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Taproot nitrogen accumulation and use in overwintering alfalfa (Medicago sativa L.)
Abstract
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.
... Процентът презимуване корелира положително с диаметъра на кореновата шийка (r = 0.750), теглото коренова маса растение -1 (r = 0.696,) и съдържанието на разтворими захари в кореновата маса (r = 0.352), установени в есенните подрасти на тревостоите. В края на 4годишния период на отглеждане и след реколтиране на последният (есенен) подраст, акумулираните растителни остатъци в почвеният слой (0-30 cm) са средно 10 Alfalfa biomass is characterized with a high content of crude protein (Dinić et al., 2005, Marković et al., 2007, vital vitamins and microelements, and is well balanced with respect to amino acids that are балансирана по отношение на аминокиселини, които са от съществено значение за нормалния растеж и развитие на животните (Marković et al., 2007). ...
... Физиологичният статус на растенията, особено съдържанието на безструктурни карбохидрати (TNC) и разтворими захари (SS), натрупани в кореновата шийка и корените, може да ограничат толерантността, особено когато те са намалени до критични нива до около 15% от сухото тегло. Установено е, че разтворимите протеини (Hendershot and Volenec, 1993), както и редуцираната абсорбция на вода, зависеща от размера на кореновата система и кореновата морфология, също имат отношение към разглежданият параметър (Jung and Larson, 1972). В настоящото проучване се установяват корелации с висока положителна стойност между Winter-and cold-resistance in alfalfa are associated with a number of physiological and chemical indicators that allow plants to overcome low-temperature stress (Jung and Larson, 1972;Avice et al., 1997;Dhont et al., 2004;Hakl et al., 2007). ...
... It has been found that soluble proteins (Hendershot and Volenec, 1993), as well as reduced water absorption, depending on the root system size and root morphology, also have relevance to the considered parameter (Jung and Larson, 1972). ...
With aim, a comparative characteristic of eight alfalfa cultivars (Europe, Prista 2, Prista 3, Prista 4, Obnova 10, Pleven 6, Dara, Multifoliate.), an estimation of main biological parameters (stand formation, overwintering and factors determining it, accumulation of plant residues and corresponding amounts of N, P and Ca in the soil) was conducted. For carrying out experimental activity, a randomized block method was used, at a row spacing of 11.5 cm and a sowing rate of 2.5 kg da-1. The analysis of variance showed a statistically significant influence of the year and cultivar on the stand formation, expressed as stems m-2 (plants m-2 × stems plant-1). The parameter was dynamic and during the particular years, different cultivars demonstrated a higher density. For the 4-year period of alfalfa development was established a general trend of decrease in stand density, with exception of a slight increase in the 3rd year determinative by the variation in the components "plants m-2" and "stems plant-1" and the compensatory mechanism between them. At the end of the 4th experimental year, the number of stems per unit area was reduced by 52.6% on average compared to the 1st year, with a variation of 25.0 (Obnova) to 69.4% (Dara). The evaluation of cultivars regarding sensitivity to unfavorable conditions during the winter period determined a higher percentage of overwintering plants in Multifoliate, Obnova 10 and Prista 4 (88.1 - 88.5%). Overwintering percentage correlated positively with the morphological parameters of root crown diameter (r = 0.750) and root weight plant-1 (r = 0.696) and soluble sugars content in the root mass (r = 0.352), established in the autumn regrowths of stands. At the end of the 4-year period of alfalfa cultivation and after harvesting of the last (autumn) regrowth, accumulated plant residues in the soil layer 0-30 cm were on average 10.7 t ha-1, providing 225.1 kg N ha-1, 15.4 kg P ha-1 and 77.1 kg Ca ha-1, respectively. Cultivars demonstrating higher values in this regard were Prista 4, Dara, Prista 3 and Pleven 6.
... The impact of temperature, photoperiod and soil water availability on the levels of taproot N reserves means that partitioning N into storage follows a seasonal pattern similar to that of taproot TNC levels, with an increase in taproot N levels though autumn and into winter (Wilding et al. 1960;Hendershot and Volenec 1992;Li et al. 1996;Dhont et al. 2006b;Teixeira et al. 2007c), followed by a decrease in spring and summer (Hendershot and Volenec 1992;Li et al. 1996;Teixeira et al. 2007c). ...
... The impact of temperature, photoperiod and soil water availability on the levels of taproot N reserves means that partitioning N into storage follows a seasonal pattern similar to that of taproot TNC levels, with an increase in taproot N levels though autumn and into winter (Wilding et al. 1960;Hendershot and Volenec 1992;Li et al. 1996;Dhont et al. 2006b;Teixeira et al. 2007c), followed by a decrease in spring and summer (Hendershot and Volenec 1992;Li et al. 1996;Teixeira et al. 2007c). ...
... For either one of these propositions to be correct, genes encoding for VSPs should be up-regulated in plants exposed to drought when compared to plants grown under fully watered conditions. The process of cold acclimation and associated winter dormancy increases the concentration of VSPs in lucerne taproots (Hendershot and Volenec 1992), as well as increasing the transcript abundance of genes belonging to the cold regulated (COR), cold acclimation-specific (CAS) and cold acclimation-responsive (CAR) gene families (Castonguay et al. 2006). The punitive proteins predicted from their gene sequences possess homology to dehydrins, nuclear signalling proteins and enzymes involved in the synthesis of simple sugars belonging to the raffinose family (Monroy et al. 1993;Cunningham et al. 2003;Haagenson et al. 2003a). ...
... Millard, 1988). This is supported by the decline in soluble protein concentrations observed during the first few days following shoot removal in M. sativa (Hendershot & Volenec, 1993), Trifolium repens (Corre et al., 1996) and L. perenne (Ourry et al., 1989). Staswick (1989Staswick ( , 1990Staswick ( , 1994 has proposed several criteria by which the involvement of specific proteins in N storage may be judged. ...
... Several polypeptides fulfilling these criteria have been identified in T. repens and M. sativa, and have been designated as functional VSPs. These are relatively abundant proteins (Hendershot & Volenec, 1993 ;Corre et al., 1996) that are hydrolysed to a greater extent than are the majority of soluble proteins, and then accumulate during the later stages of refoliation. As with many of the VSPs identified in woody species (Ste! pien et al., 1994), it is notable that these VSPs in forage legumes accumulate during autumn and winter, and are then almost entirely hydrolysed when growth resumes in spring (Bouchard et al., 1998). ...
... VSPs have been characterized in two species of forage legumes : M. sativa (Hendershot & Volenec, 1993) and T. repens (Corre et al., 1996). In both species they account for at least 20 % of total soluble protein, in taproots and stolons. ...
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.
... These authors suggested that P deficiency may have limited C export from taproot amyloplasts impairing both shoot regrowth and taproot starch use after defoliation. In this study, the higher starch in taproots of K-fertilized plants may result from stimulated activities of starch synthase and ADPglucose pyrophosphorylase, key enzymes in starch synthesis whose activities are enhanced by K (Nitros and Evans, 1969;Hawker et al., 1979). As previously reported (Bula and Smith, 1954;Li et al., 1996), taproot starch concentrations declined markedly between December and March, a response attributed in large part to respiratory losses (Avice et al., 1996). ...
... Little change in taproot protein concentration occurred after plants were defoliated September 7. This agrees with previous results (Hendershot and Volenec, 1992), whereas Haagenson et al. (2003b) observed lower protein concentrations in taproots sampled 1 month after a September defoliation. Defoliation in summer generally reduces protein concentrations in taproots sampled 10 to 20 days post-defoliation (Hendershot and Volenec, 1993;Berg et al., 2009), and 15 N labeling verified transfer of N from taproots to regrowing shoots (Avice et al., 1996;Barber et al., 1996). ...
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.
... It is well known that N reserves in the root system are an alternative source of N used by the lucerne plant when N 2 fixation and/ or mineral N uptake is reduced (Volenec, Ourry, & Joern, 1996). Hendershot and Volenec (1993) found that N concentrations in taproot bark and wood tissues gradually increased in autumn and declined in spring when growth commenced. The results from Hamilton showed no difference in N concentration between treatments but the N concentration in the taproots did increase at Rutherglen from late summer into autumn thereby supporting the Hendershot and Volenec (1993) finding. ...
... Hendershot and Volenec (1993) found that N concentrations in taproot bark and wood tissues gradually increased in autumn and declined in spring when growth commenced. The results from Hamilton showed no difference in N concentration between treatments but the N concentration in the taproots did increase at Rutherglen from late summer into autumn thereby supporting the Hendershot and Volenec (1993) finding. ...
