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Developmental Regulation and the Influence of Plant Sinks on Vegetative Storage Protein Gene Expression in Soybean Leaves
Abstract
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.
... Species variance perhaps resulted in this difference as soybean protein is an important source of plant protein while the primary nutrients in cereal or fruit were starch or carbohydrates 47,48 ; thus proteins biosynthesis is more active in soybean, especially at grain filling stage. According to previous reports, some proteins synthesized in soybean leaves, such as vegetative storage proteins (VSPs), serve as temporary storage molecules for nitrogen 49,50 . They can be rapidly synthesized and subsequently broken down; and the resulted amino acids or metabolites are presumably exported to developing seeds via the phloem transport system to support seed protein synthesis 49,50 . ...
... According to previous reports, some proteins synthesized in soybean leaves, such as vegetative storage proteins (VSPs), serve as temporary storage molecules for nitrogen 49,50 . They can be rapidly synthesized and subsequently broken down; and the resulted amino acids or metabolites are presumably exported to developing seeds via the phloem transport system to support seed protein synthesis 49,50 . In soybean, translocation from vegetative tissue (mainly leaves) contributes no less than 50% of the fixed nitrogen utilized by seeds [49][50][51] . ...
... They can be rapidly synthesized and subsequently broken down; and the resulted amino acids or metabolites are presumably exported to developing seeds via the phloem transport system to support seed protein synthesis 49,50 . In soybean, translocation from vegetative tissue (mainly leaves) contributes no less than 50% of the fixed nitrogen utilized by seeds [49][50][51] . The Kac modification could potentially influence the biosynthesis of VSPs in soybean leaves, and further mediate soybean seed protein synthesis and accumulation. ...
Protein lysine acetylation (Kac) is an important post-translational modification in both animal and plant cells. Global Kac identification has been performed at the proteomic level in various species. However, the study of Kac in oil and resource plant species is relatively limited. Soybean is a globally important oil crop and resouce plant. In the present study, lysine acetylome analysis was performed in soybean leaves with proteomics techniques. Various bioinformatics analyses were performed to illustrate the structure and function of these Kac sites and proteins. Totally, 3148 acetylation sites in 1538 proteins were detected. Motif analysis of these Kac modified peptides extracted 17 conserved motifs. These Kac modified protein showed a wide subcellular location and functional distribution. Chloroplast is the primary subcellular location and cellular component where Kac proteins were localized. Function and pathways analyses indicated a plenty of biological processes and metabolism pathways potentially be influenced by Kac modification. Ribosome activity and protein biosynthesis, carbohydrate and energy metabolism, photosynthesis and fatty acid metabolism may be regulated by Kac modification in soybean leaves. Our study suggests Kac plays an important role in soybean physiology and biology, which is an available resource and reference of Kac function and structure characterization in oil crop and resource plant, as well as in plant kingdom.
... Los polipéptidos de reserva son reservorios de proteínas altamente enriquecidas con nitrógeno que son intensamente movilizadas para la reactivación de órganos en primavera, incluyendo la expansión de las hojas y crecimiento cambial y para mantener la respiración durante la etapa de dormancia y que se almacenan en abundancia durante el otoño e invierno en órganos permanentes para ir desapareciendo paulatinamente a lo largo del ciclo vegetativo (Staswick, 1989Mason y Mullet, 1990;Mason et al. 1992). El propósito de la presente investigación es llevar a cabo un estudio detallado del comportamiento de la concentración de azúcares solubles en diferentes etapas fenológicas (desarrollo vegetativo y dormancia) en caladio, así como la presencia/ausencia de polipéptidos de reserva en tubérculos de plantas sometidas bajo tratamientos en diferentes niveles de concentración de productos orgánicos y tratamientos hormonales, con la finalidad de determinar los niveles bajo los cuales se lleva a cabo una mejor acumulación de reservas vegetativas (azúcares solubles y polipéptidos de reserva), así como su influencia en el fenotipo y de esta forma contribuír al conocimiento del manejo de esta especie ornamental bajo condiciones de invernadero. ...
... Desde entonces, las proteínas específicas pueden tener un papel similar y han sido llamados VSP. Estas proteínas se acumulan en grandes cantidades en diferentes tejidos vegetativos: hojas, tallos tubérculos, raíces y rizomas (Staswick, 1989;Mason y Mullet, 1990). Estas proteínas se almacenan en abundancia durante el otoño y el invierno en órganos permanentes para ir desapareciendo paulatinamente a lo largo del ciclo vegetativo (Staswick, 1989;Mason y Mullet, 1990, Mason et al., 1992. ...
... Estas proteínas se acumulan en grandes cantidades en diferentes tejidos vegetativos: hojas, tallos tubérculos, raíces y rizomas (Staswick, 1989;Mason y Mullet, 1990). Estas proteínas se almacenan en abundancia durante el otoño y el invierno en órganos permanentes para ir desapareciendo paulatinamente a lo largo del ciclo vegetativo (Staswick, 1989;Mason y Mullet, 1990, Mason et al., 1992. ...
Este estudio presenta la expresión fenotípica en cuanto a coloración, el com- portamiento de la concentración de azúcares solubles en dos etapas fenológicas (desarrollo vegetativo y dormancia) en caladio (Caladium bicolor), así como la presencia/ausencia de polipéptidos de reserva en tubérculos bajo tratamientos de 4 y 8 mL•L-1 de productos orgánicos (ácidos húmicos y fúlvicos) y trata- mientos hormonales con auxinas (30 ppm), con citocininas (30 ppm) y con la combinación auxinas (30 ppm)–citocininas (30 ppm), con la finalidad de con- tribuir al conocimiento del manejo de esta especie ornamental bajo condiciones de invernadero en macetas de 11 L. El muestreo se realizó con tres repeticiones por tratamiento. La determinación de la concentración de azúcares se realizó con el método colorimétrico de van Handel. La determinación de polipépti- dos de reserva se hizo en geles de poliacrilamida. Los resultados se analizaron con el Programa SPSS- Statics 8. Se presentaron seis diferentes fenotipos de acuerdo con los tratamientos. Cada tratamiento reflejó un fenotipo diferente en cuanto a expresión de color. La aplicación de 8 mL•L-1 de productos orgá- nicos fue la que favoreció la acumulación de azúcares (0.850 mg•g-1 MS). Se detectaron tres polipéptidos de 10, 20 y 35 kDa en los tratamientos con pro- ductos orgánicos y tres polipéptidos de 10, 15 y 25 kDa en los tratamientos con fitohormonas que presentan las características proteínas de reserva vegetativa.
