and their use during the initial growth in spring should enhance efforts to improve alfalfa growth and herbage yield in spring.
... Discussions about the mineral management in perennials place the focus on N resources. Specific vegetative storage proteins (VSPs) are deposited in late-season taproots of alfalfa (Medicago sativa L.) and white clover (Trifolium repens L.) [65,66] to support regrowth in spring. Non-VSPs N resources formed in red clover (T. ...
Crossing annual cereals, legumes, and oilseeds with wild rhizomatous relatives is used to create perennial lines that fruit over 2–3 seasons. Contrary to annual crops, the year-round vegetation cover should contribute to carbon sequestration, soil formation, and root mineral preservation. Soil erosion, nutrient leaching, and labor expenses may be reduced. While deep-rooted grasses actually inhibit nitrate leaching, advantages in nutrient storage and soil formation are not yet shown. Therefore, the turnover of organics and minerals in the perennial goldenrod was compared with that of winter wheat between blooming and resprouting (28 February) by gravimetry and ICP-MS. From blooming (23 August) to harvest (13 November), goldenrod stalks of 10,070 (given in kg ha−1) lost 23% by dry weight (DW) and released 14.9/9.6/65.7 in NPK and 2193 in water-soluble organics via leaching and root exudation. Apart from a transient rise of 28.8 in N around 13 November, the stubble/rhizome system held CaKMg(N)P stable at a level avoiding metal stress from 23 August to 28 February. Filling seeds in wheat excluded net losses of minerals and organics from anthesis to harvest (23 July). Stubbles (16 cm) and spilt grains of 2890 represented 41.8/2.91/62.5 in NPK and lost 905 in biomass with 25.4/1.8/59.8 in NPK to the soil by 28 February. In wheat-maize rotations, ploughing was avoided until early March. Weeds and seedlings emerged from spilt grains replaced losses in stubble biomass, N, and P but left 40.5 in K unused to the soil. In wheat-wheat rotations, organics and minerals lost by the down-ploughed biomass were replenished by the next-rotation seedlings that left only 18.3 in K to the soil. In summary, off-season goldenrod rhizomes did not store excess minerals. The rate of mineral preservation corresponded with the quantity of the biomass irrespective of its perennial habit. Released water-soluble organics should foster microbial carbon formation and CO2 efflux while soil improving gains in humate C should depend on the lignin content of the decaying annual or perennial biomass. Clues for NPK savings by perennials were not found.
... Plants that were P-deficient contained high levels of taproot starch, but these concentrations were unchanged after defoliation, suggesting that starch use is severely impaired by P deficiency in taproots of this species. Cunningham and Volenec (1996) identified three vegetative storage proteins (VSPs) that are preferentially utilized as a source of N for shoot regrowth. Understanding the role these N pools play in alfalfa shoot regrowth and how this process interacts with P nutrition is vital for alfalfa improvement efforts aimed at increasing persistence and forage yield. ...
Phosphorus (P) deficiency reduces forage yield and stand persistence of alfalfa (Medicago sativa L.). The objective of this study was to determine the influence of P nutritionand defoliation on alfalfa shoot growth, root carbohydrate and protein metabolism, and steady-state mRNA levels for high-affinity P transporters. In a greenhouse study, P-deprived plants were provided with 0, 0.25, 2, and 6 mM P beginning 7 d before shoot removal. Plants were sampled immediately (day -7) on days -5, -2, 0 (day of shootremoval), and on days 1, 2, 6, and 9 post-shoot removal. Addition of P to P-deficient plants stimulated growth of shoots but not roots. Taproot bark sugar concentrations were reduced significantly in cut plants at any rate of P, whereas only the 6 mM P treatment reduced taproot wood sugar concentrations in uncut plants. There was a significant defoliation-induced decline in both wood and bark sugar and amino-acid concentration that was enhanced at high P rates. Low P reduced utilization of starch and protein reserves in taproots. Transcripts for a high-affinity P transporter were not detected in any root or shoot tissue assayed, irrespective of defoliation or P treatment. The uncertain relationship between P availability and P-transporter transcript abundance in our greenhouse-grown plants requires additional investigation.
... Among the different soluble N pools, soluble proteins and amino acids represent the largest soluble N fractions. Additionally, four vegetative storage proteins (VSPs) (15, 19, 32 and 57 kDa) which represent up to 40% of soluble protein concentration, have been identified in alfalfa taproots (Cunningham and Volenec 1996;Gana et al. 1998). These VSPs appear to be preferentially degraded and mobilized at twice the rate observed for total soluble protein concentrations during the first 10 days of regrowth after harvest and reaccumulate rapidly as shoots begin reproductive development. ...
Key determinants of regrowth rate following harvest in alfalfa (Medicago sativa L.) are the levels of root carbon (C) and nitrogen (N) reserves. This study was undertaken to assess the impact of alfalfa cultivars differing in fall dormancy on regrowth potential in relation to the quantitative changes in root C and N reserves at the onset of regrowth. Five contrasting alfalfa cultivars were grown in the field and were harvested three times. Shoot regrowth dry weight increased significantly with decreasing fall dormancy. Alfalfa cultivars with less dormancy tended to have higher concentrations of root C and N reserves, whereas significantly greater pools of root C and N reserves were found in roots at each harvest time because of greater root dry weight. Shoot regrowth was positively correlated to the pools of root soluble sugar, starch and total non-structural carbohydrates, but not correlated to their concentrations at each harvest time. Shoot regrowth was also linearly related to the pools of root total N and soluble proteins, but not consistently related to their concentrations. Our results suggest that the total amount of C and N organic reserves in alfalfa roots rather than their concentrations are determining factors of shoot regrowth.
... For example, several forage legumes accumulate vast quantities of N in taproots during autumn that are subsequently used for shoot growth initiation in spring and shoot regrowth after defoliation in summer (Hendershot and Volenec, 1993a,b;Barber et al., 1996;Li et al., 1996); times when N from N 2 fixation is inadequate to meet plant N needs (Vance et al., 1979). In alfalfa (Medicago sativa L.) this N accumulates primarily as vegetative storage proteins (VSPs, Cunningham and Volenec, 1996) that, like seed storage proteins, are rapidly degraded and translocated to regrowing shoots to meet their N needs. Like alfalfa, white clover (Trifolium repens L.) also appears to accumulate species-specific VSPs during autumn that are mobilized when growth resumes in spring (Corre et al., 1996). ...
Nitrogen (N) reserves in vegetative tissues contribute N to regrowth of Miscanthus × giganteus shoots in spring, but our understanding of how N fertilization and plant genotype affect this process is incomplete. Our specific objectives were to: (1) determine how N fertilizer management impacts accumulation of dry matter and N among aboveground and belowground tissues and organs; (2) understand how changes in N management and tissue N concentration influence seasonal fluctuations in concentrations of buffer-soluble proteins and amino acids in putative storage organs including rhizomes and roots; and (3) characterize genotypic variability and genotype × N interactions for N reserve accumulation and use among Miscanthus × giganteus genotypes. Established plots of the IL Clone and Nagara-sib population were fertilized with 0–0, 0–150, 75–75, 150–0, and 150–150 kg N ha⁻¹ where the first numeral denotes the N rate applied in 2011 (Year 1) and the second number denotes the N rate applied in 2012 (Year 2). Rhizomes, roots, stembases, and shoots were sampled at 6-week intervals between March and August and then in November at dormancy. Concentrations of N, soluble protein and amino-N increased in all tissues with fertilizer N application. With the exception of rhizome amino-N, concentrations of these N pools in roots and rhizomes declined as plants resumed growth in spring and increased sharply between August and November as growth slowed. Losses in shoot and stembase N mass between August and November were similar to total N accumulation in roots and rhizomes during this interval. Compared to the unfertilized control, specific N managements enhanced growth of above- and belowground tissues. The IL Clone generally had greater biomass yield of all organs than the Nagara-sib; the exception being shoot biomass in November when extensive leaf senescence reduce yield of the IL Clone. High biomass yields were obtained with 75 kg N ha⁻¹ applied annually rather than semi-annual N applications of 150 kg N⁻¹ ha that depended on N recycling from roots/rhizomes as a supplemental N source.
... Electrophoretic transfer of polypeptides from SDS-PAGE gels to PVDF membrane (Immobilon-P, Proteigene, Saint-Marcel, France) was conducted using semi-dry electroblotting (2.5 mA for 20 min, Milli Blot system, Proteigene), according to the protocol described previously (Towbin et al., 1979). After blotting, PVDF membranes were treated with affinity-purified polyclonal anti-VSP (15, 19 and 32 kDa) primary antibodies as described earlier (Cunningham and Volenec, 1996). The antigen–antibody complex was visualized with alkaline phosphatase linked to goat anti-rabbit IgG as described previously (Blake et al., 1984). ...
Herbage yield of alfalfa ( Medicago sativa L.) depends on forage management or environmental conditions that change C and N resource acquisition, and endogenous plants factors such as root organic reserves and number of active meristems. The aim of this work is to study the influence of two sowing dates in summer (12 July or 9 August), N fertilization (0 or 100 kg ha ⁻¹ ) and/or irrigation applied during the first year of alfalfa establishment on (i) the accumulation of N organic reserves (soluble proteins and more specifically vegetative storage protein) in taproots during autumn, (ii) the number of crown axillary meristems present at the end of winter and (iii) the dynamics of spring shoot growth. Delaying the sowing date for one month reduced root growth and root N storage, especially vegetative storage proteins (VSP) during autumn. Irrespective of sowing dates, N fertilization did not affect root biomass, number of crown buds, total root N, root soluble protein or VSP concentrations. By contrast, water deficiency during alfalfa establishment in the early summer reduced both root growth and N reserve accumulation. When spring growth resumed, there is a significant linear relationship between leaf area development and soluble protein and VSP concentrations in taproots, and also the number of crown buds. The results showed that an early sowing date and adequate water status during the summer allowed alfalfa plants to accumulate N reserves by increasing taproot mass and soluble protein concentrations, especially VSPs. This resulted in rapid shoot regrowth rates the following spring.
... This shows that there is a close relationship between N availability in the shoot and its re-growth, and that shoot re-growth after defoliation is highly dependent on the nitrogen reserves of the roots. This may support the idea that the re-growth of alfalfa after defoliation could be accelerated by nitrogen (Cunningham and Volenec 1996;Khan et al. 1997Khan et al. /1998Dhont et al. 2003). ...
... Par ailleurs, il existe de nombreuses analogies avec les caractéristiques des VSP du soja ou d'autres espèces : i) Comme les VSP identifiées chez le soja [11,12,13] ou chez l'arabette des dames [14], la synthèse de la protéine de 23 kDa est induite par un apport externe de méthyl-jasmonate. ii) Comme la plupart des VSP étudiées notamment chez les ligneux [15] et chez la luzerne [16], les isoformes de cette protéine sont glycosylées. Le rôle de cette glycosylation serait de favoriser l'adressage de ces protéines vers des compartiments cellulaires de stockage comme la vacuole [17]. ...
Le colza (Brassica napus L.) est une culture connue pour présenter des capacités élevées d’absorption du nitrate, d’où son qualificatif de culture piège à nitrate. Cependant, la quantité d’azote présent dans les tissus récoltés est généralement faible en raison d’une part, d’une faible efficience de remobilisation de l’N foliaire, et d’autre part, de la chute de feuilles sénescentes dont la teneur en azote peut rester élevée (ce qui n’est pas sans conséquence sur le bilan environnemental de cette culture). La mise en réserve transitoire de cet azote par la plante (préalablement à la chute des feuilles) pourrait améliorer l’efficience de remobilisation de l’N des feuilles vers les graines. Il a par exemple été montré chez les espèces ligneuses et herbacées que l’N peut être stocké de façon transitoire sous forme de protéines de réserve des organes végétatifs (VSP). Ces VSP peuvent ensuite être hydrolysées et constituent alors une source d’N non négligeable pour les organes puits en croissance. Les objectifs de ce travail ont donc été de quantifier les flux d’azote au sein de la plante grâce à un marquage 15N, d’identifier les tissus « source » et de mettre en évidence l’existence d’hypothétiques protéines de réserve (VSP).