Lucerne (Medicago sativa L.) has a large taproot to store and release starch, carbohy- drates and nutrients during the plant's growth. Recommended management of the lucerne crop aims to keep the taproot stable subject to the demands for feed provi- sion and stand longevity. Field experiments were conducted in Victoria, Australia, to examine the effects of recovery period on taproot mass and nutritive status. Both experiments used established SARDI Seven lucerne crops and were either cut every 21 days (short recovery SR) or every 42 days (long recovery LR). At each defoliation, taproots were extracted for determination of DM yield and starch, water-soluble car- bohydrate (WSC) and nitrogen (N) concentration and DM yield. At both sites, WSC and N DM yields along with total taproot DM yield did not change greatly as the experiment progressed. Starch was responsive to herbage accumulation with both concentration and DM yield, increasing when herbage accumulation rates were high and decreasing when herbage accumulation rates were low. At both sites, LR tap- roots had starch levels equal to or higher than those of SR taproots. We conclude that short intervals between defoliations disrupt the energy cycling between shoots and roots. This is likely to reduce lucerne productivity, particularly during periods of rapid growth.
... Clearly, the pattern of decreased-amylase activity coincident with large declines in root starch concentration are inconsistent with the perceived role of-amylase as a starch hydrolase in roots of alfalfa and the other forage legumes. 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). ...
... Boyce and Volenec (1992b) purified a 57.5-kD-amylase protein from alfalfa taproots and found that it constituted 8% of root-soluble protein. The seasonal pattern of-amylase activity followed the trends in concentration of root VSPs, increasing in autumn and declining markedly in spring (Volenec et al., 1991;Hendershot and Volenec, 1993a;Li et al., 1996). Because defoliation reduces nitrogenase activity (Vance et al., 1979) and uptake of nitrogen from the soil (Kim et al., 1993), we speculate that-amylase, like VSPs, may be hydrolyzed to its constituent amino acids, which are then transported to regrowing shoots to provide some of the nitrogen needed for herbage growth in spring and regrowth after defoliation in the summer (Boyce and Volenec, 1992b). ...
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.
... Among proteins that accumulated in plants in response to water stress, vegetative storage proteins (VSPs) which constitute the major nitrogenous storage pool in plants [6]. VSPs are simply known to associate not only with the regrowth potential after defoliation [7] but also with winter survival and spring growth [8,9]. For example, it has been found that VSP accumulate in alfalfa (Medicago sativa L.) during autumn and early winter, then rapidly decline at the early spring to provide a source of organic N for spring growth [8]. ...
... VSPs are simply known to associate not only with the regrowth potential after defoliation [7] but also with winter survival and spring growth [8,9]. For example, it has been found that VSP accumulate in alfalfa (Medicago sativa L.) during autumn and early winter, then rapidly decline at the early spring to provide a source of organic N for spring growth [8]. Rossato et al. [10] reported that VSP accumulation in the taproot of oilseed rape during flowering stages acts transient N storage buffer in response to N requirements during N filling of grain. ...
... Different storage sites for N have been identified, depending on the plant life form studied. Within herbaceous biennials and perennials, storage is confined to mainly below-ground structures. The predominant storage organs are taproots in Medicago sativa (Hendershot & Volenec 1992), Cichorium intybus and Taraxacum officinale (Cyr & Bewley 1990); tubers in Cyperus rotundus ( Fischer, Brouquisse & Raymond 1995); roots and rhizomes in Urtica dioica (Rosnitschek-Schimmel 1985); and roots and stolons in Trifolium repens (Corre et al . 1996). ...
... Increasing protein concentrations in storage tissue over the winter and their decline in spring have been observed in a number of species (e.g. M. sativa , Hendershot & Volenec 1992;Euphorbia esula , Luster & Farrell 1996;C. intybus and T. officinale , Cyr & Bewley 1990). ...
The contribution of N storage and remobilization to the vegetative and reproductive growth of the forb Rumex acetosa was quantified using ¹⁵ N labelling techniques with plants derived from semi‐natural grasslands in Scotland.
The contribution of remobilized N to the total N in the new above‐ground tissues was highest at the beginning of the growing season at 58%. New leaves and reproductive organs contained equal amounts of remobilized N.
During early vegetative growth, the taproot was the main source of remobilized N, whereas during reproductive growth, N was additionally remobilized from fine roots and leaves.
Free amino acids (mainly arginine and glutamine) and proteins were identified as the main storage compounds in the taproots. The protein pool did not show any seasonal variations that indicated the existence of a vegetative storage protein, indicating that such proteins are not a necessary component of N storage/remobilization in all species.
The ability to store and remobilize N provides a mechanism for growth in the spring when the availability of soil N is low, and means that growth depends upon environmental conditions during more than one year.
... . In many roots carbohydrates are the primary source of reserve energy (Ho 1988;MacIsaac & Bewley 1995), although nitrogen-containing compounds are also a critical currency of storage metabolism (Suzuki & Kohno 1987;Cyr & Bewley 1989, 1990aHendershot & Volenec 1993). ...
... 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.
... Nitrogen reserve mobilization estimated using pulse-chase 15 N labelling in Medicago sativa L. (Kim et al., 1991(Kim et al., , 1993 revealed that approximately 40% of total N in roots was mobilized to supply N to shoot after defoliation. Hendershot and Volenec (1993a) identified three specific polypeptides (32, 19, and 15 kilodaltons) in the taproot, referred to as vegetative storage proteins (VSP) which represented a large proportion of the hydrosoluble proteins. These VSPs exibited a typical cycle of accumulation/mobilization which was induced by the spring regrowth (Hendershot and Volenec, 1993a) or by shoot removal (Avice et al., 1996a;Hendershot and Volenec, 1993b). ...
... Hendershot and Volenec (1993a) identified three specific polypeptides (32, 19, and 15 kilodaltons) in the taproot, referred to as vegetative storage proteins (VSP) which represented a large proportion of the hydrosoluble proteins. These VSPs exibited a typical cycle of accumulation/mobilization which was induced by the spring regrowth (Hendershot and Volenec, 1993a) or by shoot removal (Avice et al., 1996a;Hendershot and Volenec, 1993b). In conclusion, the higher starch and N accumulation found in Lodi vs. Europe taproots, or in plants allowed a longer regrowth interval in spring, suggests that these plants have a greater opportunity to re-accumulate VSP and starch reserves, and that this contributes positively to enhancing shoot regrowth. ...
The frequency of shoot removal in lucerne (Medicago sativa L.) has long been recognized as a key factor in its management and productivity. The present study was undertaken to determine the impact of cutting interval during spring (30 or 45 days) on the subsequent summer regrowth, in contrasting lucerne cultivars (cv. Europe and Lodi). In particular, the dynamics of shoot regrowth (leaf area index, radiation use efficiency, N accumulation in harvestable biomass) and its relationship with taproot organic reserves (starch and N contents) were studied. Results showed that increasing the duration of the spring regrowth had a positive effect on subsequent summer regrowth, but there were also effects of cultivars. During the first 14 days of summer regrowth, the Lodi cultivar showed higher leaf area index (LAI) and greater photosynthetic active radiation interception than the Europe cultivar. The organic reserve level was also affected by the length of the previous spring cutting treatment (45 days treatment > 30 days treatment) and cultivar (Lodi > Europe). Lodi accumulated a larger amount of starch and N reserves which were subsequently mobilized to a greater extent in the first three weeks of regrowth and this contributed to its faster initial shoot growth rate. Our results confirm the important role played by N and C taproot reserves in shoot growth rate in the first days following shoot removal. Results are discussed in relation to recent studies on the role of storage N compounds in regrowth, and the concepts of radiation use efficiency (RUE); nitrogen nutrition index (NNI), and the decline in N content seen during the accumulation of biomass in lucerne canopies.
... Several proteins were increasingly or newly synthesized during the acclimation of alfalfa to the cold (Mohapatra et al., 1987). Simultaneously, free amino acids were shown to accumulate in the taproots and crowns (Hendershot and Volenec, 1993;Dhont et al., 2003). The increase in free proline was notable, and the concentrations of arginine and histidine were also markedly increased (Dhont et al., 2003). ...
Introduction
Alfalfa (Medicago sativa L.) is a highly nutritious leguminous forage that plays an essential role in animal husbandry. In the middle and high latitudes of the northern hemisphere, there are problems with its low rates of overwintering and production. The application of phosphate (P) is an important measure to improve the cold resistance and production of alfalfa, but little is known about the mechanism of P in improving the cold resistance of alfalfa.
Methods
This study integrated the transcriptome and metabolome to explain the mechanism of alfalfa in response to low-temperature stress under two applications of P (50 and 200 mg kg⁻¹) and a control of none applied.
Results
The application of P fertilizer improved the root structure and increased the content of soluble sugar and soluble protein in the root crown. In addition, there were 49 differentially expressed genes (DEGs) with 23 upregulated and 24 metabolites with 12 upregulated when 50 mg kg⁻¹ of P was applied. In contrast, there were 224 DEGs with 173 upregulated and 12 metabolites with 6 upregulated in the plants treated with 200 mg kg⁻¹ of P compared with the Control Check (CK). These genes and metabolites were significantly enriched in the biosynthesis of other secondary metabolites and the metabolic pathways of carbohydrates and amino acids. The integration of the transcriptome and metabolome indicated that P affected the biosynthesis of N-acetyl-L-phenylalanine, L-serine, lactose, and isocitrate during the period of increasing cold. It could also affect the expression of related genes that regulate cold tolerance in alfalfa.