... The resulting fusion clone was expressed in E. coli and the protein was purified on His-BindTM resin according to procedures supplied by Novagen. Antibodies against the fusion protein were raised in a rabbit as previously described, and VSP antisera was obtained previously (Staswick, 1989). ...
... For loading the RCP gel, the amount of protein recovered in each pellet was assumed to be proportional to that in each corresponding supernatant. Proteins were separated by SDS-PAGE, electrotransferred to nitrocellulose membrane, and detected with an alkaline phosphatase-conjugated secondary antibody as previously described (Staswick, 1989). ...
... Nucleic acid electrophoresis, blotting and hybridizations were done using standard procedures (Sambrook et al., 1989) as previously described (Staswick, 1989). Genomic DNA was isolated as described previously (Staswick, 1988). ...
Vegetative storage proteins (VSPs) have been extensively studied in Glycine max, but not in perennial relatives of the cultivated soybean. The occurrence and gene expression of VSPs and a Rubisco Complex Protein (RCP) in several Glycine species was investigated by mRNA blot hybridization and protein immunoblotting. RCP had a developmental pattern of gene expression that closely paralleled that of VSP. The RCP gene was also induced by depodding, methyljasmonate treatment, wounding, and to a lesser extent by nitrogen fertilization, as was previously found for the VSPs. VSP in leaves of 13 perennial soybeans was heterogeneous in apparent size and number of bands detected by immunoblotting following SDS-PAGE. In contrast, RCP was detected as a single band of nearly identical mobility in all species. Both proteins were most abundant in young leaves of the perennials, and methyljasmonate and wounding induced both VSP and RCP gene expression in perennial soybeans. These results suggest that the VSPs in perennial soybeans function as storage reserves, as they do in G. max.
... VSPs are not confined to typical storage organs of perennial plant species. They have been shown to accumulate also in annual herbaceous plants such as soybean and Arabidopsis thaliana, in non-storage organs, namely leaves, pods or hypocotyls (Staswick 1989, Mason and Mullet 1990, Berger et al. 1995. The rapid and preferential turnover of VSPs in these organs suggests a role in temporary storage of nitrogen, for example to buffer a surplus of nitrogen. ...
... A discrepancy between transcripts and VSP protein accumulation has also been reported in soybean lamina, where large accumulation of VSPs occurred only when sink organs were removed, which affects export of N out of the sources. (Wittenbach 1983, Staswick 1989, 1991. ...
In addition to their putative role in nitrogen storage, some vegetative storage proteins (VSPs) support further roles in biotic and abiotic stress responses. Functions of the 17 kDa VSP from witloof chicory (CiVSP) in N storage and plant resistance to pathogens and its regulation by nitrogen were investigated. The N-terminal end of this protein was sequenced and the corresponding full-length cDNA was obtained. The expression of the CiVsp gene was studied in various organs of chicory grown under replete or limited nitrogen supply. A strong expression of CiVsp was observed in both taproot and fine roots of mature plants and seedlings. CiVsp transcripts were also detected in mature leaves, especially in veins. In senescing leaves CiVsp transcripts accumulated concomitantly to a decrease in RbcS transcript abundance and Rubisco small-subunit degradation. CiVSP protein accumulated significantly only in the subterranean part of the plant during late stages of development. Nitrate limitation caused a reduction in CiVsp mRNA accumulation and a delay in CiVSP storage in the taproot. It is concluded that CiVSP accumulation is regulated at the transcriptional level by N external supply and that the protein is involved in long and short-term N storage. In silico analysis indicated that CiVSP is highly homologous with several allergens and PR-10 proteins. Moreover, CiVsp transcript and protein expression were significantly higher in Erwinia carotovora-resistant chicory inbred lines compared with susceptible lines, suggesting its involvement in chicory resistance to pathogens attack.
... The sheer abundance of these proteins in the central vacuoles of specialised cells after sink removal provided support for the hypothesis that Vspa and Vspb are storage proteins and that the bundle sheath and PVM function, in part, as storage tissues. Further reports indicated that the induced accumulation of Vspa and Vspb coincides with an upregulation of their transcription (Staswick 1988(Staswick , 1989 and that Vsp transcription can be induced by treatment with methyl jasmonate Staswick 1994;Berger et al. 1995). This rapid induction has been regarded as potentially a reflection of regulation of Vsp levels in response to changes in signalling related to the dynamics of whole-plant nitrogen demands (Staswick 1989). ...
... Further reports indicated that the induced accumulation of Vspa and Vspb coincides with an upregulation of their transcription (Staswick 1988(Staswick , 1989 and that Vsp transcription can be induced by treatment with methyl jasmonate Staswick 1994;Berger et al. 1995). This rapid induction has been regarded as potentially a reflection of regulation of Vsp levels in response to changes in signalling related to the dynamics of whole-plant nitrogen demands (Staswick 1989). Tranbarger et al. (1991) employed micro-sequencing of the 94 kDa band from SDS PAGE gels of soybean leaf protein extracts and subsequent database searches to tentatively identify Vsp94 as a lipoxygenase. ...
The paraveinal mesophyll (PVM) of soybean is a distinctive uniseriate layer of branched cells situated between the spongy and palisade chlorenchyma of leaves that contains an abundance of putative vegetative storage proteins, Vspα and Vspβ, in its vacuoles. Soybean vegetative lipoxygenases (five isozymes designated as Vlx(A–E)) have been reported to co-localise with Vsp in PVM vacuoles; however, conflicting results regarding the tissue-level and subcellular localisations of specific Vlx isozymes have been reported. We employed immuno-cytochemistry with affinity-purified, isozyme-specific antibodies to reinvestigate the subcellular locations of soybean Vlx isozymes during a sink limitation experiment. VlxB and VlxC were localised to the cytoplasm and nucleoplasm of PVM cells, whereas VlxD was present in the cytoplasm and nucleoplasm of mesophyll chlorenchyma (MC) cells. Label was not associated with storage vacuoles or any evident protein bodies, so our results cast doubt on the hypothesis that Vlx isozymes function as vegetative storage proteins.