... Nitrogen is stored in the form of soluble proteins and amino acids (Ta et al. 1990; Avice et al. 1997b;Meuriot et al. 2004a;Dhont et al. 2006b), with soluble protein the primary form of stored N (Hendershot and Volenec 1993;Avice et al. 1997b). In the soluble protein pool, Cunningham and Volenec (1996) have characterised three highly abundant proteins (comprising 20% of the soluble protein pool) that exhibit a cyclic behaviour of preferential depletion and accumulation through a regrowth cycle (Barber et al. 1996), and are characteristic of vegetative storage proteins (VSPs; Staswick 1994). Vegetative storage proteins accumulate within cell vacuoles associated with starch granules in the ray parenchyma cells of the taproot (Avice et al. 1996b). ...
... These storage proteins include plant proteins such as patatin from potato, sporamin from sweet potato, VSPs and lipoxygenase from soybean, crown storage proteins from alfalfa and lectin-like proteins from the bark of certain deciduous trees (Staswick, 1994;Cunningham and Volenec, 1996). ...
... Most of the alternative functions attributed to VSPs are related to abiotic or biotic stress responses. VSPs are thought to be involved in cold tolerance or wounding (Cyr and Bewley 1989, Mason and Mullet 1990, Cunningham and Volenec 1996 as well as in plant resistance to pathogens. In elderberry, taro and several legume trees, vegetative storage proteins have been shown to be lectins and may specifically recognize and bind carbohydrate ligands of others organisms (Van Damme et al. 2002, Shewry 2003. ...
In addition to their putative role in nitrogen storage, some vegetative storage proteins (VSPs) support further roles in biotic and abiotic stress responses. Functions of the 17 kDa VSP from witloof chicory (CiVSP) in N storage and plant resistance to pathogens and its regulation by nitrogen were investigated. The N-terminal end of this protein was sequenced and the corresponding full-length cDNA was obtained. The expression of the CiVsp gene was studied in various organs of chicory grown under replete or limited nitrogen supply. A strong expression of CiVsp was observed in both taproot and fine roots of mature plants and seedlings. CiVsp transcripts were also detected in mature leaves, especially in veins. In senescing leaves CiVsp transcripts accumulated concomitantly to a decrease in RbcS transcript abundance and Rubisco small-subunit degradation. CiVSP protein accumulated significantly only in the subterranean part of the plant during late stages of development. Nitrate limitation caused a reduction in CiVsp mRNA accumulation and a delay in CiVSP storage in the taproot. It is concluded that CiVSP accumulation is regulated at the transcriptional level by N external supply and that the protein is involved in long and short-term N storage. In silico analysis indicated that CiVSP is highly homologous with several allergens and PR-10 proteins. Moreover, CiVsp transcript and protein expression were significantly higher in Erwinia carotovora-resistant chicory inbred lines compared with susceptible lines, suggesting its involvement in chicory resistance to pathogens attack.
... Studies with dormant cultivars also suggest that N-containing compounds accumulate in roots during winter hardening , and that these are used as an N source for regrowing shoots (Volenec et al., 1991; Hendershot and Volenec, 1993 a, Avice et al., 1996; Volenec et al., 1996). Three polypeptides with molecular masses of 15, 19, and 32 kD (Cunningham and Volenec, 1996) accumulate in roots of dormant cultivars in autumn. When shoot growth resumes in spring and after defoliation in summer these polypeptides are preferentially depleted from roots and used as an N source for regrowing shoots (Avice et al., 1996; Barber et al., 1996). ...
Prostrate shoot growth of fall dormant alfalfa (Medicago sativa L.) cultivars in autumn is positively associated with winter survival. Our objective was to determine how carbohydrate and nitrogen pools in roots of alfalfa cultivars exhibiting contrasting fall dormancy change during winter hardening in autumn and when shoot growth resumes in spring. Sugars, buffer-soluble protein, low molecular weight-N, and vegetative storage proteins (VSPs) increased in roots of all cultivars in autumn, while root starch concentrations declined throughout autumn and winter. Sugar, protein, low molecular weight-N, and VSPs levels declined in spring as shoot growth resumed, then re-accumulated in roots as shoots began to flower on June 2. Defoliation on June 2 resulted in a loss of starch, protein, and VSPs from roots as shoots regrew. Roots of fall dormant, winter hardy cultivars contained higher concentrations of sugars and buffer soluble protein in November and December, whereas higher concentrations of starch and low molecular weight-N were found in roots of nondormant cultivars at these times. Concentrations of total N and VSPs were similar between dormant and nondormant cultivars indicating that N deficiency caused by low dinitrogen fixation during hardening is not a factor contributing to the poor winter survival of nondormant alfalfa. Efforts aimed at understanding fall dormancy and winter hardiness of alfalfa should focus on mechanisms controlling accumulation of sugars and specific (non-VSP) soluble proteins in roots in autumn.
... Non-specific binding sites were blocked by immersing membranes in Tris-buffered saline (TBS; 10 mmol/L Tris, 150 mmol/L NaCl, pH 8.0) containing 0.15% (v/v) Tween 20 (TBST) for 30 min. Membranes were incubated for 90 min with antibodies raised to the low or middle molecular weight lucerne VSP (Cunningham and Volenec 1996). Antibodies to the VSP were diluted at 1 : 10 000 in TBST. ...
In the summer dry environment of cool temperate Tasmania, summer irrigation is used to maximise forage
production. For lucerne (Medicago sativa L.) this irrigation is likely to interact with winter-dormancy genotypes to influence
seasonal changes in taproot reserves and thus, the process of cold acclimation. To test this hypothesis four lucerne cultivars
with contrasting levels of winter dormancy (DuPuits: winter-dormant; Grasslands Kaituna: semi winter-dormant; SARDI 7:
winter-active: SARDI 10, highly winter-active) were grown in small plots at Elliott, Tasmania, under irrigated or dryland
conditions. At each defoliation taproots were sampled and assayed for the concentration of soluble sugars, starch, amino
acids, soluble protein, the abundance of vegetative storage proteins (VSP), and the abundance of mRNA transcripts
associated with cold acclimation and VSP. Taproot-soluble protein concentrations in DuPuits significantly increased from
summer to autumn when plants were grown under dryland conditions. When grown under irrigated conditions, taprootsoluble
protein concentrations decreased over summer and increased in autumn for all cultivars. The abundance of VSP
increased in summer in all cultivars grown under dryland conditions. Taproot-soluble sugar concentrations increased and
starch decreased in autumn for all cultivars grown under both water regimes. Plants grown under dryland conditions showed
little change in RNA transcript abundance of cold acclimation genes across all cultivars and sampling dates, while in those
plants grown under irrigated conditions, transcript abundance was influenced by sampling date, and for some genes,
by cultivar. There was a clear carry-over effect from the exposure of summer drought on the winter-dormancy response.
The expression of winter dormancy at an agronomic and molecular level was greater under dryland conditions.
... Plants that were P-deficient contained high levels of taproot starch, but these concentrations were unchanged after defoliation, suggesting that starch use is severely impaired by P deficiency in taproots of this species. Cunningham and Volenec (1996) identified three vegetative storage proteins (VSPs) that are preferentially utilized as a source of N for shoot regrowth. Understanding the role these N pools play in alfalfa shoot regrowth and how this process interacts with P nutrition is vital for alfalfa improvement efforts aimed at increasing persistence and forage yield. ...
Phosphorus (P) deficiency reduces forage yield and stand persistence of alfalfa (Medicago sativa L.). The objective of this study was to determine the influence of P nutrition and defoliation on alfalfa shoot growth, root carbohydrate and protein metabolism, and steady-state mRNA levels for high-affinity P transporters. In a greenhouse study, P-deprived plants were provided with 0, 0.25, 2, and 6 mM P beginning 7 d before shoot removal. Plants were sampled immediately (day -7) on days -5, -2, 0 (day of shoot removal), and on days 1, 2, 6, and 9 post-shoot removal. Addition of P to P-deficient plants stimulated growth of shoots but not roots. Taproot bark sugar concentrations were reduced significantly in cut plants at any rate of P, whereas only the 6 mM P treatment reduced taproot wood sugar concentrations in uncut plants. There was a significant defoliation-induced decline in both wood and bark sugar and amino-acid concentration that was enhanced at high P rates. Low P reduced utilization of starch and protein reserves in taproots. Transcripts for a high-affinity P transporter were not detected in any root or shoot tissue assayed, irrespective of defoliation or P treatment. The uncertain relationship between P availability and P-transporter transcript abundance in our greenhouse-grown plants requires additional investigation.
... Qualitative analysis of soluble proteins by SDS–PAGE has showed that three polypeptides of 32, 19 and 15 kDa, representing up to 40% of water-soluble proteins of taproots, exhibit a pattern of preferential mobilization and accumulation when shoot growth resumed in spring (Hendershot and Volenec 1993a) or after cutting (Hendershot and Volenec 1993b; Avice et al. 1996). Polyclonal antibodies raised against these VSPs (Cunningham and Volenec 1996 ) were used to immunolocalize them, primarily in vacuoles of parenchyma cells of wood rays and bark of alfalfa taproots (Avice et al. 1996). ...
Our objective was to study the effect of short-day photoperiod for 28, 42 and 56 d on growth, N uptake and N partitioning, particularly vegetative storage protein (VSP) accumulation in taproots of two alfalfa (Medicago sativa L.) cultivars (Lodi and Europe). For both varieties, the reduction of daylength from 16 h (long day, LD) to 8 h (short day, SD) for 28 d reduced total plant growth by decreasing shoot growth. Nitrogen uptake and N distribution within the plant was determined by 15N labeling. N uptake decreased with SD treatment duration, and was 2- and 3-fold lower for Europe and Lodi, respectively, for 56 d in SD conditions when compared with LD plants. The SD treatment resulted in preferential partitioning of N to taproots in comparison with LD conditions (19 vs 9% for Lodi and 12 vs 5% for Europe after 28 d). For both cultivars, the SD-induced changes in N allocation to taproots did not significantly affect taproot soluble protein concentrations during 42 d of daylength treatment. In contrast, VSP accumulation occurred after only 28 d for plants grown in SD conditions (6.2 vs 4.8 mg g–1 DW for Lodi and 5.1 vs 1.4 mg g–1 DW for Europe). SD exposure also increased vsp 57 and vsp 32 mRNA transcript levels in Lodi and Europe (up to 2-fold higher) taproots in SD for 28 d compared with LD conditions. Overall results indicate that photoperiod modulates taproot N accumulation in alfalfa by enhancing both β-amylase (vsp 57) and vsp 32 gene expression and accumulation. The enhanced VSP accumulation by short-day photoperiod may result from altered VSP gene expression / transcript stability or occur indirectly through altered N source–sink relationships. Additionally, when SD treatment included a night break with 15 min illumination with sodium high pressure light or red light, our results suggest that the induction of vsp 57 and vsp 32 gene expressions by SD signal is mediated by the phytochrome system.
... This latter protein from Jerusalem artichoke is antigenically related to the 18 kDa protein in dandelion roots; both species are members of the Compositae. Several low-molecular-mass proteins (15-32 kDa) are present in alfalfa (Medicago sativa) taproots (but not a similar 18 kDa protein); these accumulate during the autumn and early winter, and rapidly decline when spring growth commences (Hendershot & Volenec 1993;Cunningham & Volenec 1996). In white clover (Trifolium repens) 18 kDa proteins occur in the stolons, and two 15 kDa proteins in the roots are mobilized during early regrowth of defoliated plants (Corre et al. 1996). ...
The root of the persistent weed, dandelion (Taraxacum officinale Weber), contains a predominant 18 kDa protein which undergoes small seasonal fluctuations in amount, increasing in the late autumn months, and declining in the spring. This protein has been purified and found to consist of two major isoforms of pI 5·56 and pI 5·49. A full-length cDNA has been obtained, coding for the pI 5·56 isoform, and the 156-amino-acid sequence deduced. The protein shows homologies in amino acid composition to several allergen and intracellular pathogenesis-related proteins. The deduced protein does not contain a signal peptide nor any known organelle-targeting sequences, and thus is likely to be cytosolic. Expression of the 18 kDa protein gene is exclusive to the roots and stem tissues; transcripts accumulate during the late autumn months, and decline in the early spring. Changes in the amount of protein in the root are much less. The mRNA for the 18 kDa protein is not present in the dry seed, but appears in the roots within 16–18 h from the start of imbibition, and is expressed constitutively thereafter. Although it is the predominant protein in dandelion roots, its properties are different from those commonly associated with vegetative storage proteins.