Discussion
Our findings could contribute to a deeper understanding of the mechanism that alfalfa uses to tolerate cold and lay a theoretical foundation for breeding alfalfa that is highly efficient at utilizing phosphorus.
... In alfalfa, the genetic resources accumulating more vegetative storage proteins in the roots have enhanced winter hardiness (Cunningham et al., 2001). These storage components can also be used by plants for regrowth after harvest (Hendershot and Volenec, 1993). Even a partial harvest can impact the sink/source relationship in plants. ...
Stevia rebaudiana (Bertoni) is a perennial crop from north Paraguay (humid subtropical climate), belonging to the Asteraceae family. Stevia is an emerging crop in Europe (mild climate), cultivated for its steviol glycosides (SG), natural sweeteners that are 300 times sweeter than sucrose which is the main agronomical and industrial interest of stevia. Recent studies showed that it is possible to cultivate stevia in mild climates as a perennial and economically viable crop. However, lack of knowledge on cropping system specific to perennial plants, the duration of cultivation, the overwintering and the impact of first-year crop establishment act as a disincentive to crop expansion. Harvest management through the impact of harvesting in the first year of establishment was investigated for agronomic traits over three years of production- for 15 stevia genotypes cultivated in the south-west of France. Two harvest modalities were compared: 2H when the plant is not harvested in the first year of establishment and 3H when the plant is harvested in the first year. The genotypes performance was assessed based on: regrowth rate after winter, SG yield (g/plant) through its two components of SG content (%w/w) and dry leaf biomass (g/plant), and the SG profile. Two cumulative variables, cumulated SG yield and cumulated dry leaf biomass, were also added to the study to obtain an overview of genotype performance during cultivation time and in both harvest modalities. The tested genotypes showed a wide range of response for overwintering, but with a significant decrease of 30 % survival rate for plants harvested in first year (3H). SG yield and dry leaf biomass results presented high variability among the different genotypes. These traits were also significantly impacted by the harvest modality, and a decrease in SG yield and dry leaf biomass was identified for plants harvested in first year (3H). No clear tendency was revealed for SG content or SG profile. Cumulative variables confirmed previous results showing a better SG yield and leaf biomass production for plants non-harvested during the first year (2H), at crop-life scale. Our results, on a wide range of genotypes, shed light on the agronomic management of Stevia rebaudiana in temperate conditions. They suggest the interest of a first year without harvest, allowing a better establishment of the crop, a better overwintering and a better cumulative yield.
... Tableau 1 : Les protéines de réserve des organes végétatifs identifiées chez les plantes herbacées (d'après Volenec et al., 1996). (Hendershot et Volenec, 1993a) et une VSP de 17 kDa dans le stolon du trèfle blanc (Corre et al., 1996). Ces protéines, qui représentent jusqu'à 40% des protéines solubles totales chez la luzerne, sont fortement hydrolysées dans les premiers jours suivant la coupe avant d'être à nouveau accumulées en fin de repousse. ...
Les espèces prairiales sont soumises à des défoliations successives qui réduisent fortement la quantité de CO2 assimilé et l'absorption d'azote. Afin d'assurer sa croissance après la coupe, la plante mobilise vers les tissus aériens en croissance certains substrats carbonés et azotés stockés dans les tissus laissés en place par la coupe. Pour assurer la productivité et la pérennité du couvert par une bonne gestion, il importe de bien identifier ces mécanismes d'adaptation. Les substrats carbonés et azotés mis en réserve sont disponibles dans les tissus laissés en place par la coupe (racines, pivots, tiges, stolons, base des feuilles). Différentes expérimentations sur ray-grass anglais, trèfle blanc, luzerne ont permis d'évaluer le rôle de ces réserves mobilisées vers les tissus en repousse. Les incidences actuellement connues des modes de défoliation (selon leur rythme et leur intensité) et de certains facteurs environnementaux sur ces réserves sont abordées. Chez les légumineuses, il apparaît qu'une plus faible disponibilité en réserves azotées au moment de la coupe se traduit par une réduction du potentiel de repousse alors que chez certaines graminées une compensation intervient suite à une augmentation de l'absorption d'azote par la plante.
... These VSPs can represent up to 40% of the total soluble proteins in the taproot according to Erice et al., (2007). Furthermore, as observed by previous studies (Hendershot and Volenec, 1993;Avice et al., 1996;Corre et al., 1996;Gana et al., 1998) during regrowth, VSPs are degraded, with a rate of remobilization from 60% to 80%, in order to provide N to re-growing shoots. In our experiment, we hypothesize that PR played a similar role by remobilizing N assimilates and proteins, thereby compensating for the drop in photosynthetic input by shoots caused by water deficit. ...
Drought is one of the most limiting factors on crop productivity under Mediterranean conditions, where the leguminous species alfalfa (Medicago sativa L.) is extensively cultivated. Whereas the effect of drought on plant performance has been widely described at leaf and nodule levels, less attention has been given to plant-nodule interactions and their implication on metabolites exchange during a regrowth period, when water is limiting. For this purpose, physiological characterization and metabolite profiles in different plant organs and nodules were undertaken under water deficit, including regrowth after removal of aerial parts. In order to study in more detail how nitrogen (N) metabolism was affected by water stress, plants were labelled with N-enriched isotopic air (¹⁵N2) using especially designed chambers. Water stress affected negatively water status and photosynthetic machinery. Metabolite profile and isotopic composition analyses revealed that, water deficit induced major changes in the accumulation of amino acids (proline, asparagine, histidine, lysine and cysteine), carbohydrates (sucrose, xylose and pinitol) and organic acids (fumarate, succinate and maleic acid) in the nodules in comparison with other organs. The lower ¹⁵N-labeling observed in serine, compared with other amino acids, was related with its high turnover rate, which in turn, indicates its potential implication in photorespiration. Isotopic analysis of amino acids also revealed that proline synthesis in the nodule was a local response to water stress and not associated with a feedback inhibition from the leaves. Water deficit induced extensive reprogramming of whole-plant C and N metabolism, including when the aerial part was removedto trigger regrowth.
... Root and rhizome protein concentrations both declined in spring when growth resumed and accumulated in autumn as growth subsided and plants acclimated for winter (Figure 4). This general pattern agrees with previous research on vegetative storage proteins in perennials (Hendershot and Volenec, 1993b;Avice et al., 2003). As expected, protein concentrations of both tissues in March of Year 2 generally reflected Year 1 N management whereas protein concentrations in November of Year 2 responded to that season's N management. ...
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.
... Studies on seasonal fluctuations of N and C reserves in taproots of alfalfa (Li et al., 1996;Cunningham and Volenec, 1998) indicated that N concentrations increased markedly in autumn, and substantial reductions in root N occurred as shoot growth resumed in the early spring. Among the different pools of N, amino acids and soluble proteins significantly accumulated in autumn and were extensively mobilized from taproots when meristem reactivation occurred in the spring (Hendershot and Volenec, 1993a;Li et al., 1996). Similarly, shoot regrowth following forage harvest in the summer induced an extensive depletion of amino acids and soluble proteins pools from taproots (Hendershot and Volenec, 1993b). ...
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.
... In contrast, large changes in soluble protein and low molecular weight N pools of roots occur seasonally and after defoliation (Fig. 6-1B and 6-3). Amino Nand soluble protein N accumulate in autumn as forage legumes harden for winter (Hendershot & Volenec, 1993a;Li et al., 1996). Both of these N pools are rapidly depleted from roots beginning in March as spring shoot growth resumes. ...
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.
... Furthermore, the taproot diameter and WSC concentration of the roots of chicory varied over sampling dates, whereas that of plantain remained stable. Typically, taprooted plants utilise their carbohydrate reserves for winter survival and initiation of growth in spring (Kim et al. 1991;Hendershot & Volenec 1993) and following defoliation (Kim et al. 1993). Furthermore, carbohydrate reserves are often associated with persistence in perennial forage legumes (Smith 1962). ...
Herb and legume mixes have been shown to increase animal performance compared with perennial ryegrass/white clover. The objective of this experiment was to determine the response of a herb and legume mix containing chicory (Cichorium intybus), plantain (Plantago lanceolata), red clover (Trifolium pratense) and white clover (T. repens) to either Hard (post-grazing residual of 4 cm) or Lax (post-grazing residual of 8 cm) grazing treatments using a 3–5 week grazing cycle with sheep over 2 years. The sward produced a greater dry matter yield under Hard than Lax grazing (11.6 vs 8.9 t DM/ha/year). However, Lax grazing maintained all four species in the sward, with a greater red clover contribution to the sward. Chicory had a greater taproot diameter and root water-soluble carbohydrate concentration under Lax than Hard grazing. Overall, this study illustrates that the herb and legume mix is likely to be more persistent under Lax than Hard grazing.