... Previous studies have shown that removing pods (depodding) at the pod setting and filling stages promotes GSD occurrence 6-12 . Depodding resulted in an upregulated gene expression and accumulation of vegetative storage proteins (VSPs) in the stem and leaves [13][14][15] . Furthermore, other biotic or abiotic factors that possibly promote GSD, such as high soil moisture content and diseases during the reproductive period 16,17 and pest attack 18 , result in sink limitation at the pod setting and filling stages. ...
Green stem disorder (GSD) of soybean is characterized by delayed leaf and stem maturation despite normal pod maturation. Previous studies have suggested that GSD occurrence is promoted by a high source–sink ratio, which is produced by thinning or shade removal at the R5 growth stage (the beginning of seed filling). Here the effects of different times and durations of shade removal after the R5 stage on GSD severity were analyzed. First, shade removal for more than 28 days after R5 increased GSD severity by more than 0.4 point in GSD score. Thinning treatment at R5 increased specific leaf weight by 23%, suppressed stem dry weight reduction, and upregulated 19 genes including those encoding vegetative storage proteins at R5 + 28d, indicating excess source ability relative to sink size. On the contrary, shade removal for 14 days after R5 decreased GSD severity by 0.5 point in GSD score. In this treatment, seed size was smaller, while seed number was significantly larger than control, suggesting that shortage of source ability relative to sink size. These results implied that soybean plants regulate GSD occurrences either positively or negatively according to a source-sink ratio during the R5 to R5 + 28d growth stages.
... In literature it is known that the accumulation of storage proteins in vegetative tissues (such as potato tubers) is influenced by the plant's source and sink capacities (i.e. sink strength) (Nsimba-Lubaki and Peumans 1986;Staswick 1989;van Cleve and Apel 1993;Wetzel et al. 1989). Sink strength has been suggested to be controlled by a combination of physiological, developmental and partitioning factors (Figure 2) (White et al. 2015). ...
On a global scale, potato is an important crop for both consumption and the
starch industry. At present, the starch industry is keen to create economic
value and to reduce its impact on the environment by valorising all resources
in potatoes. One way to contribute towards this goal is to develop new potato
varieties with elite traits, i.e. a high level of tuber protein content and a reduced
level of fibre hydration. Improving these traits is however challenging due to
their complex nature. Therefore, a better understanding of the genetics and
biological processes underlying tuber protein content and fibre hydration are
relevant. The objectives of this study were to shed light on the genetic and
molecular architectures of tuber protein content and fibre hydration and to
pinpoint key factors (i.e. biological processes, molecular structures, genes and
alleles) that are involved in modulation of these traits.
... Les VSP ont d'abord été étudiées chez le pommier (Tromp, 1970, O'Kennedy et Titus, 1979. Les VSP sensu stricto ont été décrites pour la première fois chez le soja comme étant des protéines de stockage transitoire de l'azote qui s'accumulaient lorsque le transport du phloème est bloqué (Wittenbach, 1983, Staswick, 1989. Ensuite, elles ont été étudiées chez plusieurs espèces d'arbres (fruitiers, décidus et conifères): Populus (e.g. ...
Le chêne sessile et le hêtre sont deux espèces feuillues décidues tempérées, caractérisées par des phénologies foliaires et cambiales contrastées. Afin de progresser dans la compréhension de la gestion des réserves qui sont des composantes importantes des cycles internes du carbone et de l'azote, nous avons étudié la dynamique saisonnière des réserves carbonées (C) et azotées (N) chez des arbres adultes des deux espèces dans leur environnement naturel. Afin de répondre à nos objectifs, nous avons développé une approche pluridisciplinaire associant écophysiologie, biochimie et isotopie. Le suivi mensuel des variations saisonnières des réserves C et N dans le tronc a révélé chez le chêne une forte remobilisation de l'amidon à partir des cernes les plus récents au printemps pour fournir le carbone nécessaire pour la croissance du bois initial du nouveau cerne qui est concomitante à l'expansion foliaire. Chez le hêtre, la croissance printanière ne semble pas dépendante des réserves C du tronc. Chez les deux espèces, deux polypeptides de 13 et de 26 kDa s'accumulent avec la sénescence foliaire en automne, sont très abondants en période hivernale froide et sont remobilisés avec le débourrement au printemps. Cette cinétique saisonnière leur confère un rôle dans le stockage de l'azote (protéines végétatives de réserve, VSP), mais n'exclut pas un rôle dans la tolérance au froid. Chez le chêne sessile, l'étude de la source d'azote pour la croissance des feuilles et des pousses par marquage isotopique au 15N a montré que les réserves N contribuent jusqu'à 90% de l'azote total des nouveaux organes aux premiers stades de développement. La contribution de l'azote nouvellement assimilé ne devient significative que quand le débourrement est achevé. L'étude de la répartition et des quantités des composés C et N non-structuraux à l'échelle de l'arbre a été réalisée par un échantillonnage destructif d'arbres des deux espèces en hiver (Janvier) et à l'étalement complet des feuilles (Juin). Cette étude a montré i) une distribution des concentrations entre organes dépendante de leur fonction physiologique, de l'anatomie du bois et de la distance aux organes puits, ii) l'importance du tronc et des racines dans le stockage des réserves en hiver, iii) l'importance des quantités d'azote non-structural des feuilles et des pousses malgré leur faible biomasse, iv) des quantités de C et N non-structuraux plus importants chez le chêne par rapport au hêtre, pouvant refléter des besoins contrastés pour la croissance et l'entretien des tissus en hiver
... Batchelor et al. (1984) also observed greater N concentration in leaves of soybeans between the R2 and R5 growth stages. There is evidence that vegetative store proteins may be rapidly synthesized or degraded according to other plant part nutrient requeriments (Staswick, 1989). In contrast to stems and leaves, the N concentration in the roots decreased slightly throughout the reproductive growth stages. ...