... SDS-PAGE was performed using a 150 g l (1 acrylamide separation gel. Separated proteins were blotted onto PVDF membrane for Western blotting analysis using the anti-32, -19 and -15 kDa VSP antibodies [35,25]. Amino acids were analyzed using HPLC on a Beckman Gold 8.0 system (Beckman, Roissy, France). ...
Our objective was to understand how mineral N availability alters accumulation of N reserves (nitrate, amino acids, soluble proteins and vegetative storage proteins known as VSP) in alfalfa (Medicago sativa L. cv Lodi) taproots. The effects of variation in NH4NO3 availability were followed by studying non-nodulated plants grown under hydroponic conditions during 21 days with (i) different N supplies which corresponded to N-replete plants (N100%=optimal N) and N-limited plants receiving only 50% (N50%) or 25% (N25%) of optimal N (Experiment I), or (ii) decreasing concentrations of NH4NO3 (1000, 250, 100 or 50 μM, Experiment II). Regardless of the N-limitation mode (Experiments I or II), and compared with higher N treatments (N100 or 1000 μM), there was a significant reduction of total shoot dry matter per plant for lowest N treatments (N25 or 50 μM). This was accentuated by the degree of N deficiency in Experiment I only. In Experiment II, taproot biomass significantly increased for low N treatments. In both experiments, total N, nitrate and amino acid concentrations in taproots increased for high N treatments, while the concentration of soluble proteins, and particularly VSP, increased for low N treatments. These results indicated that non-nodulated alfalfa was able to accumulate N reserves (mainly as VSPs), even under N-limited conditions, while under high mineral N availability, taproot amino acid concentrations (mainly asparagine) increased without a corresponding increase in soluble protein concentration. These results show that alfalfa was capable of optimizing N cycling and storage as a function of mineral N availability. These adaptive responses to low soil N environments also allow alfalfa to go dormant and perenniate, while awaiting more favorable conditions for shoot growth.
... The amino acid sequence on the HMW VSP upstream from the box containing the N-terminal sequence has been identi®ed as a signal sequence using Signal-P (Nielsen et al., 1997 ). Immunoblot analysis of proteins extracted from M. truncatula roots using antisera raised to alfalfa taproot VSPs detected small quantities of taproot VSP in one of the two cultivars studied (Cunningham and Volenec, 1996). This indicates that proteins similar to these alfalfa taproot VSPs can be found in M. truncatula roots, but their function as a VSP in M. truncatula has not been demonstrated. ...
The objective of this paper is to assess the effectiveness of alfalfa (Medicago sativa L.) improvement efforts over the last century, and with the advent of molecular biology, identify challenges for alfalfa improvement in the future. Yield trials conducted between 1986 and 1998 from around the US were used to compare yield and persistence of older alfalfa cultivars to those released in the 1990s. First and second harvest forage yield of recently released alfalfa cultivars were not improved over those of older cultivars. New cultivars had higher forage yield at fourth harvest, in early September, possibly due to a reduction in fall dormancy. Efforts to improve alfalfa persistence by breeding for improved disease resistance and greater winter hardiness also have not been effective at most locations. Use of molecular biology for alfalfa improvement depends upon identifying genes that control important agronomic traits that translate into greater yield, improved persistence, and enhanced forage quality. Few such genes have been identified in alfalfa, and their use might be complicated by the polyploid nature of this outcrossing species. The Medicago truncatula genome project is providing large amounts of sequence information, but little is known about the regulation of these genes and the function of their protein products in planta. Uncertainty exists regarding the effectiveness of transferring these genes to alfalfa to obtain a desired phenotype. Much remains to be done to identify key genes that determine agronomic performance of crop plants, including alfalfa, and to clarify mechanisms that regulate the expression of genes and the function(s) of their protein products under field conditions. Future efforts to improve agronomic performance of alfalfa will be enhanced by partnerships between public and private scientists because companies now dominate commercial release of new alfalfa cultivars.
... Methods used for immunoblotting were based on a modification from Cunningham and Volenec (1996). Proteins from SDS-PAGE gels were transferred to a polyvinylidene fluoride (PVDF) membrane (IPVH00010, Millipore, USA) with a VE-186 electrophoresis transfer unit (Tian Neng, Shanghai, China) using a continuous buffer system [0.39 M glycine, 0.48 M Tris, 20% methanol (v/v)] at 100 V for 1 h. ...
This study was designed to identify physiological responses and differential proteomic responses to salinity stress in roots of a salt-tolerant grass species, seashore paspalum (Paspalum vaginatum), and a salt-sensitive grass species, centipedegrass (Eremochloa ophiuroides). Plants of both species were exposed to salinity stress by watering the soil with 300 mM NaCl solution for 20 d in a growth chamber. The 2-DE analysis revealed that the abundance of 8 protein spots significantly increased and 14 significantly decreased in seashore paspalum, while 19 and 16 protein spots exhibited increase and decrease in abundance in centipedegrass, respectively. Eight protein spots that exhibited enhanced abundance in seashore paspalum under salinity stress were subjected to mass spectrometry analysis. Seven protein spots were successfully identified, they are peroxidase (POD, 2.36-fold), cytoplasmic malate dehydrogenase (cMDH, 5.84-fold), asorbate peroxidase (APX, 4.03-fold), two mitochondrial ATPSδ chain (2.26-fold and 4.78-fold), hypothetical protein LOC100274119 (5.01-fold) and flavoprotein wrbA (2.20-fold), respectively. Immunblotting analysis indicated that POD and ATPSδ chain were significantly up-regulated in seashore paspalum at 20 d of salinity treatment while almost no expression in both control and salt treatment of centipedegrass. These results indicated that the superior salinity tolerance in seashore paspalum, compared to centipedegrass, could be associated with a high abundance of proteins involved in ROS detoxification and energy metabolism.
... Electrophoretic transfer of polypeptides from SDS-PAGE gels to PVDF membrane (Immobilon-P, Proteigene, Saint-Marcel, France) was conducted using semi-dry electroblotting (2.5 mA for 20 min, Milli Blot system, Proteigene), according to the protocol described previously (Towbin et al., 1979). After blotting, PVDF membranes were treated with affinity-purified polyclonal anti-VSP (15, 19 and 32 kDa) primary antibodies as described earlier (Cunningham and Volenec, 1996). The antigen–antibody complex was visualized with alkaline phosphatase linked to goat anti-rabbit IgG as described previously (Blake et al., 1984). ...
Herbage yield of alfalfa (Medicago sativa L.) depends on forage management or environmental conditions that change C and N resource acquisition, and endogenous plants factors such as root organic reserves and number of active meristems. The aim of this work is to study the influence of two sowing dates in summer (12 July or 9 August), N fertilization (0 or 100 kg ha(-1)) and/or irrigation applied during the first year of alfalfa establishment on (i) the accumulation of N organic reserves (soluble proteins and more specifically vegetative storage protein) in taproots during autumn, (ii) the number of crown axillary meristems present at the end of winter and (iii) the dynamics of spring shoot growth. Delaying the sowing date for one month reduced root growth and root N storage, especially vegetative storage proteins (VSP) during autumn. Irrespective of sowing dates, N fertilization did not affect root biomass, number of crown buds, total root N, root soluble protein or VSP concentrations. By contrast, water deficiency during alfalfa establishment in the early summer reduced both root growth and N reserve accumulation. When spring growth resumed, there is a significant linear relationship between leaf area development and soluble protein and VSP concentrations in taproots, and also the number of crown buds. The results showed that an early sowing date and adequate water status during the summer allowed alfalfa plants to accumulate N reserves by increasing taproot mass and soluble protein concentrations, especially VSPs. This resulted in rapid shoot regrowth rates the following spring.
... These results showed that, like most vegetative storage proteins identified, for example, in woody species (Stépien et al., 1994), soybean (Wittenbach, 1983) or alfalfa (Cunningham and Volenec, 1996), the two 23 kDa isoforms of Brassica napus L. taproot are also glycosylated . The role of these oligosaccharide chains remains unclear. ...
In taproot of oilseed rape (Brassica napus L.), a 23 kDa polypeptide has been recently identified as a putative vegetative storage protein (VSP) because of its accumulation during flowering and its specific mobilization to sustain grain filling when N uptake is strongly reduced. The objectives were to characterize this protein more precisely and to study the effect of environmental factors (N availability, daylength, temperature, water deficit, wounding) or endogenous signals (methyl jasmonate, abscisic acid) that might change the N source/sink relationships within the plant, and may therefore trigger its accumulation. The 23 kDa putative VSP has two isoforms, is glycosylated and both isoforms share the same N-terminal sequence which had been used to produce specific polyclonal antibodies. Low levels of an immunoreactive protein of 24 kDa were found in leaves and flowers. In taproot, the 23 kDa putative VSP seems to accumulate only in the vacuoles of peripheral cortical parenchyma cells, around the phloem vessels. Among all treatments tested, the accumulation of this protein could only be induced by abscisic acid and methyl jasmonate. When compared to control plants, application of methyl jasmonate reduced N uptake by 89% after 15 d, induced a strong remobilization of N from senescing leaves and a concomitant accumulation of the 23 kDa putative VSP. These results suggested that, in rape, the 23 kDa protein is used as a storage buffer between N losses from senescing leaves promoted by methyl jasmonate and grain filling.
... Protein analysis was adapted from Cunningham and Volenec (1996). Proteins were extracted by suspending 300 mg of ground, freeze-dried taproot with 5 ml of extraction buffer (100 mM sodium phosphate, 10 mM b-mercaptoethanol, pH 7) at 4 C. ...
This study presents the effects of methyl jasmonate (MeJA) on growth, N uptake, N partitioning, and N storage in taproots of non-nodulated alfalfa (cv. Lodi). When compared to untreated plants, addition of 100 micro M MeJA to the nutrient solution for 14 days reduced total growth and modified biomass partitioning between shoots and roots in favour of taproots and lateral roots. MeJA decreased N uptake (after 7 days) and increased N partitioning towards roots after 14 days. This preferential N partitioning to roots was accompanied by increased N storage in taproots as soluble proteins. Compared to total soluble proteins, VSP accumulation occurred earlier (7 days), and was greater (2-fold increase) in plants treated with 100 micro M MeJA. Steady-state transcript levels for two VSPs (32 and 57 kDa) also increased markedly (about 4-fold) in roots of plants treated with 100 micro M MeJA. This suggests that MeJA could act directly (transcriptional regulation) or indirectly (via the changes of N partitioning among alfalfa organs) on N storage as soluble proteins and in particular, VSPs. Because the deduced amino acid sequence of the 32 kDa VSP clone reveals high homology with Class III chitinases, we propose that the 32 kDa VSP may have a role in pathogen defense, in addition to its function as a storage protein.
The adverse effect of fall harvesting alfalfa (Medicago saliva L.) during a critical rest period on persistence and the following spring regrowth has been historically attributed to a reduction in the concentrations of organic reserves, especially total nonstrudural carbohydrates. However, recent reports highlight the determinant role of N reserves in overwintering and spring regrowth of alfalfa. This study was undertaken to assess the impact of fall harvest management on regrowth potential in relation to the quantitative changes in N reserves in alfalfa taproots throughout fall and winter. The experiment was conducted under simulated winter conditions in an unheated greenhouse with two alfalfa cultivars (AC Caribou and WL 225). The fall harvest treatments were no additional fall harvest (two harvests = control) or a third fall harvest applied at 400, 500, or 600 growing degree days (GDD) after the second harvest. Total N concentrations were significantly reduced in plants harvested at 400 or 500 GDD as compared with plants harvested at 600 GDD or harvested only twice. The striking accumulation of proline, arginine, and histidine observed in fall and winter was depressed by a fall harvest, especially in plants harvested at 400 or 500 GDD. The abundance of a major soluble protein of 32 kDa was reduced by harvesting at 400 or 500 GDD. Concentrations of major N components were correlated with shoot regrowth in spring in AC Caribou, but not in WL 225. However, the total amounts of major N components in taproots were correlated with spring regrowth In both cultivars. Our results point out that N reserves available in roots are determinant for spring regrowth in alfalfa under various fall harvest treatments.
Our long-term goal is to improve persistence and yield of alfalfa (Medicago sativa
L.) and other forage legumes by identifying and manipulating genes that affect these traits.