... In spring, roots lose weight as stored carbohydrates are either lost in respiration and remobilised for the initiation of the new basal buds after defoliation (Kim et al. 1993). In contrast, autumn shoot growth is reduced because of increased assimilate partitioning to roots as plants replenish reserves for overwintering and spring regrowth (Hendershot & Volenec 1992; Kim et al. 1991). Figure 1 Linear growth rates of irrigated lucerne in relation to mean temperature at Lincoln University, Canterbury, New Zealand. ...
Successful lucerne stand management requires balancing animal and plant requirements to produce crops of high quality and yield at times of high animal demand. Understanding the impact of environmental signals on crop growth and development can aid management decisions throughout the season. In spring, crops remobilise reserves from the roots to shoots and expand nodes accumulated through the winter, producing rapid stem extension and canopy closure as temperatures increase. The timing of spring defoliation should be based on crop yield and animal requirements rather than any specific developmental stage. Through spring and summer, crops should be rotationally grazed, with highest lamb live-weights achieved from 6–8 weeks grazing solely on lucerne. Summer crop production is dependent on rainfall and the plant available water content. During summer, grazing at the appearance of open flowers or basal buds is recommended as a compromise between maximum yield and quality. In autumn, the priority of assimilates allocation in the crop changes from above to below ground growth. To enhance the recharge of root reserves, an extended period of flowering is recommended in February or March. The time of flowering is dependent on the accumulation of thermal time and increases as photoperiod shortens. In periods of prolonged drought, lucerne herbage should be hard grazed and then spelled until the end of late autumn regrowth. A final hard grazing in June or early July, to remove overwintering aphids, should be followed by spraying 7–14 days later. Crops continue to develop nodes through the winter, and stands should be spelled until spring to ensure nodes are not removed by grazing, as this delays regrowth and reduces dry matter production.
... Carbohydrate reserves in perennial forage plants are an important source of energy necessary for winter survival and initiation of growth in spring (Kim et al. 1991, Hendershot andVolenec 1993). Furthermore, they can provide the necessary energy to support shoot growth following defoliation (Kim et al. 1993). ...
Plants are often subjected to periods of water stress. There are little data examining the effect of water stress on the forage species Plantago lanceolata and Cichorium intybus. In two pot experiments with P. lanceolata and C. intybus, morphological responses under optimum, dry, and very-dry water treatments with weekly, fortnightly and 3-weekly defoliation intervals and physiological responses under optimum and very-dry water treatments were measured. A third experiment compared the rooting depths of P. lanceolata and C. intybus under field conditions. These findings suggest that both P. lanceolata and C. intybus can survive and continue to grow under water stress conditions with the main differences between the two species being attributable to morphological characteristics (root mass, taproot diameter and shoot mass fraction) rather than differences at a physiological level. Overall, the results suggest plantain may be more productive under moderate drought due to its greater shoot mass fraction, whereas chicory may be more productive and persistent under severe drought due to its greater root mass, taproot diameter and root depth under field conditions.
... Three VSPs of 15, 19 and 32 kda were identified as a major component of the soluble proteins in the taproots of alfalfa. These proteins are accumulated during fall, when the plants are dormant, and degraded during spring or after defoliation (Hendershot and Volenec, 1993). Thus, it was observed that their level was 28 % of the total soluble protein before defoliation, decreased after defoliation but it was recovered 30 days later (Avice et al., 1996). ...
... In our experiments, VSP did not accumulate under low temperatures even in association with SD exposure (SD/5°C , Figs 4 and 5). Although it appears that the effect of late autumn environment on VSP accumulation initially observed by Hendershot and Volenec (1993a) was not easy to reproduce under controlled conditions, our results are consistent with data obtained in field conditions (Li et al. 1996;Cunningham and Volenec 1998). By studying the seasonal changes of taproot N accumulation in different alfalfa cultivars, Cunningham and Volenec (1998) reported that concentration of VSPs (including β-amylase) increased 4-fold in autumn when photoperiod was decreasing and soil temperatures progressively declined, but always exceeded 4°C. ...
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.
... There are indications that the bark tissues of the taproot may contain more mobilisable protein than the wood tissues. The bark not only contained consistently higher concentrations of N than the wood tissue, but also had twice the level of proteolytic activity in early spring (Hendershot and Volenec 1993b). However, other studies have suggested that more VSP might be stored in the wood parenchyma cells than in the bark (Avice et al. 1996b). ...
Growing dryland crops after lucerne is known to be risky because of the lack
of residual soil water. We investigated ways of reducing this risk by removing
portions of a lucerne pasture, using either herbicides or cultivation, at
monthly intervals between November and April, before sowing a wheat crop in
May, followed by a canola crop in the following year. The experimental site
was on a red-brown earth in southern New South Wales. Lucerne removal was
incomplete when the wheat was sown, so all lucerne plants were removed from
half of each plot with a post-emergence herbicide, to allow comparisons of
intercropped wheat–lucerne and wheat monoculture. Measurements were made
on crop growth, yield, grain quality, soil water, and soil mineral nitrogen
(N) before and after both crops.
On average, each additional month between lucerne removal and wheat sowing led
to a yield increase of 8% and a grain protein increase of 0.3
percentage units. The main reason for the increases was additional soil
mineral N, associated with a longer period of mineralisation. The soil water
content at the time of wheat sowing was greater with early lucerne removal but
the growing season rainfall did not limit yields, and there was more residual
soil water at the time of wheat maturity where lucerne had been removed late
and yields were lower. Method of lucerne removal did not significantly affect
wheat yield, grain protein, soil water, or soil mineral N. The portions of the
plots containing lucerne plants that survived the initial removal attempt
produced similar wheat yields to the portions where lucerne had been totally
removed, but grain protein was lower.
The following growing season was drier, but despite less residual soil water
where lucerne had been removed earlier in the previous year, the average
canola yield was 2.5% greater for each additional month of fallow. The
increase again appeared to be due to more residual mineral N. The seed oil
concentration also decreased in response to later lucerne removal but seed
protein increased. Where lucerne plants had been retained in the previous
wheat crop, canola yield was lower than where they had been totally removed,
apparently because of less soil water at sowing.
Over the 2 years of the experiment, the net supply of mineral N was 374 kg
N/ha, equivalent to an annual net mineralisation of 2% of the total
soil N. The initial mineralisation rate was slow, suggesting that the soil may
be deficient in mineral N soon after lucerne removal.
... In spring, roots lose weight as stored carbohydrates are either lost in respiration and remobilised for the initiation of the new basal buds after defoliation (Kim et al. 1993). In contrast, autumn shoot growth is reduced because of increased assimilate partitioning to roots as plants replenish reserves for overwintering and spring regrowth (Hendershot & Volenec 1992;Kim et al. 1991). ...
Successful lucerne stand management requires balancing animal and plant requirements to produce crops of high quality and yield at times of high animal demand. Understanding the impact of environmental signals on crop growth and development can aid management decisions throughout the season. In spring, crops remobilise reserves from the roots to shoots and expand nodes accumulated through the winter, producing rapid stem extension and canopy closure as temperatures increase. The timing of spring defoliation should be based on crop yield and animal requirements rather than any specific developmental stage. Through spring and summer, crops should be rotationally grazed, with highest lamb live-weights achieved from 6–8 weeks grazing solely on lucerne. Summer crop production is dependent on rainfall and the plant available water content. During summer, grazing at the appearance of open flowers or basal buds is recommended as a compromise between maximum yield and quality. In autumn, the priority of assimilates allocation in the crop changes from above to below ground growth. To enhance the recharge of root reserves, an extended period of flowering is recommended in February or March. The time of flowering is dependent on the accumulation of thermal time and increases as photoperiod shortens. In periods of prolonged drought, lucerne herbage should be hard grazed and then spelled until the end of late autumn regrowth. A final hard grazing in June or early July, to remove overwintering aphids, should be followed by spraying 7–14 days later. Crops continue to develop nodes through the winter, and stands should be spelled until spring to ensure nodes are not removed by grazing, as this delays regrowth and reduces dry matter production.
... In alfalfa. a 15 and 19 kDa protein accumulates in taproots (Hendershot andVolenec. 1991. ...
In the present work we evaluate the accumulation and further remobilization of vegetative storage proteins (VSP) in chicory. A protein with molecular weight of 17kDa, corresponding to 7 isoforms with pi ranging between 5 and 7, accumulated dramatically over the vegetative phase from spring to autumn and was extensively depleted during the flowering period in the following summer, a pattern typical for a VSP.