Under greenhouse condition an experiment was carried out to evaluate trifluralin, chlorimuron and clomazone effects on nitrogen content and chlorophyll concentration in soybeans (Glycine max (L.) Merr. cv Uberlandia), throughout the plant cycle. The samples were collected at 14-day intervals being the first sample collected at 14 days after emergence. Herbicides affected N concentration of plant parts only during the vegetative growth stages of soybeans. Trifluralin and clomazone applications reduced N concentrations in the stems, leaves and roots. Chlorimuron, however, tended to increase leaf and nodule N concentrations. Pod, pod wall and seed N concentrations were not influenced by the herbicides. Leaves, stems and roots had greater N concentrations at the beginning of the vegetative cycle. There were no effects of herbicides on chlorophyll concentrations. It was estimated that the soil gained 4.7 g/m 2 of N during the plant cycle, as result of nitrogen fixation. Additional index terms: Glycine max, N2-fixation, pigment, chlorimuron, clomazone, trifluralin. CONTEÚDO DE NITROGENIO E TEOR DE CLOROFILA FOLIAR EM PLANTAS DE SOJA TRATADAS COM HERBICIDAS RESUMO-Um experimento foi conduzido sob condições de casa-de-vegetaçao para avaliar os efeitos de trifluralina, clorimuron e clomazone sobre o acúmulo de nitrogênio na planta e teor de clorofila nas folhas, em soja (Glycine max (L.) Merr. cv. Uberlândia), ao longo do ciclo da cultura. As amostras foram coletadas a intervalos regulares de 14 dias, a partir da emergência. O teor de N nos órgaos foi máximo nos estádios iniciais da fase vegetativa. Somente nesta fase houve efeito dos herbicidas nos teores de N. Os tratamentos com trifluralina e clomazone reduziram o teor de N em caules, folhas e raízes. Clorimuron, entretanto, tendeu a aumentar o teor de N em folhas e nódulos. Os herbicidas nao influenciaram o teor de N dos legumes, pericarpos e sementes, nem a concentraço de clorofila nas folhas. Estimou-se que o solo teve um ganho de 4.7 g/m 2 de N durante o ciclo da cultura, como resultado da fixaçao de nitrogênio. Termos adicionais para indexaçâo: Glycine max, fixaçao de N, pigmento, clorimuron, clomazone, trifluralina.
... VSPs localize themselves in vacuoles and bundle sheath cells of soybean (Franceschi et al., 1983). Staswick (1989) demonstrated that accumulation of VSPs occurs in pods and developing reproductive parts excluding seeds, so it can be suggested that the VSPs are temporary deposits of amino acids obtained from disassembly of leaf proteins and Rubisco, that become active for seed formation. JA along with sugars, phosphate, nitrogen and auxin regulate these proteins . A. thaliana AtVSPs and soybean VSPs show high levels of expression in developing fruit as well as flowers (Bell et al., 1995). ...
Jasmonates (JAs) [Jasmonic acid (JA) and methyl jasmonates (MeJAs)] are known to take part in various physiological processes. Exogenous application of JAs so far tested on different plants under abiotic stresses particularly salinity, drought, and temperature (low/high) conditions have proved effective in improving plant stress tolerance. However, its extent of effectiveness entirely depends on the type of plant species tested or its concentration. The effects of introgression or silencing of different JA- and Me-JA-related genes have been summarized in this review, which have shown a substantial role in improving crop yield and quality in different plants under stress or non-stress conditions. Regulation of JAs synthesis is impaired in stressed as well as unstressed plant cells/tissues, which is believed to be associated with a variety of metabolic events including signal transduction. Although, mitogen activated protein kinases (MAPKs) are important components of JA signaling and biosynthesis pathways, nitric oxide, ROS, calcium, ABA, ethylene, and salicylic acid are also important mediators of plant growth and development during JA signal transduction and synthesis. The exploration of other signaling molecules can be beneficial to examine the details of underlying molecular mechanisms of JA signal transduction. Much work is to be done in near future to find the proper answers of the questions like action of JA related metabolites, and identification of universal JA receptors etc. Complete signaling pathways involving MAPKs, CDPK, TGA, SIPK, WIPK, and WRKY transcription factors are yet to be investigated to understand the complete mechanism of action of JAs.
... Wittenbach proposed that pod removal might exert an important influence on the leaf senescence progress in soybean [19]. Previous studies also showed that some physiological parameters in soybean were influenced by pod removal [20][21], while the molecular changes of leaves by seed regulation remains rarely reported. In the current study, we tracked leaf development process and measured the physiological parameters and expression of senescence-related and flowering-timing genes under the treatments of pod removal and seed injury to evaluate the effects of pod/seed status on leaf development, to understand the relationship between source and sink in soybean and to know the cause for the outbreak of "Zhengqing". ...
Leaves provide substances and signals for pod and seed development in soybean. However, the regulatory feedbacks of pod and seed on leaf development remain unclear. Here, we investigated the effects of pod/seed on leaf senescence by conducting pod removal and seed injury experiments. Our results showed that pod removal and seed injury delayed leaf senescence and caused the stay green phenotype of leaves. There were dosage effects of pod number on the extent of stay green in the depodded plants. The concentrations of chlorophyll (SPAD value, the index of relative chlorophyll content), soluble protein, and soluble sugar in the leaves of depodded plants were higher than those of intact plants. During seed development, the content of IAA decreased while that of ABA increased. This trend was more pronounced in the intact plants compared with the depodded and seed-injured ones. The ratio of GA3/ABA decreased gradually in all treatments. The content of GA3 was relatively stable, and it was higher in the intact plants than in the depodded plants. The expression levels of the four senescence-related genes, GmSARK, GmSGR1, GmCYN1 and GmNAC, declined in the depodded or seed-injured treatments and were positively correlated with the number of leaves retained in the plants. GmFT2a, the major flowering promoting gene, was expressed at a higher level while E1, a key flowering inhibitory gene, was expressed at a lower level in depodded plants than in intact plants. We postulated that pod or seed can regulate leaf development. When the seed is aborted due to disease infection or pest attack, the leaves stay green because of the absence of the seed signals for senescence.