Future improvements by genetic manipulation depend, however, on new insights into
basic physiological and biochemical plant processes. Currently we lack knowledge of discrete
traits controlling agronomic performance that can serve as targets for manipulation
using modern genetic techniques. Our work, and recent work of others, has failed to show
a positive association between root total nonstructural carbohydrate (TNC) levels and
genetic variation in regrowth and winterhardiness of forage legumes. We are exploring
alternatives to the conventional thinking that root TNC reserves control alfalfa regrowth
and persistence. Recent results indicate that root N declines during herbage regrowth after
defoliation, and again in spring when shoot growth resumes. Labeling studies have proven
that much of the N found in shoots during early regrowth is derived from root N pools. In
alfalfa, certain root N pools, especially root vegetative storage proteins (VSPs) are preferentially
used as N reserves during the early stages of shoot regrowth. The VSPs represent
25% of the root protein pool. They are unique to alfalfa roots, and their synthesis is developmentally
regulated. Work is underway to isolate and characterize the cDNAs for the
VSPs to learn more about regulation of VSP synthesis and degradation in alfalfa roots.
Unlike grain crops, the economic yield of alfalfa (Medicago sativa L.) is herbaceous, composed predominantly of leaves and stems. Understanding physiological control of alfalfa yield, therefore, requires careful evaluation of factors influencing leaf and stem initiation and growth. Alfalfa yield is determined by the total amount of dry matter produced and the partitioning of dry matter. Partitioning of dry matter to crowns and roots also influences the survival of this perennial species. Natural selection may favor alfalfa survival over herbage production in most ecosystems. Finally, recent emphasis on production of high-quality alfalfa forage results in frequent defoliation at immature stages of development. As a result, increasing yield of high-quality forage without compromising alfalfa survival presents a challenging opportunity.
The ST (ShooT Specific) proteins are a new family of proteins characterized by a signal peptide, tandem repeats of 25/26 amino acids, and a domain of unknown function (DUF2775), whose presence is limited to a few families of dicotyledonous plants, mainly Fabaceae and Asteraceae. Their function remains unknown, although involvement in plant growth, fruit morphogenesis or in biotic and abiotic interactions have been suggested. This work is focused on ST1, a Cicer arietinum ST protein. We established the protein accumulation in different tissues and organs of chickpea seedlings and plants and its subcellular localization, which could indicate the possible function of ST1. The raising of specific antibodies against ST1 protein revealed that its accumulation in epicotyls and radicles was related to their elongation rate. Its pattern of tissue location in cotyledons during seed formation and early seed germination, as well as its localization in the perivascular fibres of epicotyls and radicles, indicated a possible involvement in seed germination and seedling growth. ST1 protein appears both inside the cell and in the cell wall. This double subcellular localization was found in every organ in which the ST1 protein was detected: seeds, cotyledons and seedling epicotyls and radicles.
Several studies have previously shown that shoot removal of forage species, either by cutting or herbivore grazing, results in a large decline in N uptake (60%) and/or N2 fixation (80%). The source of N used for initial shoot growth following defoliation relies mainly on mobilisation of N reserves from tissues remaining after defoliation. To date, most studies investigating N-mobilisation have been conducted, with isolated plants grown in controlled conditions. The objectives of this study were for Lolium perenne L., grown in a dense canopy in field conditions, to determine: 1) the contribution of N-mobilisation, NH4+ uptake and NO3- uptake to growing shoots after defoliation, and 2) the contribution of the high (HATS) and low (LATS) affinity transport systems to the total plant uptake of NH4+ and NO3-. During the first seven days following defoliation, decreases in biomass and N-content of roots (34% and 47%, respectively) and to a lesser extent stubble (18% and 43%, respectively) were observed, concomitant with mobilisation of N to shoots. The proportion and origin of N used by shoots (derived from reserves or uptake) was similar to data reported for isolated plants. Both HATS and LATS contributed to the total root uptake of NH4+ and NO3-. The Vmax of both the NH4+ and NO3- HATS increased as a function of time after defoliation, and both HATS systems were saturated by substrate concentrations in the soil at all times. The capacity of the LATS was reduced as soil NO3- and NH4+ concentrations decreased following defoliation. Data from 15N uptake by field-grown plants, and uptake rates of NH4+ and NO3- estimated by excised root bioassays, were significantly correlated, though uptake was over-estimated by the later method. The results are discussed in terms of putative mechanisms for regulating N uptake following severe defoliation.
Growth of alfalfa (Medicago sativa L.) following the correction of water deficits will depend, in part, on the amount of plant constituents available to support regrowth and the availability of crown buds for the development of new shoots. To investigate if water deficit affected the accumulation of taproot constituents and crown bud development of alfalfa, two alfalfa cultivars were exposed to increasing levels of water deficit (100, 75, 50 25 and 0% of the replacement water requirement) during a regrowth period. Shoot growth was reduced in proportion to the level of water deficit. Taproot starch concentrations decreased while taproot soluble sugar increased in plants exposed to a water deficit. Thirty‐five d after initiation of treatments, taproot protein and amino acid concentrations were similar in all treatments that received water. Water deficit increased the relative concentration of low and middle molecular weight taproot vegetative storage proteins (VSP) and water deficits reduced the number and mass of green crown buds. Both cultivars had a similar response to water deficit for all taproot constituents and crown bud development. Further studies are required to determine the influence of increasing VSP abundance and decreasing numbers of crown buds on plant recovery following the removal of a water deficit.
Genotypic variation in winter injury exists among zoysiagrasses (Zoysia spp.), but the physiological basis for these differences is not understood. Our objective was to determine the relationships between protein accumulation, polypeptide composition, and freeze tolerance of zoysiagrass. Thirteen genotypes of zoysiagrass with contrasting cold hardiness were identified. Cold acclimation was induced with 4 wk of 8/2°C day/night cycles and a 10‐h photoperiod of 300 μmol m⁻² s⁻¹ Rhizomes and stolons of zoysiagrass were harvested from nonacclimated and cold‐acclimated plants and used for protein analysis. Protein composition was analyzed using sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS‐PAGE) and immunoblotting with an antidehydrin polyclonal antibody. Buffer‐soluble protein concentrations were higher among cold‐acclimated (7.3 g kg⁻¹ dry wt.) than nonacclimated (5.1 g kg⁻¹ dry wt.) plants. The SDS‐PAGE analysis indicated few differences in polypeptide composition among genotypes irrespective of cold acclimation. Immunoblotting indicated that dehydrin polypeptides (23 and 25 kDa) increased during cold acclimation. Abundance of the 23‐kDa dehydrin polypeptide was positively associated (r² = 0.41) with genetic variation in freezing tolerance. Our results suggest that dehydrins are associated with zoysiagrass cold acclimation, but that only the 23‐kDa dehydrin plays a role in improving freeze tolerance.
Fertilization with K and P impacts alfalfa (Medicago sativa L.) yield, but how these nutrients influence taproot reserves and gene expression is unknown. Our objectives were to determine how P and K impact (i) alfalfa yield and yield components, (ii) accumulation and use of taproot carbon (C) and nitrogen (N) pools, and (iii) transcript levels for beta-amylase, sucrose synthase, and the high molecular weight vegetative storage protein in alfalfa taproots. Yield and yield components were determined at 30-d intervals beginning in late May. Roots were sampled after the late June harvest (Day 0) and 1, 3, 6, 10, 14, 21, and 30 d thereafter. Addition of P and K increased forage yield by enhancing mass per shoot. High P resulted in rapid starch use, while taproots of plants fertilized with K had low sugar concentrations. Transcripts decline by Day 6 and by Day 10 were below detection limits. Transcripts for beta-amylase and sucrose synthase accumulated rapidly after Day 10 in taproots of plants fertilized with both P and K. Balanced P and K nutrition resulted in the accumulation and effective utilization of C and N reserves and in improved alfalfa adaptation to defoliation.
Harvesting alfalfa (Medicago sativa L.) after mid-September in the North-Central USA often reduces plant winter survival, but the physiological mechanisms associated with poor winter survival are not understood. Our objective was to determine how autumn harvesting affects alfalfa root physiology, gene expression, and plant winter survival. In Exp. 1, seven fall harvest dates were used to identify 1 to 15 October as a critical interval where significant changes in alfalfa winter survival and root physiology occur in Indiana. In Exp. 2, rows of six alfalfa cultivars possessing contrasting fall dormancy (FD) were established in May. Plants in one-half of each row were defoliated in mid-October, and roots were sampled at this defoliation and again in December. Winter injury was determined in mid-April. Shoot removal in mid-October increased winter injury and reduced plant vigor in spring. As expected, the October defoliation reduced root protein and starch concentrations in December, but unexpectedly increased root sugar concentrations. In addition, defoliation did not reduce the steady state transcript levels of several cold-acclimation responsive (car) genes that are associated with genetic variation in winter survival. Although positively associated with genetic differences in winter hardiness, factors other than root sugar accumulation and expression of these car genes regulate defoliation-induced changes in winter survival of these alfalfa cultivars.
Fall dormancy is positively associated with alfalfa (Medicago sativa L.) winter survival, but the physiological bases for this association are not understood. Our objective was to determine how incremental changes in fall dormancy due to genetic selection influenced autumn height and winter survival, root physiology, and expression of a cold acclimation responsive gene family. Seed from each of three cycles of selection for contrasting (greater or less) fall dormancy using 'Mesilla' and 'CUF 101' as parents were planted in rows in the field (Starks-Fincastle, fine-silty, mixed, mesic, Aeric Ochraqualf) in West Lafayette, IN, in May 1997 and 1998. Plant height was measured in October and roots were sampled in December. Plant survival was determined in March of the year following seeding. Fall dormancy (reduction in shoot height in October) increased in a linear manner over the three cycles of selection for both Mesilla and CUF 101. A positive linear relationship was observed between fall height and winter injury in both years. Root sugar and protein concentrations increased as fall dormancy increased in populations derived from both Mesilla and CUF 101. Expression of the cold acclimation-responsive gene, RootCAR1, was positively associated with winter survival, and may be useful as a molecular marker for identifying winter hardy plants among semi-dormant or nondormant alfalfa germplasm in December of the seeding year.
The regrowth interval between the last summer harvest and the fall harvest is a major determinant of alfalfa (Medicago sativa L.) persistence and spring regrowth. This study assessed the impact of the timing of a fall harvest on persistence, dry matter yield (DMY), and root carbohydrate and N reserves of field-grown alfalfa. Two cultivars (AC Caribou and WL 225) were harvested either only twice during the summer or three times with the third harvest taken 400, 500, or 600 growing degree days (GDD) after the second summer harvest. Plant density was not affected by a fall harvest across three production years (1997 to 1999). The DMY of the first harvest in the second production year (1998) was significantly reduced by prior fall harvests taken at 400 or 500 GDD. However, the seasonal DMY in 1998 was higher with the 500 or 600 GDD harvest management than with the two harvest system. Root dry weight (DW) was generally reduced by a third harvest in the fall, especially when taken at 400 GDD. Amounts of root carbohydrate reserves (raffinose family oligosaccharides [RFO], sucrose, and starch), as well as the amounts of total amino acids and soluble proteins were significantly reduced by harvesting at 400 or 500 GDD. Untimely fall harvest reduced alfalfa spring regrowth by impeding the accumulation of both carbohydrate and N reserves. Seasonal DMY remained advantageous as long as an interval of 500 or 600 GDD was kept between the last summer and the fall harvests.
In perennial forage legumes such as alfalfa (Medicago sativa L.) and white clover (Trifolium repens L.), vegetative storage proteins are extensively mobilized to meet the nitrogen requirements of new shoot growth in spring or after cutting in summer. The 32-kDa alfalfa storage protein possesses high homology with class III chitinases, belonging to a group of pathogenesis-related proteins that possess antifreeze protein properties in some species and exhibit chitinolytic activity in vitro. This protein and the corresponding mRNA accumulate in taproots of cold-hardy culti vars during acclimation for winter, and in response to short-day conditions in controlled environments. The 17.3-kDa storage protein of white clover possesses high homology with pathogenesis-related proteins and abscisic- acid-responsive proteins from several legume species and has characteristics common to stress-responsive proteins. Low temperature enhances accumulation of this 17.3-kDa protein and its corresponding transcript. Exogenous abscisic acid stimulates the accumulation of vegetative storage proteins and their transcripts in both legume species. These observations suggest that vegetative storage proteins do not exclusively serve as nitrogen reserves during specific phases of legume development, but may play important adaptive roles in plant protection against abiotic (low temperature) and biotic (pathogen attack) stresses.Key words: nitrogen reserves, vegetative storage proteins, regulation, cold tolerance, chitinase, pathogenesis-related proteins.