When mature tuberized roots of chicory are harvested in autumn and forced in darkness, an etiolated bud (chicon) grows: this is the salad known as Belgian endive. In our experiments plants were fed, during the forcing process, nutrient solutions containing 1.5 or 18 mmol/L 15NO3 (1.79 % atom excess 15N) or with demineralized water (control). We determined the cycling of endogenous nitrogen (14N), protein (VSP) and amino acids, and the movement of concurrently absorbed nitrogen (15N). Soluble proteins were remobilized at the onset of forcing as a primary response of nitrogen cycling in chicory root. Amino acid remobilization took place only when the chicon growth began with arginine remobilized first. Although 14N flux into the chicon was similar in all three treatments, indicating that NO3 supply did not effect endogenous N remobilization, VSP use was effected by NO3 supply. SDS-PAGE and 2-D gel electrophoresis analyses showed an extensive depletion of VSP (especially five isoforms) only in the control. We suggested that extensive and specific depletion of VSP was delayed by NO3 supply; with higher NO3 availability, there was lower VSP remobilization. Furthermore, neo-synthesis of VSP could occur during the forcing process. The finding that 15N was incorporated into the protein pool during this period supports this hypothesis. The chicon constituted a very strong sink for absorbed nitrogen. Either in high or low NO3 supply, 15N was translocated to the chicon almost without mixing with the bulk nitrogen of the root.
... There are indications that the bark tissues of the taproot may contain more mobilisable protein than the wood tissues. The bark not only contained consistently higher concentrations of N than the wood tissue, but also had twice the level of proteolytic activity in early spring (Hendershot and Volenec 1993b). However, other studies have suggested that more VSP might be stored in the wood parenchyma cells than in the bark (Avice et al. 1996b). ...
In the mixed cropping regions of Australia, the perennial fodder legume lucerne (Medicago sativa L.) is being utilised to improve the sustainability of farming systems through improving soil fertility and reducing groundwater recharge. However, the full rotational benefits of lucerne cannot be realised without a reliable approach to terminate the lucerne phase before cropping. Farmer surveys have identified difficulties in successfully removing lucerne before cropping as a significant problem with lucerne-based phase-farming systems. In 1 survey up to 40% of the respondents were not satisfied with the level of lucerne control they were achieving, while in another survey 49% indicated that lucerne removal issues were a constraint to further adoption of lucerne on their farms. Most growers used herbicides to kill lucerne, usually in conjunction with cultivation or grazing. Respondents estimated that herbicides were capable of killing 80% of the lucerne, but most regarded this level of control unsatisfactory. Lucerne removal using herbicides is ineffective when lucerne is able to regenerate from buds in the crown that have not received a lethal herbicide dose. Systemic herbicides such as glyphosate and the auxinic herbicides are predominantly translocated to those parts of the plant with a high demand for photosynthate, often the apical meristems. Hence, effective control of lucerne can only be achieved through targeted application of systemic herbicides to lucerne plants that are actively translocating photosynthate to the crown and taproots. Evidence presented in this review suggests that this is most likely to occur when the storage reserves in the crown and taproot are being replenished several weeks after defoliation. The importance of timing of removal and the potential for intercropping are also discussed.
... Taproot soluble proteins and, to a lesser extent, amino acids, represent the quantitatively most important N reserve pools with more than 50 and 25% of total taproot soluble N compounds, respectively (Hendershot and Volenec 1993b;Meuriot et al. 2003Meuriot et al. , 2004). Moreover, four vegetative storage proteins (15,19,32 and 57 kD VSPs), which can represent up to 40% of taproot total soluble proteins, were identified as having a key role in N storage in alfalfa taproots (Hendershot and Volenec 1993a;Avice et al. 1996b;Gana et al. 1998). These VSPs are mobilised at twice the rate observed for other taproot soluble proteins 10 d after shoot removal, and re-accumulate rapidly as shoots begin reproductive development (Avice et al. 1996b). ...
We studied the effects of stubble carbon/nitrogen (C/N) reserves or residual leaf area (RLA) on the contribution of taproot C/N reserves to shoot regrowth of Medicago sativa L. after cutting. The study assessed the effects of two cutting heights (6 and 15 cm), two RLAs (0 or 100%), and two initial C/N reserve levels (high N or low N) on forage production, nitrogen (N) distribution, and C/N reserve dynamics within stubble and taproot. Alfalfa forage production was mainly affected by the initial taproot C/N reserve levels. However, stubble initial organic reserves (and to a lesser extent the RLA) were also of particular importance during early regrowth. The increase of cutting height led to increased stubble C/N supply to regrowing shoots, which partly offset the negative effect on forage production and on taproot C/N reserve depletion. Unlike taproot reserves, the positive contribution of stubble organic reserves to shoot C/N supply was effective for a single defoliation-regrowth cycle. Alfalfa management strategies that increase cutting height (and RLA) during the penultimate harvest in autumn should be considered in cold regions with significant winter stress in order to improve alfalfa winter survival and persistence, as well as spring herbage regrowth.
... Soluble proteins accumulated in taproots are extensively mobilized to sustain shoot regrowth in early spring and after defoliation (Hendershot and Volenec 1993a, b; Avice et al. 1996). 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.
... Covariate analysis showed RUE * shoot was highest (1.0 g DM/MJ) in early spring and late autumn periods and lowest (0.47 g DM/MJ) in early autumn period (Table 2). This change in RUE * shoot was probably caused by seasonal changes in biomass partitioning between shoot and perennial organs (Hendershot and Volenec, 1992;Khaiti and Lemaire, 1992;Cunningham and Volenec, 1998). This analysis also showed increased temperature gave an increase in RUE shoot (Table 2). ...
This research quantifies the influence of seasonal variations in solar radiation, temperature and biomass partitioning on lucerne production in a temperate climate. Above ground biomass (shoot) production of fully irrigated ‘Kaituna’ lucerne was measured in the field over 5 years and 33 regrowth cycles in Canterbury, New Zealand. Shoot production increased linearly (R2 of 0.93±0.07) with intercepted total radiation within each regrowth cycle but radiation use efficiency (RUEshoot, in g DM/MJ total radiation) ranged from 0.29 to 1.09gDM/MJ. Covariate analysis showed season and temperature both influenced RUEshoot with temperature adjusted RUEshoot(RUEshoot*) decreasing from 1.01gDM/MJ in September to 0.77gDM/MJ between October and February, decreasing again to 0.47gDM/MJ in March and April before increasing back to 0.99gDM/MJ in May. A second fully irrigated experiment with lucerne plants grown in plastic columns under near field conditions investigated the seasonality of biomass partitioning between shoots and perennial biomass (roots and crowns). The proportion of total biomass partitioned to shoots (Pshoot) was 0.90 in September, ∼0.67 from October to February but only 0.35 in March and could be related to photoperiod. These Pshoot values were closely correlated with RUEshoot* showing seasonal changes in RUEshoot were partly caused by changes in biomass partitioning. Field data for RUEshoot were divided by Pshoot to estimate the RUE for total biomass production (RUEtotal). The RUEtotal increased linearly from 0.60 to 1.60gDM/MJ as mean air temperatures increased from 6 to 18°C. These results quantified the effects of solar radiation and mean temperature on total lucerne biomass production and its seasonal partitioning between shoots and perennial biomass. The influence of regrowth duration on this partitioning was also investigated.
... However, fall harvests have a major impact on the accumulation of the stress-related VSP of 32kDa and amino acids such as proline, arginine, and histidine during cold acclimatation (Dhont et al. 2003). It has been suggested that these free amino acids play an important osmoregulatory role in response to abiotic stresses (Rai 2002) while contributing N for the regrowth in the spring (Hendershot and Volenec 1993). ...
2006. Winter damage to perennial forage crops in eastern Canada: Causes, mitigation, and prediction. Can. J. Plant Sci. 86: 33–47. Harsh winter climate results in frequent losses of stands and yield reduction in many forage-growing areas of Canada and other parts of the world. Climatic conditions and crop management both affect the winter survival of perennial for-age crops. In this review, we present the main causes of winter damage in eastern Canada and we discuss crop management prac-tices that help mitigate the risks of losses. Predictive tools available to assess the risks of winter damage both spatially and temporally are also presented. Our understanding of the causes of winter damage and of the plant adaptation mechanisms to win-ter stresses, particularly the role of N and C organic reserves, has improved. Forage species commonly grown in eastern Canada differ in their tolerance to subfreezing temperatures and to anoxia caused by the presence of ice on fields. Some improvement in winter hardiness of forage legume species has been achieved through breeding in eastern Canada but new technologies based on laboratory freezing tests and the identification of molecular markers may facilitate the future development of winter-hardy culti-vars. Crop management practices required for good winter survival are now better defined, particularly those involving cutting management and the interval between harvests. Simulation models and climatic indices derived from our current knowledge of the causes of winter damage provide general indications on the risk of winter damage but their degree of precision and accuracy is still not satisfactory. Further improvements in winter survival require a more thorough understanding of the different causes of winter damage and, primarily, of their complex interactions with genetic, climatic, and management factors.