... Photosynthesis during the seed filling period (SFP) provides both the carbon and nitrogen requirement for seed growth. Sucrose forms the major carbon component while nitrogen assimilated via NO 3 , contributes to 50% or more of the nitrogen in the seeds (Layzell and LaRue, 1982;Rainbird et al., 1984;Shibles et al., 1987;Harper, 1987;Staswick, 1989;Wardlaw, 1990). A positive correlation exists between sucrose concentrations in the source and seed growth rate (Thompson et al., 1977;Egli et al., 1989). ...
Soybean (Glycine max (L.) Merrill) is the most widely cultivated oilseed crop accounting for more than 50% of the world’s oilseed production. Yield gain in soybean estimated to be 0.5-0.7% per year in North America has been driven by the adoption of agronomic or management practices and genetic improvement. While genetic improvement through breeding will continue to play a significant role in enhancing yield by the development of cultivars adapted to a wide range of latitudes, biotech traits such as enhanced insect protection and weed control contribute indirectly to yield improvement. An understanding of physiological traits associated with genetic gain in yield offers vast opportunities for further advances in yield improvement. Potential targets for genetic improvement include source capacity (leaf area index, leaf area duration, carbon and nitrogen assimilation, and dry matter partitioning), sink strength (number of primary and secondary yield components, seed-filling rate and duration), and tolerance to suboptimal conditions (water limitation and high/low temperature). Manipulating single or multiple traits using breeding and biotechnology approaches will help to improve intrinsic yield potential and yield stability traits in soybean. Application of multiple technologies to improve yield gain is vital, with the changing climatic conditions and increasing global demand for food and feed.
... Previous studies have shown that these two abundant glycoproteins accumulate in leaves, stems, pods, flower petals, and germinated cotyledons (Staswick 1989a, b). It is estimated that these proteins can account for about 6-15% of soluble leaf protein (Wittenbach 1983a;Staswick 1989a). Because of their abundance they have been designated as vegetative storage proteins. ...
Xylem sap plays a major role in long-distance transport of water, nutrients, and metabolites. However, there is little information on the behavior of metabolites in mineral-deficient xylem sap. For this reason, the time-dependent changes in selected metabolites (amino acids, organic acids, and soluble sugars) from tomato xylem sap in response to nitrogen (N), phosphorus (P), or potassium (K)-deficient condition were investigated. Tomato plants (Solanum lycopersicum L.) were grown hydroponically in liquid culture under three different mineral regimes: N-deficient [0.5 mM Ca(NO3)2 and 0.5 mM KNO3], P-deficient (0.05 mM KH2PO4), and K-deficient (0.5 mM KNO3), respectively. Xylem sap was collected at 10:00 am after 1, 5, 15, and 30 d, and the selected metabolites were analyzed with liquid chromatography. All N, P, or K deficiencies led to a substantial increase in metabolites in the xylem sap. The predominant amino acid in the xylem sap was glutamine and, interestingly, all mineral deficiencies resulted in a substantial amount of γ-aminobutyric acid (GABA). Additionally, organic acids (citrate and malate) and soluble sugars were strongly increased in all mineral deficiencies, and, in particular, the level of shikimate was greatly affected by N deficiency. Based on these data, it is necessary to clearly elucidate an unknown event taking place in xylem loading in a variety of environmental impacts, and we are now studying to expand our knowledge on metabolic and proteomic responses using GC-MS and LC-MS.
... The soybean (Glycine max L. Merr. cv Williams 82) vegetative storage proteins VSP␣ and VSP are vacuolar acid phosphatases that accumulate in hypocotyl hooks and plumules during seedling development, and in leaves, stems, and pods during the reproductive phase (Wittenbach, 1982;Staswick, 1989a;DeWald et al., 1992). Sink deprivation by depodding or petiole girdling also causes a massive accumulation of the Vsp mRNAs and proteins (Wittenbach, 1982;Staswick, 1989b). ...
The soybean (Glycine max L. Merr. cv Williams 82) genes VspA and VspB encode vacuolar glycoprotein acid phosphatases that serve as vegetative storage proteins during seed fill and early stages of seedling growth.VspB expression is activated by jasmonates (JAs) and sugars and down-regulated by phosphate and auxin. Previous promoter studies demonstrated that VspB promoter sequences between −585 and −535 mediated responses to JA, and sequences between −535 and −401 mediated responses to sugars, phosphate, and auxin. In this study, the response domains were further delineated using transient expression of VspBpromoter-β-glucuronidase constructs in tobacco protoplasts. Sequences between −536 and −484 were identified as important for phosphate responses, whereas the region from −486 to −427 mediated sugar responses. Gel-shift and deoxyribonuclease-I footprinting assays revealed four DNA-binding sites between −611 and −451 of the soybeanVspB promoter: one in the JA response domain, two in the phosphate response domain, and one binding site in the sugar response domain. The sequence CATTAATTAG present in the phosphate response domain binds soybean homeodomain leucine zipper proteins, suggesting a role for these transcription factors in phosphate-modulated gene expression.
... In addition , soybean leaves accumulate a vegetative storage protein (VSP), a member of the vacuolar acid phosphatase family (DeWald et al., 1992; Staswick et al., 1994 ). VSP accumulation is highly responsive to nitrogen availability ( Staswick, 1989a,b), and it increases with depodding, i.e., removal of the seed sink. This observation led to proposals that VSP is a necessary adjunct that provides additional nitrogen resources for the seed. ...
The soybean seed’s protein content and composition are regulated by both genetics and physiology. Overt seed protein content is specified by the genotype’s genetic framework and is selectable as a breeding trait. Within the genotype-specified protein content phenotype soybeans have the capacity to rebalance protein composition to create differing proteomes. Soybeans possess a relatively standardized proteome, but mutation or targeted engineering can induce large-scale proteome rebalancing. Proteome rebalancing shows that the output traits of seed content and composition result from two major types of regulation: genotype and post-transcriptional control of the proteome composition. Understanding the underlying mechanisms that specifies the seed proteome can enable engineering new phenotypes for the production of a high-quality plant protein source for food, feed, and industrial proteins.