The presence of storage proteins has been reported in roots of several perennial and biennial weed and crop species, and particularly in members of the Compositae, Euphorbiaceae, and Leguminosae. In some species the amount of these root proteins fluctuates seasonally, increasing in the fall and winter months and declining in the spring and early summer. Also, the root proteins may decline during regrowth of decapitated plants. The evidence that these proteins play a role as storage proteins is frequently only circumstantial; moreover, they are usually only a relatively minor component of the total nitrogen pool within the root. Only one root protein, that from the dandelion taproot, has been extensively characterized, and it has no properties in common with known vegetative storage proteins. The literature on root proteins is reviewed, with particular emphasis on those present in taproots. The paucity of definitive data allows few conclusions to be reached, and more research is required to establish the role, nature, and importance of root proteins.Key words: taproots, perennial weeds, root proteins, nitrogen pools, storage proteins.
In a pot experiment the responses of two alfalfa cultivars differing in salt tolerance were evaluated in terms of root nitrogen remobilization rates (RNRR) and their relationship with the ionic status of the plants. A split-plot design with factorial treatments in three replications was used. Three levels of salinity stress with electrical conductivities (ECs) of 1.2, 7 and 12 ds m−1 were established in irrigation water by using tap water with and without NaCl. The average data taken from plant materials at three defoliations were used for statistical analysis. Each time, plant materials were harvested at the 10 % flowering stage and then 10 days later. From the results observed, it was found that alfalfa shoot growth is highly dependent on RNRR under salinity stress. However, the total N reserves within the roots do not appear to be a limiting factor. The high positive correlation coefficient between shoot K+/Na+ and RNRR (r = 0.77; P = 0.01) indicates that lower demands for N because of diminished metabolic activities within the shoot sink may have reduced the rates of root N utilization. Unlike in some other species, the shoot K+ concentration and contents of alfalfa plants were significantly reduced by increasing salt stress. However, a relatively suitable K+/Na+ ratio of 7.1 is maintained in the shoots at the second level of salinity, as lowering the rates of salt induced an increase in Na+ uptake (Na exclusion). The salt tolerance recognized in the Bami cultivar may be attributed to the 339 % increase in its selectivity rates of K+ over Na+ in ion transport from the soil to the shoots, as the shoot Na+ content did not increase with increasing salt levels.
Nitrogen (N) allocated to leaf growth in forage grasses and legumes following severe defoliation is predominately mobilized from the remaining root and leaf sheath tissues, since both N uptake from the soil and N2 fixation are severely down-regulated for several days. The hypothesis that a low N reserve status at the time of defoliation limits N remobilization and leaf regrowth was tested with contrasting cultivars of Lolium perenne (cvs Aberelan and Cariad) in flowing solution culture. Plants were grown under ‘high’ or ‘low’ (uptake of N decreased by 50%) regimes of N supply for 10 d before a single severe defoliation. Labelling with 15N was used to assess the importance of N reserves, including putative vegetative storage proteins, relative to N translocated from concurrent uptake, as a source of leaf N during regrowth. Leaf regrowth, N uptake and N mobilization were all affected by previous N supply. Low plant N status at the time of defoliation increased regrowth dry weight of ‘Aberelan’ by 10% and translocation of N absorbed from the medium by 23%, while mobilization of N reserves was decreased by 56%. On the contrary, regrowth dry weight of ‘Cariad’ was decreased by 23%, and translocation of N absorbed by 21% in low plant N status, compared with high plant N status. Concentrations of soluble protein in roots and remaining leaf sheaths decreased after defoliation in plants only under optimal N supply. Analysis of soluble proteins in sheath material by SDS–PAGE suggested that three polypeptides (55, 36.6 and 24 kDa) might function as vegetative storage proteins, although they were of low abundance in plants, subjected to monthly harvests, grown in controlled conditions and in the field. The apparent antagonism between uptake of NH4+ or NO3− by roots and mobilization of N reserves is discussed together with evidence for functional vegetative storage proteins in L. perenne.
Soybean vegetative storage proteins (S-VSPs) are a group of high-lysine proteins. These proteins can accumulate up to 15% of the soluble leaf proteins in young shoots, as well as in shoots of depodded mature plants. Closely related proteins are found in forage crops, such as alfalfa. We have expressed the S-VSPα gene, fused to the strong constitutive cauliflower mosaic virus 35S promoter, in transgenic tobacco as a model plant to study the potential of producing high levels of S-VSPs for the nutritional improvement of forage crops. S-VSPα was detected in the soluble (albumin) fraction of the transgenic plants. The transgenic protein migrated in sodium dodecyl sulfate-polyacrylamide gel electrophoresis similarly to the natural S-VSPα of soybean, suggesting that it was correctly processed in tobacco and accumulated in the vacuoles. The S-VSPα ranged between 2 and 6% of the soluble proteins in leaves of the transgenic plants and was present in various organs and in mature leaves. Our results suggest that S-VSPs can serve as an excellent protein source for improving the nutritional quality of crop plants, particularly cereal forage and grains, which contain limiting levels of lysine.
Oilseed rope (Brassica napus L.) is known for its high capacity to absorb mineral N from the soil profile and therefore can be grown as a catch crop to reduce nitrate leaching from arable cropping systems. However, the N harvest index of this crop is usually low, due in part to a low ability to remobilize N from leaves and to the fall of N-rich leaves which allows a significant amount of N to return to the environment. Our hypothesis of work was that the efficiency with which leaf N is mobilized to the seeds might be improved by increasing N storage within the plant through alternative sinks. It has, for example, been shown in both herbaceous and woody species that N con be transiently stored as vegetative storage proteins (VSP), which con be hydrolyzed to supply developing sinks with N. The objectives of this study were first, to quantify and distinguish by N-15 labelling between the origin of the N used for pod filling, namely from uptake or from vegetative tissue mobilization, secondly to describe the kinetic behaviour of different source tissues and, lastly, to identify some proteins that might be involved in N storage.
Lea, P. J.; Morot-Gaudry, J. F. (eds.) This book covers all aspects of the transfer of nitrogen from the soil and air to a final resting place in the seed protein of a crop plant. It describes the physiological and molecular mechanisms of ammonium and nitrate transport and assimilation, including symbiotic nitrogen fixation by the Rhizobiacea. Amino acid metabolism and nitrogen traffic during plant growth and development and details of protein biosynthesis in the seeds are also extensively covered. Finally, the effects of the application of nitrogen fertilisers on plant growth, crop yield and the environment are discussed. Written by international experts in their field, Plant Nitrogen is essential reading for all plant biochemists, biotechnologists, molecular biologists and physiologists as well as plant breeders, agricultural engineers, agronomists and phytochemists.
Plants store amino acids for longer periods in the form of specific storage proteins. These are deposited in seeds, in root and shoot tubers, in the wood and bark parenchyma of trees and in other vegetative organs. Storage proteins are protected against uncontrolled premature degradation by several mechanisms. The major one is to deposit the storage proteins into specialized membrane-bounded storage organelles, called protein bodies (PB). In the endosperm cells of maize and rice prolamins are sequestered into PBs which are derived from the endoplasmic reticulum (ER). Globulins, the typical storage proteins of dicotyledonous plants, and prolamins of some cereals are transported from the ER through the Golgi apparatus and then into protein storage vacuoles (PSV) which later become transformed into PBs. Sorting and targeting of storage proteins begins during their biosynthesis on membrane-bound polysomes where an N-terminal signal peptide mediates their segregation into the lumen of the ER. After cleavage of the signal peptide, the polypeptides are glycosylated and folded with the aid of chaperones. While still in the ER, disulfide bridges are formed which stabilize the structure and several polypeptides are joined to form an oligomer which has the proper conformation to be either deposited in ER-derived PB or to be further transferred to the PSV. At the trans-Golgi cisternae transport vesicles are sequestered which carry the storage proteins to the PSV. Several storage proteins are also processed after arriving in the PSVs in order to generate a conformation that is capable of final deposition. Some storage protein precursors have short N- or C-terminal targeting sequences which are detached after arrival in the PSV. Others have been shown to have internal sequence regions which could act as targeting information. In some cases positive targeting information is known to mediate sorting into the PSV whereas in other cases aggregation and membrane association seem to be major sorting mechanisms.
An experiment with lucerne plants (Medicago sativa L.) previously labelled with 15N and grown in hydroponic culture, was undertaken to define sink and source behaviour of different organs in defoliated and intact plants. Changes in 15N contents of plants during 24 days of regrowth on an unlabelled medium after defoliation were used to estimate flows of exogenous and endogenous nitrogen. The 15N content of regrowing stems and leaves increased as a result of remobilisation mainly from lateral and tap roots which, therefore, acted as source organs. Nitrogen remobilisation reached a plateau after 10 days of regrowth and, until this time, nearly all N for shoot regrowth came from endogenous N in roots and crown. Between 25 and 35% of N reserves were translocated to regrowing stems, the remainder to regrowing leaves. Amino acid-N was the most readily available form of N while protein-N was the largest storage pool. Nitrogen uptake from the medium and accumulation in source organs (roots and crown) was significant only between days 6 and 14, and almost all was subsequently translocated to regrowing tissues. Defoliation induced changes in source-sink relationships for N. Whereas stems and tap roots were the main sink organs in intact plants, regrowing shoots exerted a stronger sink behaviour in defoliated plants.
Alfalfa (Medicago sativa L.) accumulates organic reserves in taproots that are thought to be used as substrates for newly developing shoots after defoliation. Two experiments were conducted to determine if specific N pools in taproot tissues undergo depletion and reaccumulation following defoliation. In Exp. 1, bark tissues of taproots of ‹Hi-Phy› alfalfa had higher concentrations of total N, soluble NH2-N and buffer-soluble protein than did wood tissues. Concentrations of these N pools declined in both tissues after defoliation and then reaccumulated after 21 d of regrowth. In Exp. 2, two genotypes differed in concentration of N-containing pools, although trends following defoliation of both genotypes were similar to those observed in Exp. 1. ASP + ASN were the most prevalent of the amino acids found in bark and wood tissues, together comprising approximately 50% of the total amino acid pool. Concentration of the ASP + ASN pool declined markedly in roots following defoliation, while concentrations of other amino acids (LEU, ILE, TYR, and PHE) increased. Characterization of buffer-soluble proteins using SDS-PAGE indicated that specific proteins with molecular masses of 15 and 19 kDa were depleted, especially in bark tissues, as soluble protein concentrations declined. The depletion of specific amino acids and certain buffer-soluble proteins from taproots during regrowth of defoliated alfalfa suggests that these N-pools may be utilized as a source of N during foliar regrowth after defoliation.
Nitrogen re-mobilization during ryegrass (Lolium perenne L.) re-growth was studied as a function of time. Roots and stubble compounds were labelled with 15N before clipping. Experimental results clearly showed that ryegrass re-growth involves two periods of differing physiological
significance. The first occurs during the first 6 d when nearly all the nitrogen of the new leaves comes from organic nitrogen
re-mobilized from roots and stubble. At the same time, the uptake of nitrogen from the medium is very low, no reduction of
endogenous nitrate occurs and proteolysis and amino amido nitrogen re-mobilization in stubble are not fully compensated for
by synthesis.Thus, the change in protein-N content, expressed per plant, in stubble decreases from 998 μg (uncut plant) to
866 μg and 661 μg for 2 d and 4 d respectively after cutting. The second period (after the sixth day) may be more representative
of stable behaviour of ryegrass, with assimilation of mineral nitrogen from the medium providing the predominant source of
nitrogen for re-growth.