... Free amino acids were shown to accumulate in taproots and crowns during cold acclimation of alfalfa (Dhont et al., 2003; Hendershot and Volenec, 1993). A marked increase in free Pro in roots of alfalfa during cold acclimation has long been documented (McKenzie et al., 1988; Paquin, 1984). ...
Insufficient cold hardiness is a major impediment to reliable alfalfa (Medicago sativa L.) production in northern regions experiencing harsh winter conditions. Numerous studies have documented the morphological and physiological traits associated with the acquisition of freezing tolerance and winter survival in alfalfa. Use of this information as selection criteria to breed cultivars with superior winter hardiness has thus far been met with limited success. This can be attributed to many factors including: the large number of traits affecting winter survival; the multigenic nature of most traits, large environmental interactions, and an undesirable linkage between acquisition of freezing tolerance and fall growth cessation (fall dormancy). In the last two decades, the advent of molecular biology and quantitative genetic techniques has markedly increased our knowledge of the molecular and genetic bases of superior alfalfa winter hardiness. Our understanding of the mechanisms underlying the perception of the low temperature signal and its transduction into morphological and physiological responses leading to cold hardiness has progressed, but still remains fragmentary. Current evidence indicates that cold hardiness of alfalfa relies on tolerance to extensive freeze‐induced desiccation. Low temperature‐induced accumulation of soluble sugars and stress‐related translation products were found to be, in some instances, more abundant in cold‐tolerant cultivars and to be under some level of genetic control. Limited stability of these traits and conflicting reports on their relationship with freezing tolerance preclude their adoption as molecular screening tools. The development of robust screening techniques will require a more complete knowledge of the genetic bases of freezing tolerance. Heritability estimates suggest that independent selection for winter hardiness, freezing injury and autumn growth is possible, and that winter hardiness and autumn growth could be manipulated independently. This creates the opportunity to develop high‐yielding cultivars with improved winter hardiness. A screening test for freezing tolerance performed under controlled conditions recently led to the development of populations with increased freezing tolerance and led to significant improvement in alfalfa winter survival. Unique genetic material, combined with novel gene discovery approaches, could be lead to the identification of genetic polymorphisms associated with freezing tolerance in alfalfa and pave the way to marker‐assisted selection. Based on the current knowledge, we propose a conceptual framework for the genetic determination of cold adaptation of alfalfa.
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.
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.
Forage legumes benefit pastures and hay crops by fixing N, improving seasonal distribution of growth, and enhancing animal performance, but their lack of persistence is viewed as a major limitation. Stand persistence depends largely on plant persistence in crown-forming perennials that do not spread by stolons or rhizomes, but depends on seed production, timely germination, and seedling survival in annuals, biennals, and many short-lived perennials. Stolon- and rhizome-forming perennials can colonize unoccupied areas if management is favorable. Conversely, differentials in seed production, seed dispersal mechanisms, and seed survival allow reseeding annuals, biennials, and short-lived perennials to colonize areas that are more widely dispersed [...]
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.
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.
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.
An investigation was conducted to determine the effect of potassium (K) nutrition on alfalfa (Medicago sativa L.) growth and metabolism of root total nonstructural carbohydrates (TNC) and proteins, and to study whether nitrogen (N) fertilization overcomes N deficiency and low root protein concentrations caused by K deficiency. In Experiment 1, nodulated alfalfa plants were grown in plastic pots containing washed quartz sand and provided minus‐N Hoagland's solution containing 0, 0.6, or 6.0 mM K. Shoot and root K concentrations increased with increasing solution K. Root N concentrations were higher in plants receiving 6.0 mM K than in plants receiving 0.6 or 0 mM K, but shoot N concentrations were similar for all treatments. Plant persistence, shoots per plant, and shoot mass increased as solution K levels increased. Root starch concentration and utilization were positively associated with K nutrition. Total amylase activity was higher, but endoamylase activity was lower in roots of plants receiving 6.0 mM K compared to plants receiving 0.6 or 0 mM K. Root soluble protein concentrations were significantly higher in plants receiving 6.0 mM K than in plants receiving 0 or 0.6 mM K. In Experiment 2, plants were supplied with Hoagland's solution containing 10 mM N as ammonium (NH4 ) or nitrate (NO3) with 0,3, or 6.0 mM K. The addition of N increased root N concentrations only in plants receiving 0 mM K. Plant persistence was reduced by NH4 application, especially in plants receiving 0 or 3 mM K. Root starch concentrations were markedly reduced in plants receiving NH4 at all K levels. The addition of NO3 had little effect on alfalfa root carbohydrate and protein metabolism and subsequent shoot growth. Potassium deficiency reduced starch and protein concentrations in roots; factors that were associated with poor persistence and slow shoot regrowth of alfalfa.
Alfalfa (Medicago sativa L.) root reserves are thought to provide nutrients to regrowing shoots, enhance stress tolerance, and improve plant persistence. Factors affecting carbohydrate and protein accumulation and metabolism in roots are important in alfalfa production. Our objectives were to determine 1) the influence of phosphorus (P) nutrition on alfalfa shoot growth and root carbohydrate and protein metabolism after defoliation and 2) how quickly growth and root carbohydrate and protein metabolism of P‐deficient alfalfa plants responds to supplemental P. In Experiment 1, nodulated alfalfa was grown in quartz sand with minus‐nitrogen (N) Hoagland's solution containing 0,1,2, or 6 mM P. Root P concentrations increased with increasing solution P levels. Phytate P in roots of plants grown with 6 mM P was greater than that of plants grown in 0, 1, or 2 mM P. Shoot mass and shoots per plant were reduced by 67 and 43%, respectively, in plants grown with 0 mM P as compared to plants grown with 6 mM P. Root starch and protein concentrations did not decline after defoliation in plants provided 0 mM P. Defoliation increased endoamylase activity in roots of plants that received P. Root total amylase activity declined as P supply increased, and was not associated with root starch degradation. In Experiment 2, P‐deficient alfalfa plants were provided with minus‐N Hoagland's solution containing 6 mM P either at defoliation (Day 0), or at Day 14, and these plants were compared to others provided 0 or 6 mM P throughout the study. Root P concentrations increased within 7 days of P application. Shoot regrowth of P‐deficient plants recovered to equal that of plants provided 6 mM P within 7 to 21 days after P application. Application of P at defoliation enhanced root starch use, whereas P application on Day 14 had no effect on root starch depletion. Normal root starch utilization occurred in plants provided 6 mM P when plants were defoliated a second time 35 days after the initial harvest. Normal root protein utilization was not restored by P application during regrowth. Roots of P‐deficient alfalfa contained high concentrations of organic reserves, but use of these reserves after defoliation was impaired.
Elevated levels of atmospheric carbon dioxide (CO 2 ) can directly affect the cold hardening process in evergreens through their effect on the accumulation of carbon and nitrogen reserves. This study investigated the biochemical responses of black spruce [ Picea mariana (Mill.) B.S.P.] seedlings to CO 2 enrichment during growth, cold hardening and dehardening. Seedlings were grown under 350 (ambient) or 710 (elevated) ppm of CO 2 for 12 months in eight mini-greenhouses. Photoperiod and temperature were gradually lowered in autumn to induce cold hardening, and the conditions were reversed in spring to promote dehardening. At regular intervals, cold tolerance was assessed and sugars, starch and amino acid concentrations were measured. The freezing tolerance differed between the two treatments only in early autumn, with seedlings growing under high CO 2 being more tolerant. The northern ecotype was more cold tolerant with concomitant higher concentrations of sucrose, fructose, pinitol, glucose and total soluble sugars. The concentration of soluble sugars increased in needles and roots of black spruce along with cold hardening, and the concentrations of the cryoprotective sugars sucrose and raffinose were lower under elevated CO 2 . Amino acid concentrations were also lower under elevated than under ambient CO 2. The lower level of reserve did not translate into a lower level of freezing tolerance.
in nondormant, nonhardy cultivars when compared with fall dormant plants (Volenec, 1985; Boyce and Volenec, amino-N concentrations when compared with three of the other four dormant cultivars and germplasms. Germplasm 98-132 with an inter- exhibited little change in amino and non-amino N con- mediate FD, incurred relatively low winter injury, similar to that of fall centrations between August and December, winter dormant Vernal, when compared with other intermediate dormancy hardy cultivars increased root amino and non-amino N cultivars and germplasms. This germplasm had root sugar concentra- levels by 20 and 31%, respectively (Wilding et al., 1960). tions that were similar to plants with FD ratings of 1 to 3. Creation Bula et al. (1956) found a positive correlation between of even less FD germplasms that possess high winter hardiness would increased levels of soluble protein in autumn and en- facilitate our understanding of the physiological and molecular mecha- hanced winter hardiness of dormant alfalfa cultivars. nisms controlling these two very important agronomic traits of alfalfa. Hendershot and Volenec (1993) reported that soluble amino-N and buffer-soluble protein increased in au- tumn and early winter, and declined in spring as herbage T
The adverse effect of fall harvesting alfalfa (Medicago sativa 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 nonstructural 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.