... de saccharose. II est par ailleurs possible que l'effet du saccharose soit expliq~e par une atteinte a I' integrite membranatre et partant aux capacites d'importation des precurseurs azotes des proteines de reserve, dont I' expression pourrait etre so us le con-tr61e metabolique d'acides amines (comme suggere dans le cas des proteines VSP du soja,Staswick, 1988). Chez Ia pomme de terre, le rapport saccharose/acides amines semble determinant pour !'expression des proteines de reserve(Pefia-Cortes et a!., 1992). ...
In potato, starch is synthesized with two different tempos in the leaf and in the tuber. In both cases, the enzyme ADPglucose pyrophosphorylase achieves the production of the activated glucosydic precursors of starch and represents the major regulatory step of that biosynthetic pathway. Its allosteric control by intermediate metabolites of photosynthesis explains a large part of the diurnal rythmicity of starch synthesis in the leaf. In the developing tuber however, active starch synthesis is essentially supported by a high expression level of the ADPglucose pyrophosphorylase. Starch accumulation being apparently coordinated with that of storage proteins in the developing tuber, the nature of the relevant regulatory mechanisms constitutes a fundamental aspect of the physiology of tuberization. A significant achievement in the identification of these mechanisms came from the cloning and structural analysis of the ADPglucose pyrophosphorylase B-subunit promoter and its comparison with promoters of genes encoding storage proteins of the patatin group.
... VSP accumulation by water deficit was also observed in white clover during low temperature stress in nodules [19]. These results suggest that VSP might be involved in the transient storage of N that occurred during N recycling processes provoked under conditions of numerous abiotic and biotic constraints [9,26], especially dehydration stress. Indeed, this VSP accumulation may allow plants not only to give stress tolerance but also to subsequently grow effectively by sequestering the endogenous N protein form until more favorable conditions for growth arrive. ...
... Protein complement of root wood tissues changed in a similar manner (data not shown). While abundance of with molecular masses of 25 and 27 kDa are hydrolyzed to provide N to developing seeds (Staswick, 1989). Others (Wetzel et al., 1989) have reported that proteins accumulated and formed protein bodies in parenchyma cells of bark phloem tissues of Acer, Salix, and Populus spp. ...
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.
... growth ( Fig. 3 B). Specific proteins accumulated in vegetative tissues of soybean ( Staswick, 1989) and potato (Solanum tuberosum L.) ( Park, 1984) that are used as sources of N. Vegetative storage proteins from soybean have molecular masses of 25 and 27 kDa, while the storage proteins of potato have molecular masses of ca. 40 kDa. ...
Alfalfa (Medicago sativa L.) accumulates organic reserves in taproots that are thought to be used as substrates for newly developing shoots after defoliation. Two experiments were conducted to determine if specific N pools in taproot tissues undergo depletion and reaccumulation following defoliation. In Exp. 1, bark tissues of taproots of ‹Hi-Phy› alfalfa had higher concentrations of total N, soluble NH2-N and buffer-soluble protein than did wood tissues. Concentrations of these N pools declined in both tissues after defoliation and then reaccumulated after 21 d of regrowth. In Exp. 2, two genotypes differed in concentration of N-containing pools, although trends following defoliation of both genotypes were similar to those observed in Exp. 1. ASP + ASN were the most prevalent of the amino acids found in bark and wood tissues, together comprising approximately 50% of the total amino acid pool. Concentration of the ASP + ASN pool declined markedly in roots following defoliation, while concentrations of other amino acids (LEU, ILE, TYR, and PHE) increased. Characterization of buffer-soluble proteins using SDS-PAGE indicated that specific proteins with molecular masses of 15 and 19 kDa were depleted, especially in bark tissues, as soluble protein concentrations declined. The depletion of specific amino acids and certain buffer-soluble proteins from taproots during regrowth of defoliated alfalfa suggests that these N-pools may be utilized as a source of N during foliar regrowth after defoliation.
... Plants may store nitrogen as protein for protracted periods. Vegetative storage proteins can be assembled rapidly and broken down rapidly for transport to N-demanding sinks via phloem (Staswick, 1989). Amino acid nitrogen may be stored for months or years in stems, roots or foliage as vegetative storage proteins or in active proteins such as RuBisCO (Millard and Grelet, 2010). ...
Plants are organisms composed of modules connected by xylem and phloem transport streams. Attack by both insects and pathogens elicits sometimes rapid defense responses in the attacked module. We have also known for some time that proteins are often reallocated away from pathogen-infected tissues, while the same infection sites may draw carbohydrates to them. This has been interpreted as a tug of war in which the plant withdraws critical resources to block microbial growth while the microbes attempt to acquire more resources. Sink-source regulated transport among modules of critical resources, particularly carbon and nitrogen, is also altered in response to attack. Insects and jasmonate can increase local sink strength, drawing carbohydrates that support defense production. Shortly after attack, carbohydrates may also be drawn to the root. The rate and direction of movement of photosynthate or signals in phloem in response to attack is subject to constraints that include branching, degree of connection among tissues, distance between sources and sinks, proximity, strength, and number of competing sinks, and phloem loading/unloading regulators. Movement of materials (e.g., amino acids, signals) to or from attack sites in xylem is less well understood but is partly driven by transpiration. The root is an influential sink and may regulate sink-source interactions and transport above and below ground as well as between the plant and the rhizosphere and nearby, connected plants. Research on resource translocation in response to pathogens or herbivores has focused on biochemical mechanisms; whole-plant research is needed to determine which, if any, of these plant behaviors actually influence plant fitness.
... In plant families, vegetative storage proteins accumulate in leaves prior to anthesis , decline during pod filling and then accumulate again after seed maturation (Staswick, 1989). In woody species such as poplar trees, a similar set of proteins termed bark storage proteins accumulate in the autumn months in the protein storage vacuoles of the inner bark parenchyma and xylem ray cells (Coleman et al., 1994 ). ...