The glycoproteins of three different cell surface membranes of the rat have been investigated by acrylamide gel electrophoresis
with the use of discontinuous buffers in the presence of sodium dodecyl sulfate (SDS). Liver, kidney brush border, and erythrocyte
ghost membranes are solubilized in SDS and after brief exposure to dilute base are reduced with βmercaptoethanol. The gels,
after washing free of SDS, are Schiff-stained, and it is demonstrated, by the use of glycoproteins of known molecular weight
and subunit composition, that the stained patterns represent size separations of glycoprotein subunits. Each membrane is found
to have an identifiable glycoprotein subunit pattern composed of at least 6 to 11 different sized subunits. Most of the staining
intensity is present in one to three subunits. Although each membrane has its own unique pattern, liver and kidney brush border
have four identically sized subunits. These subunits have molecular weights of 250,000, 195,000, 130,000, and 96,000 when
estimated by interpolations of relative mobilities between protein markers. The erythrocyte ghost also contains a 96,000 molecular
weight glycoprotein subunit. Sialic acid determinations made on cut gel slices show that all prominent Schiff-positive glycoproteins
contain sialic acid.
Nodul¯ted alfalfa plants were grown hydroponically. In order to quantify N2 fixation and remobilization of N reserves during regrowth the plants were pulse-chase-labelled with 15N. Starch and ethanol-soluble sugar contents were analysed to examine changes associated with those of N compounds. Shoot
removal caused a severe decline in N2 fixation and starch reserves within 6 d after cutting. The tap root was the major storage site for metabolizable carbohydrate
compounds used for regrowth; initially its starch content decreased and after 14 d started to recover reaching 50% of the
initial value on day 24. Recovery of N2 fixation followed the same pattern as shoot regrowth. After an initial decline during the first 10 d following shoot removal,
the N2 fixation, leaf area and shoot dry weight increased so rapidly that their levels on day 24 exceeded initial values. Distribution
of 15N within the plant clearly showed that a significant amount of endogenous nitrogen in the roots was used by regrowing shoots.
The greatest use of N reserves (about 80% of N increment in the regrowing shoot) occurred during the first 10 d and then compensated
for the low N2 fixation. The distribution of N derived either from fixation or from reserves of source organs (tap roots and lateral roots)
clearly showed that shoots are the stronger sink for nitrogen during regrowth. In non-defoliated plants, the tap roots and
stems were weak sinks for N from reserves. By contrast, relative distribution within the plant of N assimilated in nodules
was unaffected by defoliation treatment.
A method has been devised for the electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets. The method results in quantitative transfer of ribosomal proteins from gels containing urea. For sodium dodecyl sulfate gels, the original band pattern was obtained with no loss of resolution, but the transfer was not quantitative. The method allows detection of proteins by autoradiography and is simpler than conventional procedures. The immobilized proteins were detectable by immunological procedures. All additional binding capacity on the nitrocellulose was blocked with excess protein; then a specific antibody was bound and, finally, a second antibody directed against the first antibody. The second antibody was either radioactively labeled or conjugated to fluorescein or to peroxidase. The specific protein was then detected by either autoradiography, under UV light, or by the peroxidase reaction product, respectively. In the latter case, as little as 100 pg of protein was clearly detectable. It is anticipated that the procedure will be applicable to analysis of a wide variety of proteins with specific reactions or ligands.
An affinity-purification method has been developed for the rapid, efficient, and precise elution of antibodies specifically bound to antigens immobilized on nitrocellulose after blot transfer from SDS polyacrylamide gels. The applicability of this technology has been demonstrated using antisera raised against the nuclear matrix-pore complex-lamina fraction prepared from Drosophila melanogaster embryos. In so doing, we have established the existence in whole embryo lysates, of two nearly identical forms of the predominant 74-kilodalton polypeptide previously identified in lower resolution studies of the nuclear matrix-pore complex-lamina fraction. These species, distinguishable on the basis of a slight difference in SDS PAGE mobilities on low concentration polyacrylamide gels, are immunochemically cross-reactive and have been localized exclusively to the nuclear periphery (nuclear envelope) by indirect immunofluorescence analyses of cryosections. The steady-state levels of these two polypeptides have been examined in total embryo lysates both as a function of embryogenesis and in response to heat shock. The larger species is not detectable in early embryos but approaches levels approximately equal to that of the smaller form by about the temporal midpoint of embryonic development. In response to heat shock, this larger form appears to be converted nearly quantitatively into the lower molecular weight polypeptide. These results, as well as the general reliability of the nitrocellulose blot immunoaffinity-purification methodology, have been substantiated through the use of monoclonal antibodies.
Nitrogenase-dependent acetylene reduction, nodule function, and nodule regrowth were studied during vegetative regrowth of harvested (detopped) alfalfa (Medicago sativa L.) seedlings grown in the glasshouse. Compared with controls, harvesting caused an 88% decline in acetylene reduction capacity of detached root systems within 24 hours. Acetylene reduction in harvested plants remained low for 13 days, then increased to a level comparable to the controls by day 18.Protease activity increased in nodules from harvested plants, reached a maximum at day 7 after harvest, and then declined to a level almost equal to the control by day 22 after harvest. Soluble protein and leghemoglobin decreased in nodules from harvested plants in an inverse relationship to protease activity.Nitrate reductase activity of nodules from harvested plants increased significantly within 24 hours and was inversely associated with acetylene reduction. The difference in nitrate reductase between nodules from harvested plants and control plants became less evident as shoot regrowth occurred and as acetylene reduction increased in the harvested plants.No massive loss of nodules occurred after harvest as evidenced by little net change in nodule fresh weight. There was, however, a rapid localized senescence which occurred in nodules of harvested plants. Histology of nodules from harvested plants showed that they degenerated at the proximal end after harvest. Starch in the nodule was depleted by 10 days after harvest. The meristem and vascular bundles of nodules from harvested plants remained intact. The senescent nodules began to regrow and fix nitrogen after shoot growth resumed.
Changes in the rate and pattern of protein synthesis and in translatable mRNA population during cold acclimation of alfalfa (Medicago falcata cv Anik) seedlings have been examined. There appears to be a positive correlation between the increase in ability to synthesize proteins at 4 degrees C and the increase in freezing resistance (survival at -10 degrees C). Results obtained with three different approaches using sodium dodecyl sulfate-polyacrylamide gel electrophoresis pattern visualized by (a) staining, (b) immunoblotting and autoradiography, and (c) fluorography of in vivo labeled proteins, show that at least eight polypeptides are newly synthesized during cold acclimation. Results of analysis of in vitro translation products of mRNA from nonacclimated and acclimated seedlings show the appearance of new translatable mRNAs. It is concluded that changes in gene expression occur during cold acclimation, most probably at the transcriptional level.
Bark, wood, and root tissues of several Populus species contain a 32- and a 36-kilodalton polypeptide which undergo seasonal fluctuations and are considered to be storage proteins. These two proteins are abundant in winter and not detectable in summer as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunodetection. An antibody raised against the 32-kilodalton storage protein of Populus trichocarpa (T. & G.) cross-reacts with the 36-kilodalton protein of this species. The synthesis of the 32- and 36-kilodalton proteins can be induced in micropropagated plants by short-day conditions in the growth chamber. These proteins are highly abundant in structural roots, bark, and wood and combined represent >25% of the total soluble proteins in these tissues. Nitrate concentration in the leaves and nitrate uptake rate decreased dramatically when LD plants were transferred to short-day conditions; the protein content in leaves was unaffected. A decrease of the 32- and 36-kilodalton polypeptides occurs after transferring induced plants back to LD conditions. Both polypeptides are glycosylated and can be efficiently purified by affinity chromatography using concanavalin A-Sepharose 4B. The 32- and the 36-kilodalton polypeptides have identical basic isoelectric points and both consist of at least three isoforms. The storage proteins show a loss in apparent molecular mass after deglycosylation with trifluoromethanesulfonic acid. It is concluded that the 32- and 36-kilodalton polypeptides are glycoforms differing only in the extent of glycosylation. The relative molecular mass of the native storage protein was estimated to be 58 kilodalton, using gel filtration. From the molecular mass and the elution pattern it is supposed that the storage protein occurs as a heterodimer composed of one 32- and one 36-kilodalton subunit. Preliminary data suggest the involvement of the phytochrome system in the induction process of the 32- and 36-kilodalton polypeptides.
Our objective was to identify amylases that may participate in starch degradation in alfalfa (Medicago sativa L.) taproots during winter hardening and subsequent spring regrowth. Taproots from field-grown plants were sampled at intervals throughout fall, winter, and early spring. In experiment 1, taproots were separated into bark and wood tissues. Concentrations of soluble sugars, starch, and buffer-soluble proteins and activities of endo- and exoamylase were determined. Starch concentrations declined in late fall, whereas concentrations of sucrose increased. Total amylolytic activity (primarily exoamylase) was not consistently associated with starch degradation but followed trends in soluble protein concentration of taproots. This was especially evident in spring when both declined as starch degradation increased and shoot growth resumed. Activity of endoamylase increased during periods of starch degradation, especially in bark tissues. In experiment 2, a low starch line had higher specific activity of taproot amylases. This line depleted its taproot starch by late winter, after which taproot sugar concentrations declined. As in experiment 1, total amylolytic activity declined in spring in both lines, whereas that of endoamylase increased in both lines even though little starch remained in taproots of the low starch line. Several isoforms of both amylases were distinguished using native polyacrylamide electrophoresis, with isoforms being similar in bark and wood tissues. The slowest migrating isoform of endoamylase was most prominent at each sampling. Activity of all endoamylase isoforms increased during winter adaptation and in spring when shoot growth resumed. Endoamylase activity consistently increased at times of starch utilization in alfalfa taproots (hardening, spring regrowth, after defoliation), indicating that it may serve an important role in starch degradation.
The major seed storage proteins in alfalfa are medicagin (a legumin-like globulin), alfin (a vicilin-like globulin) and a family of Lower Molecular Weight albumins (LMW13). These comprise 30%, 10% and 20%, respectively, of the total extractable protein from cotyledons of mature seeds. Alfin
is a heterogeneous oligomeric protein (Mr∼ 150 kD) composed of polypeptides ranging in size from Mr 50 to 14 kD (α1,-α6; 50, 38, 32, 20, 16 and 14 kD, respectively). Medicagin is also a high molecular weight oligomeric protein, but requires
high concentrations of salt for solubilization. It is comprised of a family of individually distinct subunits, each composed
of an acidic polypeptide (A1–A9; Mr 49 to 39 kD) linked via disulphide bond(s) to a basic polypeptide (B1, B2, B3; Mr 24, 23 and 20 kD, respectively). This pairing is highly specific and two families are recognizable on the basis of the
B polypeptide (B3 or B1/B2). Subunits (Mr∼ 50–65 kD) are assembled as trimers (8S) or larger oligomers (12S–15S) in mature seeds. The lower molecular weight albumins
(LMW1–3) are acidic (pl<6), and consist of sets of disulphide-bonded polypeptides (Mr 15 and 11 kD).
We report here the cloning and sequence analysis of cDNAs for a pair of closely related proteins from soybean (Glycine max [L.] Merr. cv. Williams 82) stems. Both proteins are abundant in soluble extracts of seedling stems but not of roots. One of these proteins (M r=28 kDa) is also foundd in the cell wall fraction of stems and actumulates there when seedlings are exposed to mild water deficit for 48 h. The mRNA for these proteins is most abundant in the stem region which contains dividing cells, less abundant in elongating and mature stem cells, and rare in roots. Using antiserum against the 28 kDa protein, we isolated cDNA clones encoding it and an antigenically related 31 kDa protein. The two cDNAs are 80% homologous in nucleotide and amino acid coding sequence. The predicted proteins have similar hydropathy profiles, and contain putative NH2-terminal signal sequences and a single putative N-linked glycosylation site. The two proteins differ significantly in calculated pI (28 kDa=8.6; 31 kDa=5.8), and the charge difference is demonstrated on two-dimensional gels. The proteins described here may function as somatic storage proteins during early seedling development, and are closely related to glycoproteins which accumulate in vacuoles of paraveinal mesophyll cells of fully expanded soybean leaves when plants are depodded.
The bark of some young woody stems contains storage proteins which are subject to an annual rhythm: they accumulate in the autumn and are mobilized in the spring. We show here that the bark phoem-parenchyma cells of Sambucus nigra L. contain numerous protein bodies, and that the bark lectin (S. nigra agglutinin) which undergoes an annual rhythm is localized in these protein bodies. The protein bodies in the cotyledons of legume seeds also contain lectin, indicating that lectins may be storage compounds themselves or may have a function in storage and-or mobilization processes.