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.
Carbohydrate accumulation and utilization during shoot regrowth after defoliation and winter has been studied extensively in most species used as forage. However, recent work suggests that N reserves found in vegetative tissues also are important for defoliation tolerance and winter hardiness. Results suggest that these N reserves constitute an alternative N source used when N2 fixation and/or mineral N uptake are reduced. 15N labelling experiments indicate that a large proportion of herbage N is derived from N reserves mobilized from stem bases or roots to developing leaves and shoots. Amino acids and specific proteins (i.e. vegetative storage proteins, VSPs) are deposited in roots and stem bases and, in the case of VSPs, are degraded rapidly after defoliation. Identification and characterization of VSPs will increase our understanding of the role N reserves play in stress tolerance and may lead to innovative approaches for improving forage persistence and productivity.
Previous evidence indicates that differences in the concentration of underground organic reserves can drive the survival and growing ability of lucerne under cold and defoliation stresses. In order to provide the selection process with further information on compounds that may influence plant performance under grazing, we assessed variations in cold-season concentrations of nitrogen and carbon reserves on genotypes that had been identified for morphological features that possibly enhance grazing tolerance. The selected genotypes encompassed distinct morphological patterns (defined as ‘models’) and different taxa within the Medicago sativa complex, as shown by different flower colours. Crown concentrations of reserves were determined on 90 genotypes, whereas root concentrations were measured on a subsample of 15 genotypes. Wide intergenotypic variation was observed for all reserve substances. Comparisons among models and among flower colour classes highlighted the high concentrations of crown carbohydrates and root and crown-soluble proteins of the model coded as ‘D1’, characterized by prostrate, rhizomatous habit and long dormancy, which largely corresponded to plants with yellow or variegated flowers, typical of ssp. falcata and × varia, respectively. There was a strong ‘flower colour × storage organ’ interaction for sugar concentration, and the results suggested a preferential compartment of sugars in the roots of purple-flowered genotypes that belonged to the ssp. sativa. A rank correlation analysis indicated a positive relationship between persistence after two years under grazing of half-sib progenies deriving from 19 genotypes out of the 90 and crown concentrations of carbohydrates of the 19 mother plants.
A major factor limiting persistence of alfalfa (Medicago sativa L.) in the northern US is poor winter hardiness. Our hypothesis is that suspension cell cultures derived from dormant, winter-hardy
alfalfa cultivars would cold acclimate and survive sub-zero temperatures better than cell cultures derived from non-dormant,
non-hardy cultivars. Our objectives were (1) to determine if genetic differences in winter hardiness between dormant and non-dormant
alfalfa were retained by suspension cells derived from these contrasting cultivars; and (2) to determine the physiological
and biochemical bases for differences in freezing tolerance of suspension cells. Cell suspensions derived from `5262' (fall
dormant) and `5929' (fall non-dormant) were cold hardened at 2 °C for 14 days. Cells were frozen in a cooling bath and cell
survival determined by measuring 2, 3, 5-triphenyltetrazolium chloride (TTC) reduction. Cold acclimation improved cell survival
of both cultivars to −5 °C when compared to unacclimated cells. Only acclimated cells of 5262 survived temperatures of −10
°C to −25 °C. The freezing tolerance of cold-acclimated 5262 cells was associated with high sugar and starch concentrations,
lower α-amylase activities and slightly lower cell protein levels when compared to 5929. No differences in polypeptide composition
were evident when comparing acclimated and unacclimated cells of 5929, but polypeptide composition did change with acclimation
of 5262 cells. As expected, expression of RootCAR1 in 5262 cells increased with cold acclimation, but high levels of RootCAR1
transcript were unexpectantly found in both cold acclimated and unacclimated 5929 cells. With the exception of the RootCAR1
expression, many of the physiological responses of these alfalfa cell lines to cold acclimation were similar to those that
have been reported for field-grown plants.
The physiological processes associated with genetic differences in shoot elongation rate (SER) and leaf area expansion rate (LAER) of alfalfa (Medicago sativa L.) are poorly understood. Our objective was to examine the relationships among SER, LAER, and the concentration and utilization of nonstructural carbohydrates in roots and meristematic shoot tissues of alfalfa. Two genotypes selected for contrasting SER were sampled after 7 d of herbage regrowth in 15-h photoperiods. Remaining plants were placed in continuous darkness and sampled periodically during the next 144 h (Exp. 1) or 33 h (Exp. 2) of regrowth. In Exp. 1，LAER, concentrations of total nonstructural carbohydrate (TNC), and dark respiration rates (Rd) of expanding leaves decreased during the first 48 h of regrowth in continuous darkness. In Exp. 2, LAER declined to 40% of midphotoperiod values after 17 h of continuous darkness, while RD of expanding leaves declined to 75% of mid-photoperiod rates. Concentrations of TNC in expanding leaves declined by Hour 17 of darkness to 19% of mid-photoperiod concentrations, reflecting trends in RD and especially LAER. In expanding leaves, TNC concentrations greater than 28 g kg⁻¹ dry wt. were necessary for maximum LAER and RD to occur. Averaged over experiments, root TNC concentrations were 185 g kg⁻¹ dry wt. during the initial 17 h of continuous darkness and did not decline significantly. Although roots contained high concentrations of TNC, LAER decreased rapidly within 17 h of continuous darkness. This indicates that expanding leaves may rely upon sources of carbohydrate other than that in roots for growth under C-limited conditions.
Electrophoretic and immunological properties of the soluble root protein complement of 6 Medicago sativa L. genotypes in the cold hardened and non-hardened physiological condition were compared. These 6 genotypes were chosen to represent a range of abilities to survive exposure to subfreezing temperatures when in the cold hardened condition.A zone of highly charged and/or low molecular weight protein components were found to be more prevalent in the protein complements of the cold-hardened material than the non-hardened material.Immunodiffusion plate tests were not so definitive as the electrophoretic patterns for identifying the genotypes or physiological conditions, but did corroborate the electrophoretic interpretations.
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.
Defoliation of alfalfa (Medicago sativa L.) results in a cyclic pattern of starch degradation followed by reaccumulation in taproots. Characterization of changes in anatomical distribution of starch grains in taproots will aid our understanding of biochemical and physiological mechanisms involved in starch metabolism in taproots of this species. Our objectives were to determine the influence of defoliation on starch grain distribution and size variation in taproots of two alfalfa lines selected for contrasting concentrations of taproot starch. In addition, we used electron microscopy to examine the cellular environment of starch grains, and computer-based image optical analysis to determine how cross-sectional area of tissues influenced starch accumulation. Taproots of field-grown plants were sampled at defoliation and weekly thereafter over a 28-day period. Taproot segments were fixed in glutaraldehyde and prepared for either light or electron microscopy. Transverse sections were examined for number and size of starch grains and tissue areas were measured. Starch grains were located throughout bark tissues, but were confined primarily to ray parenchyma cells in wood tissues. During the first week of foliar regrowth after defoliation, starch grains in ray cells near the cambium disappeared first, while degradation of those near the center of the taproot was delayed. During the third and fourth weeks of regrowth, there was a uniform increase in number of starch grains per cell profile across the rays, but by 28 days after defoliation there were more starch grains in ray cells near the cambium than in cells near the center of the taproot (low starch line only). Bark tissues from both lines showed synchronous degradation and synthesis of starch grains that was not influenced greatly by cell location. Diameter of starch grains varied with cell location in medullary rays during rapid starch degradation, but was not influenced by cell position in bark tissues. Therefore, during foliar regrowth there is a spatial separation in starch degradation and synthesis in alfalfa taproots. Amyloplasts from alfalfa taproots contained numerous starch grains, prolamellar-, and electron-dense bodies. The high starch line had 23% more cross-sectional area as ray cells in wood tissues when compared to the low starch line, which may explain part of the difference in starch accumulation between these alfalfa lines.
Free amino acids in 40 herbaceous perennial plants were analyzed under natural conditions. From the major amino acid content at the wintering stage, the pools were separated into the following five types: 1) a group which accumulated arginine (20 plants out of 40); 2) a group which accumulated arginine and proline (9 plants); 3) a group which accumulated glutamate and glutamine (3 plants); 4) a group which accumulated asparagine (4 plants); and 5) a group which accumulated proline (4 plants). Changes in the amino acid pools in the plants occurred under snow during wintering for about five months. Particularly, asparagine was no longer the major amino acid in the group which had accumulated it in fall. There was a tendency for the glutamine content to increase, suggesting that NH3 is utilized for the synthesis of the amide. Also, the relative concentrations of almost all the free amino acids increased several-fold, which was indicative of the occurrence of biosynthetic processes of general amino acids during wintering. As the mobile fractions of stored nitrogen, the amino acids appeared to contribute to the initial stage of rapid growth in early spring.