Green stem disorder (GSD) is an important agronomical problem in soybean production because it delays leaf and stem senescence and complicates the harvest. However, a rapid and precise diagnosis of GSD has not yet been established. In this study, the effect of depodding on GSD and vegetative storage protein (VSP) accumulation was investigated in GSD-susceptible cultivar ‘Tachinagaha (Tc)’ and GSD-resistant experimental line ‘Touhoku 129 (Th)’ under two different (early and late) sowing dates in 2020 and 2021. Intact Tc plants showed relatively severe GSD at early sowing in 2020 and late sowing in 2021, whereas intact Th plants showed little GSD at both sowing dates and in both years. Meanwhile, depodding reproducibly induced GSD and increased stem weight for both Tc and Th. The relative VSP content peaked 14–21 days after R3 (DAR3) in intact plants and increased afterward in depodded plants. The relative VSP content differed at 28 DAR3 between intact and depodded plants, which was earlier than the timing when SPAD (soil plant analysis development) values differed, suggesting that VSP accumulation might be a better indicator of GSD than the SPAD value. The present study will contribute to the development of tools for diagnosing GSD.
Green stem disorder (GSD), characterized by delayed stem senescence during seed maturation, complicates harvesting in soybean production. Although GSD is associated with a sink – source imbalance, a rapid and precise evaluation of GSD has not been established. In sink-limited soybean plants, vegetative storage protein (VSP) accumulates. In this study, pot and field experiments were conducted to reevaluate the relationship between GSD, sink – source imbalance caused by soil moisture change, and VSP accumulation as a possible indicator of GSD in Kyoto, Japan over two years. Drought treatment for four weeks from R1 (beginning flowering), R3 (beginning pod), or R5 (beginning seed) growth stage in pots using the short growth-period cultivar Yukihomare reduced sink size in both years, but reduced relative sink mass (pod weight/shoot weight) and increased GSD severity only in 2017, suggesting that sink-source imbalance, affected by soil moisture, can induce GSD. Soil moisture change from around R3 or R5 to maturity in fields using trench-filled or unfilled water tended to change GSD severity but not VSP accumulation in the uppermost fully expanded leaves from R5 (2018) or 15 days before R5 (2019) to 28 days after R5. GSD and VSP responses, however, differed between the two contrasting cultivars, Tachinagaha and Touhoku 129, suggesting the potential usability of VSP for GSD evaluation.
Jasmonic acid (JA) and its methyl ester, methyl jasmonates (MeJAs), is categorized under phytohormones. It is ubiquitously found all over the plant kingdom but varies in concentration from species to species. Chemically, it is known as derivatives of the fatty acid metabolism. JAs are synthesized from α-linolenic acid (α-LeA/18:3) via the octadecanoid pathway. JAs attached to its receptor, CORONATINE INSENSITIVE1 (COI1) triggers the signaling cascade and enables the expression of genes and generate various responses under stress and stress-free conditions. Moreover, JAs are known to regulate a wide range of physiological processes in plants such as plant growth, reproductive development and senescence. It also induces plant defense responses against various biotic stresses such as herbivore attack or pathogen infection. In this chapter, a summary of recent advances in our understanding of JA synthesis and signaling along with its role in regulating physiology of plant in presence or absence of biotic stress.
Soybean (Glycine max L. Merr.) contains two related and abundant proteins, VSPα and VSPβ, that have been called vegetative storage proteins (VSP) based on their pattern of accumulation, degradation, tissue localization, and other characteristics. To determine whether these proteins play a critical role in sequestering N and other nutrients during early plant development, a VspA antisense gene construct was used to create transgenic plants in which VSP expression was suppressed in leaves, flowers, and seed pods. Total VSP was reduced at least 50-fold due to a 100-fold reduction in VSPα and a 10-fold reduction in VSPβ. Transgenic lines were grown in replicated yield trials in the field in Nebraska during the summer of 1999 and seed harvested from the lines was analyzed for yield, protein, oil, and amino acid composition. No significant difference (α = 0.05) was found between down-regulated lines and controls for any of the traits tested. Young leaves of antisense plants grown in the greenhouse contained around 3% less soluble leaf protein than controls at the time of flowering. However, total leaf N did not vary. Withdrawing N from plants during seed fill did not alter final seed protein content of antisense lines compared with controls. These results indicate that the VSPs play little if any direct role in overall plant productivity under typical growth conditions. The lack of VSPs in antisense plants might be partially compensated for by increases in other proteins and/or non-protein N. The results also suggest that the VSPs could be genetically engineered or replaced without deleterious effects.
La glutamine synthétase (gs) des racines du douglas a été purifiée à homogénéité electrophoretique. Ses principales propriétés physico-chimiques ont été analysées. Ainsi, cette enzyme présente une masse moléculaire d'environ 460 kda et est constituée de deux sous-unités de 64 et 54 kda. De plus, elle possède une très forte affinité pour l'ammonium. L'étude de la gs aux stades précoces de développement du douglas a montré une forte augmentation de son activité et l'existence d'une seule isoforme de masse moléculaire identique à celle de la gs racinaire. A ces stades, la gs est affectée in vivo, mais pas in vitro, par des concentrations croissantes en ammonium ou nitrate. Cependant, à des stades de croissance plus avancés, l'absence d'azote et la source d'azote présente affectent non seulement la croissance du végétal mais aussi la répartition de l'activité gs au sein de la plante. La meilleure croissance est obtenue en présence d'ammonium qui a surtout une influence sur l'activité gs racinaire. Lors de la mycorhization du douglas par laccaria bicolor, les gs fongiques et racinaires restent fonctionnelles. En tenant compte de la proportion de la biomasse fongique (=30%) au sein des ectomycorhizes de douglas il apparait que l'activité gs fongique représente 1/3 et l'activité racinaire 2/3 de l'activité enzymatique totale.