The inner bark tissues of three temperate hardwoods contain specific proteins which undergo seasonal fluctuations. Increases in particular proteins, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, occur within the bark of several Acer, Populus and Salix spp. during late summer and early autumn. These proteins are abundant in the bark throughout the winter and their levels decline the following spring. Light and electron microscopy showed that the parenchyma cells of the inner bark are packed with spherical organelles throughout the overwintering period. These organelles are rich in protein and analogous to protein bodies found in cells of mature seeds. The protein bodies of the parenchyma cells are replaced by large central vacuoles during spring and summer, presumably as a result of the mobilization of the storage protein and fusion of the protein bodies. The high levels of specific proteins in inner bark tissues and the presence of protein bodies within the parenchyma cells indicate that the living cells of the bark act as a nitrogen reserve in overwintering temperate hardwoods.
Roots are the overwintering structures of herbaceous perennial weeds growing in temperate climates. During the fall they accumulated reserves which are remobilized when growth resumes in the spring. An 18kDa (kilodalton) protein increases in both chicory and dandelion roots during the fall months. The proteins in both species are antigenically similar, and are recognized also by an antibody to a storage-protein deposited in Jerusalem artichoke (Helianthus tuberosus) tubers. In chicory, the protein is root-specific, but in dandelion it is detectable in the flowers, vestigial stem and the seed. Electrophoretic characterization of the 18-kDa protein shows that it is a single polypeptide, without subunits, with charge isomers of pI values close to pH 6.5. The major protein present in chicory and dandelion roots is unlike the vegetative storage proteins recently found in soybean or the storage proteins in the bark of trees.
The possibility that added NO3- affects the path of N assimilation from N2 was tested in situ by following the assimilation and partitioning of 15N from 15N2 or 15NO3- in 8-week-old alfalfa (Medicago sativa L. cv. Saranac) seedlings. The labeling patterns of different N compounds indicated that there was no effect of NO3- on the assimilation pathway of N from N2, even though 2 mol · m-3 of NO3- inhibited N2 fixation 38 %. NO3- supplied at this concentration appropriate to field soil solutions, did not cause any significant accumulation of NO2- in plant organs including nodules. Some of the absorbed NO3 - was reduced and assimilated in the nodules. Ammonium produced from N2 fixation and NO3 reduction was metabolized in a similar manner. The reduced N from both sources was transported chiefly in the form of asparagine to the shoots where it was rapidly metabolized and incorporated into protein. Unlike N from N2, that from NO3- was predominantly distributed to mature leaves.
Alfalfa (Medicago sativa L.) taproots accumulate organic reserves that are important for winter survival and subsequent growth in spring. Our objective was to determine if specific nitrogen (N) pools accumulate in taproot tissues prior to winter that may subsequently be used during initiation of herbage growth in spring. Taproots were obtained at approximately monthly intervals during fall and winter, and biweekly in early spring. Taproots were separated at the cambium into bark and wood tissues. Bark tissues consistently contained higher N concentrations than did wood tissues. N concentrations of both tissues gradually increased between early and late fall and declined in early spring when growth was initiated. Both soluble amino-N and buffer-soluble proteins increased during autumn and declined extensively during early spring in both tissues. A nonwinterhardy alfalfa line accumulated less soluble protein in taproot tissue when compared to a hardy genotype. Specific proteins with molecular masses of 32, 19, and 15 kDa were identified as major components of the buffer-soluble protein pool. These proteins rapidly disappeared from taproot tissues in spring as buffer-soluble protein concentrations declined. Protease activity in bark tissues declined gradually during late autumn and winter before increasmg over two-fold in early spring. Protease activity in wood tissues was approximately one-half that of bark tissues and also increased in spring when growth resumed. Our results indicate that high concentrations of soluble amino compounds and specific proteins accumulate in taproots during autumn and early winter. These N pools decline markedly in spring coincident with the onset of herbage growth.
This investigation was conducted to determine the effects of hardening conditions (low temperature, short photoperiod) on root respiration, nodulation, and in vitro activities of soluble enzymes in roots of alfalfa ( Medicago sativa L.) cultivars which harden or fail to harden. Under hardening conditions in growth chambers root sections of Vernal alfalfa (high hardiness) had higher rates of respiration than did those from Sonora alfalfa (low hardiness). Arrhenius plots of root respiration showed that temperature kinetics for Vernal and Sonora were similar between 4 and 40 C regardless of growth conditions although Vernal had a slightly lower Arrhenius energy of activation (11.2 to 12.6 and 13.8 to 14.4 kcal mole ⁻¹ , for Vernal and Sonora respectively). Roamer and Vernal (both with high hardiness) grown under hardening conditions nodulated very well and had significant rates of acetylene reduction, whereas Sonora and Caliverde (low and moderate hardiness, respectively) grown under the same conditions nodulated poorly and did not reduce acetylene. Root NAD‐malate dehydrogenase, NADP‐malate dehydrogenase, lactate dehydrogenase, and glutamate oxaloacetatetransaminase activities were highest in hardy plants grown under hardening conditions in growth chambers. Alcohol dehydrogenase activity was usually much higher in all cultivars, hardy and nonhardy, grown under hardening conditions. Studies with six field‐grown cultivars with various degrees of hardiness yielded data similar to that from growth‐chamber plants for the in vitro activities of soluble root enzyme activities. Of 10 enzymes assayed, NAD‐malate dehydrogenase and NADP‐malate dehydrogenase activities correlated best with hardiness and levels of soluble protein. These data indicate that root functions (respiration, the ability to nodulate, and levels of various enzyme activities) increase to a greater extent or are more pronounced in hardy cultivars compared to those with lesser hardiness during the hardening process.
Synopsis
Synopsis
Cold resistance increased from late September to mid-December. A high level was maintained until February or early March. Resistance was lost slowly until early April and rapidly thereafter. In general, each nitrogen and carbohydrate fraction increased in content during the fall, was at its highest level sometime during the fall or winter, and then decreased during the spring. Correlation coefficients of the trends of the chemical fractions with the trend of cold resistance were significant in many cases.
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Synopsis
Synopsis
Eleven sweetclover varieties were evaluated for winter survival, carbohydrate reserves, trends of cold resistance development, and nitrogen content of root tissue. Arctic, Brandon dwarf, Erector, and Alpha were the only varieties to give any appreciable winter survival. Carbohydrate reserves were adequate in all varieties. Arctic, Brandon dwarf, and Erector showed significantly higher cold resistance and content of various nitrogen fractions. The possible role of various nitrogen fractions in cold resistance development is discussed. Water soluble protein appeared most closely associated with cold resistance.
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Nitrogen re-mobilization and changes in free amino acids were studied as a function of time in leaves, stubble, and roots during ryegrass (Lolium perenne L.) re-growth. Experiments with N-15 labelling clearly showed that during the first days nearly all the nitrogen in new leaves came from organic nitrogen re-mobilized from roots and stubble. On the days of defoliation, stubble had the highest content of free amino acids with 23 mg per g dry weight against 15 mg and 14 mg in leaves and roots, respectively. The major amino acids in leaves were asparagine (23% of total content in free amino acids), aminobutyrate, serine, glutamine, and glutamate (between 7% and 15%) whereas in roots and stubble the contribution of amides was high, especially asparagine (about 50%). Re-growth after cutting was associated with a rapid increase of the free amino acid content in leaves, with a progressive decrease in roots while stubble content remained virtually unchanged. In leaves, asparagine increased from the first day of re-growth, while the aspartate level remained unchanged and glutamine increased strongly on the first day but decreased steadily during the next few days of re-growth. Asparagine in stubble and roots changed in opposite directions: in stubble it tended to increase whereas in roots it clearly decreased. In contrast, stubble and roots showed a similar decrease in glutamine. In these two plant parts, as in leaves, aspartate remained at a low level. Results concerning free amino acids are discussed with reference to nitrogen re-mobilization from source organs (stubble and roots) to the sink organ (regrowing leaves).
Nitrogen remobilization from roots and pseudostems during regrowth of Lolium perenne L. was studied in miniswards grown with contrasting levels of (NH4)2SO4 in solution culture. Growth with a high N supply (5.0 mol m−3) increased the weight of leaf laminae recovered at each of five weekly clippings, and decreased the proportion of photosynthate
used for root growth. Clipped plants growing in a steady-state were supplied with 15N for 48 h and the recovery of labelled N in laminae measured after five weekly cuts. Recovery of labelled N in the laminae
from the second clipping onwards was derived only from remobilization of N from roots and pseudostem. Miniswards grown with
low N (0.5 mol m−3) relied more upon remobilization of N for lamina growth than did high N plants. Thus after 14 d 20% of lamina N was labelled
in low N plants but only 3% was labelled in the high N treatment. Thereafter, N remobilization declined until at the final
clipping after 35 d, labelled N represented only 4% and 1 % of the lamina N in the low and high N plants. When plants were
not clipped before the labelling period, they took up more 15N if grown with high N than cut plants. Thereafter, the remobilization of N followed a similar pattern as in the cut plants.
Exponential models were used to calculate the rate of N transfer from roots and pseudostem to laminae. When grown with low
N, the half-life of remobilization was 1.56 weeks. High N miniswards had an initial rapid remobilization with a half-life
of 0.66 weeks, and a slower phase with a half-life of 2.98 weeks.
The net mobilization of nitrogen compounds (total N, soluble protein, amino N, membrane-bound protein) from different plant parts after defoliation, and the extent to which mobilized nitrogen contributed to new leaf growth, were studied in small swards of Trifolium subterraneum L. cv. Seaton Park (subterranean clover) either completely dependent on nitrogen fixation (-N treatment) or receiving abundant nitrate at defoliation (+ N treatment).
Net mobilization of nitrogen from nodulated roots (75 % of mobilized nitrogen) and branches (25 % of mobilized nitrogen) accounted for most of the nitrogen in new leaf growth for up to 5 d in – N swards and for at least 2 d in +N swards. In – N swards, nodules degenerating as a result of defoliation were a major source of mobilized nitrogen for 2 d (50 % of mobilized nitrogen), after which the fine roots became the largest net contributor of mobilized nitrogen. Overall, a net 17% of the nitrogen present in residual organs was mobilized.
Soluble protein was the major form of mobilized nitrogen for which assays were performed, accounting for 50 % of the decline in total N in the – N treatment. The relative loss of soluble protein from sward components declined in the order, nodules (47 % over 5 d), fine roots (44 % over 9 d), branches (38 % over 9 d) and taproots (37 % over 5 d). The amino N pool increased sharply in size for two days after defoliation, possibly indicating breakdown of soluble protein in excess of the nitrogen demand for regrowth or to provide a respiratory substrate.
It was concluded that, in plants of adequate nitrogen status, leaf growth was unlikely to be nitrogen-limited during the first few days after defoliation.
Abstract Chicory (Cichorium intybus L.) and dandelion (Taraxacum officinale L.) are persistent weeds, the aerial portions of which do not survive in winter. However, subterranean tissues remain viable and facilitate the rapid resumption of growth in early spring. The source of nutrients for growth prior to the establishment of foliage is the roots. Carbohydrate and N reserves are accrued during late summer and autumn, respectively. Hydrolysis of fructans during late autumn occurs coincidentally with increments in sucrose, the latter providing a readily accessible C pool. Nitrate, free amino acids and soluble protein all play substantial roles in nitrogen storage. Asparagine is the predominant amino acid in the free pool during winter, followed by glutamine, ornithine, serine, aspartic acid and glutamic acid. Storage reserves remain at peak levels throughout winter and deeline prior to the resumption of growth. The patterns observed here provide evidence that N is an important currency of storage metabolism and, thus, a framework has been provided for the examination of regulation of N storage in perennial weeds.
Leafy spurge (Euphorbia esula L.), a serious perennial weed of temperature range and pasture lands, has continued to colonize despite various control strategies. The persistence of this species can be attributed in part to the presence of an extensive root system containing abundant organic reserves. These components, established towards the end of the growing season, are remobilized to support early spring growth. Carbohydrates comprise the bulk of reserve material with late fall incrents in free sugars being associated with reductions in starch content. Nitrogenous components undergo significant seasonal fluxes, with free amino acids and soluble proteins reaching maxima during late fall. Asparagine, glutamic acid, serine, ornithine, proline, arginine and aspartic acid all contribute significantly to the storage of nitrogen. Changes in nitrate content are associated with the overwintering process. These observations are indicative of the role that nitrogen plays in the overwintering strategy and regenerative capacity of leafy spurge roots.