Light- and electron-microscopical investigations revealed distinct intravacuolar protein aggregates of 0.3-0.8 μm in diameter in ray cells of poplar during the dormant season. In semi-thin sections, these bodies showed positive protein staining and enzymatic digestibility with pepsin, indicating their proteinaceous nature. Morphometric measurements showed such protein bodies in 7-13% of the area of the ray-cell lumen. This amount corresponded with the protein content of the wood determined biochemically, e.g. 2.0-5.0 μg·mg(-1) dry weight. Polyacrylamide gel electrophoresis of the total protein fraction extracted from wood showed prominent polypeptide species with an apparent molecular weight of 30-32 kilodaltons. The results indicate considerable protein storage in ray cells, especially in the form of protein-storage vacuoles.
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.
One endopeptidase was present in alfalfa (Medicago sativa L. cv. Messe) leaves, as detected by gel-filtration, ion-exchange chromatography, and high pressure liquid chromatography.
The endopeptidase found, as judged by phenylmethylsulfonyl fluoride inhibition, behaves as a serine endopeptidase, with a functional histidyl residue as judged by diethyl pyrocarbonate inhibition.
The enzyme digested hemoglobin and casein at optimum pH 3.5 and 4.5 respectively; however it retains 50% of the activity at higher pH. The molecular weight, estimated by gel filtration, was 117,000.
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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Faster regrowth of a stressed alfalfa (Medicago sativa L.) crop compared to an unstressed crop after rewatering has been reported. The bases of this compensatory response are unknown, but they may be important to understanding adaptation to water stress and to developing crop water management strategies. The authors objectives was to determine the effect of stress induced by water deficit on photoassimilate partitioning and the utilization of stored assimilates during regrowth of alfalfa. Field and greenhouse experiments were conducted using cultivars differing in winterhardiness. Plants were subjected to water stress, pulse-labeled with ¹â´COâ, and sampled following 0, 1, 14, 21, and 28-d translocation periods. Following the 14-d sampling, herbage was harvested and water stress was removed. Cultivars contrasting in winterhardiness responded similarly to water stress. Stressed plant roots contained 73 and 114% more total plant radioactivity (TPR) than the control at the 1 and 14-d translocation periods, respectively. Water stress significantly increased root starch and TPR percentage in the starch fraction, but had much smaller effects on root soluble-sugar concentration and TPR percentage of the root sugar fraction. Herbage regrowth mass following harvest and rewatering of the water-stressed plants was similar to that of the control. Compared to the control, water-stressed alfalfa has greater total nonstructural carbohydrates in the roots, apparently due to increased photoassimilate partitioning to the roots. However, the greater root carbohydrate concentrations did not result in compensatory herbage regrowth following rewatering.
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.
Growth and nitrogen partitioning were investigated in the biennial monocarp Arctium tomentosum in the field, in plants growing at natural light conditions, in plants in which approximately half the leaf area was removed and in plants growing under 20% of incident irradiation. Growth quantities were derived from splined cubic polynomial exponential functions fitted to dry matter, leaf area and nitrogen data.
Main emphasis was made to understanding of the significance of carbohydrate and nitrogen storage of a large tuber during a 2-years' life cycle, especially the effect of storage on biomass and seed yield in the second season. Biomass partitioning favours growth of leaves in the first year rosette stage. Roots store carbohydrates at a constant rate and increase storage of carbohydrates and nitrogen when the leaves decay at the end of the first season. In the second season the reallocation of carbohydrates from storage is relatively small, but reallocation of nitrogen is very large. Carbohydrate storage just primes the growth of the first leaves in the early growing season, nitrogen storage contributes 20% to the total nitrogen requirement during the 2nd season. The efficiency of carbohydrate storage for conversion into new biomass is about 40%. Nitrogen is reallocated 3 times in the second year, namely from the tuber to rosette leaves and further to flower stem leaves and eventually into seeds. The harvest index for nitrogen is 0.73, whereas for biomass it is only 0.19.
In trees, nutrient reserves built up in the previous year are of primary importance for early spring growth. Despite the relatively great importance of roots for nutrient storage, the root system should not be regarded as a special storage organ. Quantitatively, carbohydrates predominate in these reserves, but qualitatively N and other minerals are of more than minor significance. In roots carbohydrates are usually stored in insoluble form, mainly as starch; sorbitol is the predominant soluble compound in apple and peach. For nitrogen reserves, the soluble form predominates in roots, especially arginine in apple and peach, followed by asparagine. The level of reserves usually becomes maximal early in the winter. During leafing-out the reserves are drawn on until, later in the season, the supply of newly produced or absorbed nutrients exceeds the demand and replenishment occurs. The initial carbohydrate reserves do not determine the amount of new growth, whereas reserve nitrogen is of decisive importance for shoot growth vigour. Environmental factors such as light intensity and temperature affect the level of carbohydrates in roots; the concentration can be reduced by defoliation and summer pruning and increased by ample supply of nitrogen fertilizer in the autumn. The main cultural factors that influence nitrogen reserves are the amount and the time of nitrogen fertilization.
M26 apple rootstocks were grown in sand culture and supplied with three rates of nitrogen (N) with the irrigation: none, 0·8 mol N m⁻² or 8·0 mol N m⁻². All the N supplied to the trees was labelled with ¹⁵N at 5·0 atom percent enrichment. The effect of N supply on tree growth, N uptake and the remobilization of N from stems for the annual growth of the trees was measured. Increasing the N supply increased leaf growth, but had no effect upon root mass and so altered the root/leaf dry matter ratio
Plants receiving no fertilizer N had to rely entirely upon stored reserves of N for their seasonal growth. Initially this N was used for leaf growth, which stopped after a few weeks. Thereafter the N-deficient plants retranslocated some of the N from their leaves to support root growth. Increasing the N supply had little effect upon the amount of N remobilized for growth, although well-fertilized plants accumulated N in their leaves and did not retranslocate any to support root growth. The partitioning of N between roots and shoots was, therefore, altered by increasing the N supply. Amino acid analysis of stems showed that the major forms of N remobilized during growth were protein rich in asparagine and arginine
The results show the importance of internal N cycling for the growth of young apple trees, and are discussed in relation to other studies of N cycling in deciduous trees
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.
A protein determination method which involves the binding of Coomassie Brilliant Blue G-250 to protein is described. The binding of the dye to protein causes a shift in the absorption maximum of the dye from 465 to 595 nm, and it is the increase in absorption at 595 nm which is monitored. This assay is very reproducible and rapid with the dye binding process virtually complete in approximately 2 min with good color stability for 1 hr. There is little or no interference from cations such as sodium or potassium nor from carbohydrates such as sucrose. A small amount of color is developed in the presence of strongly alkaline buffering agents, but the assay may be run accurately by the use of proper buffer controls. The only components found to give excessive interfering color in the assay are relatively large amounts of detergents such as sodium dodecyl sulfate, Triton X-100, and commercial glassware detergents. Interference by small amounts of detergent may be eliminated by the use of proper controls.
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 method for the highly efficient desalting of 50–200 μl volumes of solution containing from 10 ng to 5 mg of protein has been developed. Large numbers of samples may be rapidly and quantitatively desalted. The technique should be especially useful for the removal of unbound ligand from a rapidly dissociating ligand-binding protein complex.
A colorimetric method for the quantitative analysis of pure amino acids is described. It is a modification of one reported by Yemm and Cocking, which employs cyanide and ninhydrin. The present method avoids the necessity for the preparation of solutions of reduced ninhydrin, which is an unstable reagent difficult to prepare and impracticable to store.The method is applicable to amino acid mixtures when allowances are made for slight variability of color yields, and the presence of interfering compounds.
Soluble proteins extracted from alfalfa roots of hardy and nonhardy varieties were studied in relation to cold hardiness with polyacrylamide gel electrophoresis and quantitative enzyme analysis. Soluble protein content of alfalfa roots increased during hardening in all varieties. Two new isoenzymes with peroxidase activities were found in the fully hardened samples but no large shifts in the electrophoretic pattern were detected with polyacrylamide gel electrophoresis. Peroxidase and catalase activities increased during hardening in all varieties, but only small differences among hardy and nonhardy varieties were detectable. The studies indicated that protein metabolism was altered during the hardening process.
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.
Total nitrogen Methods of Soil Analysis
- J M Bremner
BREMNER, J. M.: Total nitrogen. In: BLACK, C. A., D. D. EVANS, J. L. WHITE, L. E. ENSMINGER, and F. E. CLARK (eds.): Methods of Soil Analysis. pp 1149-1178. American Society of Agronomy, Ma-dison WI (1965).
Methods of Soil Analysis. pp 1149-1178
- L E Ensminger
WHITE, L. E. ENSMINGER, and F. E. CLARK (eds.): Methods of Soil
Analysis. pp 1149-1178. American Society of Agronomy, Madison WI (1965).