We analyzed changes in populations of translatable mRNAs occurring in crowns of the cold-tolerant alfalfa ( Medicago sativa L.) cv. Apica (CT) and the cold-sensitive cv. CUF-101 (CS) after their acclimation at low nonfreezing temperatures and at subzero temperatures. Both cultivars showed very similar translation profiles under all treatments. Low temperatures induced significant changes in the populations of translatable mRNAs. We observed a relationship between the accumulation of cold-regulated (COR) translation products and freezing tolerance within cultivars. Moreover, at least three COR translation products were specific to the CT and might be related to hardiness potential in alfalfa. Whereas extension of the cold acclimation period at 2�C reduced cold tolerance, incubation at subzero temperatures increased or maintained freezing tolerance. This increased hardiness was associated with enhanced translation of COR polypeptides and also with the appearance of new translatable mRNAs. This is, to our knowledge, the first report of altered gene expression in plants incubated at subzero temperatures. Marked changes in populations of translatable mRNAs at temperatures below freezing might be related to previous reports that alfalfa achieves maximum hardiness under snow cover when the soil has frozen. Translation in the presence of [3H]glycine showed that a large proportion of the COR genes encode for glycine-rich proteins (GRPs) and that some of the GRPs are specific to the CT.
Two cultivars of alfalfa (Medicago sativa L.), cold-tolerant Vernal and cold-sensitive Sonora, were grown under summer, winter, and dehardening environments to determine the characteristics and relationships of several hydrolytic enzymes to cold tolerance.Soluble enzymatic proteins, extracted from lyophilized crown and root tissues with three different solvents, were separated by polyacrylamide disc-gel electrophoresis and evaluated on the basis of equal dry weights of tissue and equal quantities of protein.Gels assayed for amylases, acid phosphatases, esterases, leucine aminopeptidases, and adenosine triphosphatases exhibited mainly quantitative differences in isoenzymes depending upon extractant, cultivar, and environmental differences. The qualitative differences detected were generally due to differential solubilities of isoenzymes in the three extractants and, to a lesser extent, were related to environmental, cultivar, or stability differences.While activities of esterases, acid phosphatases, and leucine aminopeptidases increased in winter samples, as soluble protein increased, only slight decreases in these enzymes occurred during dehardening. Conversely, activities of amylases were slightly lower in winter samples than in the other samples, and adenosine triphosphatase activity decreased in the most coldtolerant sample.The measured levels of total nonstructural carbohydrate, total soluble sugar, and starch indicated differences between cultivars in starch-sugar conversion. Further, the differential heat stabilities of the isoamylases also provided some information as to the nature of “protected activity” of diastatic enzymes.Differential cryostabilities of peptidases and adenosine triphosphatases detected between cultivars and environments also demonstrated the influence of the extraction medium in maintaining enzyme activity, and these observations may be important to an understanding of cold tolerance in alfalfa. The obvious speculations regarding enzyme stability and the factors involved as related to the cold tolerance of alfalfa require further examination.
We present a new method for visualizing proteins electrophoresed in sodium dodecyl sulfate-polyacrylamide gels. After electrophoresis, gels are incubated in CuCl2 to produce a negative image of colorless protein bands against a semiopaque background. Gels are stained completely within 5 min, do not require destaining, and can be stored indefinitely without loss of the image. Because proteins are not permanently fixed within the gel, they can be quantitatively eluted after chelation of Cu with EDTA. The sensitivity of the CuCl2 stain falls between that of Coomassie blue and silver. We anticipate that CuCl2 will be useful in the rapid analysis of proteins by polyacrylamide gel electrophoresis and in the preparation of purified polypeptides by elution from gel slices.
All cysteine and cystine in a protein are derivatized during hydrolysis in hydrochloric acid containing 3,3'-dithiodipropionic acid. The resulting derivative can be separated from other amino acids and used for quantitation of cysteine plus half-cystine. A procedure is presented for accurate determination by ion exchange chromatography and postcolumn derivatization of all amino acids from acid hydrolysis of a protein, including the Cys-derivative.
Ovomucoids were isolated from egg whites of 100 avian species and subjected to limited proteolysis. From each an intact, connecting peptide extended third domain was isolated and purified. These were entirely sequenced by single, continuous runs in a sequencer. Of the 106 sequences we report (five polymorphisms and chicken from the preceding paper [Kato, I., Schrode, J., Kohr, W. J., & Laskowski, M., Jr. (1986) Biochemistry (preceding paper in this issue)]), 65 are unique. In all cases except ostrich (which has Ser45), the third domains are either partially or fully glycosylated at Asn45. The majority of the third domain preparations we isolated are carbohydrate-free. Alignment of the sequences shows that their structurally important residues are strongly conserved. On the other hand, those residues that are in contact with the enzyme in turkey ovomucoid third domain complex with Streptomyces griseus proteinase B [Read, R., Fujinaga, M., Sielecki, A. R., & James, M. N. G. (1983) Biochemistry 22, 4420-4433] are not conserved but instead are by far the most variable residues in the molecule. These findings suggest that ovomucoid third domains may be an exception to the widely accepted generalization that in protein evolution the functionally important residues are strongly conserved. Complete proof will require better understanding of the physiological function of ovomucoid third domains. This large set of variants differing from each other in the enzyme-inhibitor contact area and augmented by several high-resolution structure determinations is useful for the study of our sequence to reactivity (inhibitory activity) algorithm. It is also useful for the study of several other protein properties. In the connecting peptide fragment most phasianoid birds have the dipeptide Val4-Ser5, which is absent in most other orders. This dipeptide is often present in only 70-95% of the molecules and appears to arise from ambiguous excision at the 5' end of the F intron of ovomucoid. Connecting peptides from the ovomucoids of cracid birds contain the analogous Val4-Asn5 peptide. In laughing kookaburra ovomucoid third domain we found (in 91% of the molecules) Gln5A, which we interpret as arising from ambiguous intron excision at the 3' end of the F intron.
Using an improved method of gel electrophoresis, many hitherto unknown
proteins have been found in bacteriophage T4 and some of these have been
identified with specific gene products. Four major components of the
head are cleaved during the process of assembly, apparently after the
precursor proteins have assembled into some large intermediate
structure.
This chapter describes the silver staining methods for polyacrylamide gel electrophoresis and presents an overview to develop a highly sensitive and simple to perform silver stain by the utilization of chemical photoreversal procedures and formaldehyde as the developer. In this procedure either oxidizing agents and reducing agents, or a strong light source can be used to obtain photoreversal. By soaking the gel in one of these reagents prior to exposure to silver nitrate, full photoreversal can be obtained during image development. The image obtained by silver staining a polyacrylamide gel is similar to a developed black and white photograph. The purity of the water used in the stain and rinsing solutions is critical for maximum sensitivity. Deionized water is required. Formaldehyde can present a problem because it is sometimes not actually a 37% solution. Silver stains used for PAGE gels detect most proteins in crude cell lysates that can be detected with 14C-labeled proteins by autoradiography.
A rapid, sensitive method has been developed to detect antibody-antigen complexes on "Western blots." The methods of H. Towbin, T. Staehlin, and J. Gordon were used to separate and blot the antigens onto nitrocellulose. The remaining sites of attachment were blocked and the nitrocellulose was washed with polyoxyethylenesorbitan monolaurate (Tween 20). The blot was then reacted with the antiserum or hybridoma supernate to be tested. After the antigen-antibody reaction was completed, the blot was washed and treated with anti-antibody which had been conjugated to alkaline phosphatase. The alkaline phosphatase was detected by the reduction of the tetrazolium salt to diformazan by the hydrogen ions released in the formation of indigo by the reaction of the phosphatase on the indoxyl phosphate. The advantages of this method over previously described techniques are (1) use of Tween 20 allows the blot to be stained with Coomassie blue, (2) the substrates of the alkaline phosphatase reaction are stable for long periods of time, (3) the reaction products form an intense blue color which does not fade, (4) the resolution is extremely good with little to no band broadening, (5) the reaction is sensitive to picogram quantities of antigen, and (6) the reaction is quantitative.
Subterranean clover (Trifolium subterraneum L. cv Woogenellup) and soft chess grass (Bromus mollis L. cv Blando) were grown in monocultures with (15)NH(4)Cl added to the soil to study nitrogen movement during regrowth following shoot removal. Four clipping treatments were imposed. Essentially all available (15)N was assimilated from the soil prior to the first shoot harvest. Measurements of total reduced nitrogen and (15)N contained within that nitrogen fraction in roots, crowns, and shoots at each harvest showed large, significant (P </= 0.001) declines in excess (15)N of crowns and roots in both species between the first and fourth harvests. There was no significant decline in total reduced nitrogen in the same organs over that period. Similar responses were evident in plants defoliated three times. The simplest interpretation of these data is that reduced nitrogen compounds turn over in plant roots and crowns during shoot regrowth. Calculations for grass and clover plants clipped four times during the growing season indicated that 100 to 143% of the nitrogen present in crowns and roots turned over between the first and fourth shoot harvest in both species, assuming nitrogen in those organs was replaced with nitrogen containing the lowest available concentration of (15)N. If other potential sources of nitrogen were used for the calculations, it was necessary to postulate that larger amounts of total nitrogen flowed through the crown and root to produce the measured dilution of (15)N compounds. These data provide the first quantitative estimates of the amount of internal nitrogen used by plants, in addition to soil nitrogen or N(2), to regenerate shoots after defoliation.
Soybeans (Glycine max L.) accumulate a storage glycoprotein which is abundant in vegetative tissues, but is only a minor component of seeds. Changes in vegetative storage protein gene expression in leaves of control and depodded plants were monitored throughout plant development. Western and Northern blot hybridization analysis of protein and mRNA levels, respectively, showed that expression of these genes was highly regulated during development. Expression correlated with periods when expected demand for mobilized leaf reserves by other plant sinks was low. Vegetative storage protein mRNA comprised about 0.5% of the total mRNA in immature leaves and declined at least 20-fold by flowering. Depodding or blockage of leaf petiole phloem transport increased these mRNAs to about 16% of the total mRNA. Transcript levels also increased dramatically after seed maturation, just before leaf senescence. Protein levels followed a similar pattern and were inversely related to the number of seed pods allowed to develop on the plants. The results support the role for these proteins as temporary storage molecules which can be rapidly synthesized or degraded according to the need for nutrients by other plant tissues.
Bark storage proteins (BSPs) accumulate in the inner bark parenchyma of many woody plants during autumn and winter. We investigated the effect of a short-day (SD) photoperiod on the accumulation of the 32-kilodalton bark storage protein of poplar (Populus deltoides Bart. ex Marsh.) under controlled environmental and natural growing conditions. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and protein gel blot analysis revealed that 10 days of SD exposure (8 hours of light) resulted in a 20% increase in the relative abundance of the 32-kilodalton bark storage protein of poplar. After 17 days of SD exposure, the 32-kilodalton bark storage protein accounted for nearly one-half of the soluble bark proteins. In natural field conditions, accumulation of the 32-kilodalton bark storage protein was observed to start by August 18 (daylength 14.1 hours). Immunoprecipitation of in vitro translation products with anti-BSP serum revealed that the SD protein accumulation was correlated with changes in the pool of translatable mRNA. A survey of poplar clones from different geographic origins revealed the presence of the 32-kilodalton BSP in the dormant bark of all the clones tested. These results demonstrate that a SD photoperiod induces, whether directly or indirectly, rapid changes in woody plant gene expression, leading to the accumulation of BSP.
BEWLEY: Identification and characteriza-tion of the seed storage proteins from alfalfa (Medicago sativa) Cleavage of structural proteins during the assembly of the head of bacteriophage T4 lANGHElNRICH, U. and R. TISCHNER: Vegetative storage proteins in poplar
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VOLENEC: Utilization of alfalfa taproot nitrogen reserves during regrowth
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- B C Joelln
BARBEll, L. D., B. C. JOEllN, and J. J. VOLENEC: Utilization of alfalfa taproot nitrogen reserves during regrowth (abstract No. 883).
- R M Zachailius
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Vegetative storage proteins in poplar
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