Leaves from 12 legume species representing two subtribes were examined by various techniques for the presence of vegetative storage proteins (VSPs) similar to the 27, 29, and 94 kD VSPs of soybean. Polyacrylamide gel electrophoresis (PAGE) of leaf protein followed by western immunoblotting using antibody that recognizes soybean VSP94, a lipoxygenase, demonstrated that this protein is present in six of the nine species tested. Blotting with antibody to soybean VSP27/29, which are glycoproteins, gave labelling in seven species and glycoprotein affino-blots showed that glycosylated proteins ranging around 27 to 29 kD were present in all nine species examined. Immunocytochemical localization studies of eight species demonstrated that proteins antigenically similar to VSP94 and VSP27/29 are specifically accumulated in the vacuole of paraveinal mesophyll (PVM) cells. They were not detectable at significant levels in other mesophyll cells using this technique. Comparisons of protein compositions of isolated PVM and mesophyll protoplasts from seven species further confirmed the specialized nature of the PVM. VSP94 and proteins ranging from 25 to 35 kD molecular mass were the major proteins of PVM of all but one species while Rubisco was quite low in amount compared to mesophyll protoplasts. The results show that VSP synthesis and accumulation is a general feature of legume leaves containing a PVM layer and indicate that the PVM plays a specialized role in nitrogen metabolism and partitioning in these species.
The vacuoles of plant cells contain a variety of proteins including acid hydrolases, storage proteins and plant defense proteins. During seed development, the central vacuoles of the storage parenchyma cells accumulate large amounts of all three classes of these proteins. We have studied the biosynthesis, transport, posttranslational modifications and accumulation in developing legume cotyledons of acid hydrolases (e.g. α-mannosidase), storage proteins (e.g. phaseolin), and plant defense proteins (e.g. phytohemagglutinin and α-amylase inhibitor). Transport of proteins to vacuoles is mediated by the secretory system (endoplasmic reticulum and Golgi apparatus) and correct targeting of protein to vacuoles requires positive sorting information. This information is contained within the polypeptide domain of the vacuolar glycoprotein phytohemagglutinin (PHA). When the gene for PHA is introduced into yeast ( Saccharomyces cerevisiae) cells, the resulting protein is targeted to yeast vacuoles. By expressing in yeast, chimeric genes consisting of the signal peptide and various portions of the PHA coding region with the gene for yeast invertase, we were able to show that the vacuolar targeting domain of PHA is in an amino- proximal region between amino acids 14 and 43 of the mature protein. Experiments are now under way to determine whether the same domain of PHA can target yeast invertase to plant vacuoles (protein bodies in tobacco seeds).
The soybean vegetative storage protein genes (vspA, and vspB) are regulated in a complex manner developmentally and in response to external stimuli such as wounding and water deficit. The proteins accumulate to almost one-half the amount of soluble leaf protein when soybean plants are continually depodded and have been identified as storage proteins because of their abundance and pattern of expression in plant tissues. We have shown that purified VSP homodimers (VSP-alpha and VSP-beta) and heterodimers (VSP-alpha/beta) possess acid phosphatase activity (alpha = 0.3-0.4 units/mg; beta = 2-4 units/mg; alpha/beta = 7-10 units/mg). Specific activities were determined by monitoring o-carboxyphenyl phosphate (0.7 mM) cleavage at pH 5.5 (VSP-alpha) or pH 5.0 (VSPa/beta and VSP-beta) in 0.15 M sodium acetate buffer at 25-degrees-C. These enzymes are active over a broad pH range, maintaining greater than 40% of maximal activity from pH 4.0 to 6.5 and having maximal activity at pH 5.0-5.5. They are inactivated by sodium fluoride, sodium molybdate, and heating at 70-degrees-C for 10 min. These phosphatases can liberate P(i) from several different substrates, including napthyl acid phosphate, carboxyphenyl phosphate, sugar-phosphates, glyceraldehyde 3-phosphate, dihydroxyacetone phosphate, phosphoenolpyruvate, ATP, ADP, PP(i), and short chain polyphosphates. VSP-alpha/beta cleaved phosphoenolpyruvate, ATP, ADP, PP(i), and polyphosphates most efficiently. Apparent K(m) and V(max) values at 25-degrees-C and pH 5.0 were 42-mu-M and 2.0-mu-mol/min/mg, 150-mu-M and 4.2-mu-mol/min/mg, and 420-mu-M and 4.1-mu-mol/min/mg, for tetrapolyphosphate, pyrophosphate, and phosphoenolpyruvate, respectively.
From its origins in the Andes, the cultivated potato has spread to most parts of the world in the last 400 years. It now is a major source of nutrition for people from a wide variety of cultural and ethnic backgrounds. In the developing countries of Asia and Africa, the rate of increase in potato production has been higher than that of most other crops, and presently potato ranks fourth in terms of total global food production (Bajaj and Sopory, 1986). When grown under optimal conditions, the yield of tubers can be as high as 100 tonnes/ha, and as such is second only to soybean in production of protein on a per acre basis (Johnson and Lay, 1974). Nutritionally, the tuber is a good source of protein, minerals and vitamin C but is an inefficient supplier of energy compared with grain crops such as rice. Commercial cultivars of potato are highly heterozygous tetraploids that are self-incompatible. These factors contribute to a very slow breeding cycle, and today the European and US markets are dominated by a few varieties that have existed for many years, each of which has substantial weaknesses. However, the ability to transfer single defined genes into potato using Agrobacterium vectors promises to have a disproportionately large effect on the improvement of potatoes.
To reveal the action of plant storage proteins in stress tolerance, 6 major storage proteins and 2 vegetative storage proteins were identified from the leaves of Bruguiera gymnorrhiza by 2-D and MS. The molecular weight of 181, 182, 188, 194, 198 and 204 is about 33000 to 34000Da, and their isoelectric point is about 5.5 to 6.45. While at the same time, the molecular weight of 266 and 274 is about 23500Da, and their isoelectric point is about 5.1 and 5.3, respectively. Further analysis showed that, among these storage proteins, 182, 198 and 204 were positively correlated with the mean annual temperature, while 274 was mainly correlated with the soil pH. So far, we suggest that as the temporary nitrogen sources, the contents of storage proteins in B. gymnorrhiza leaves are mainly affected by the mean annual temperature and soil pH.
Immunomodulatory activity of extracted lectins from Illicium Verum was evaluated on phagocytic activity by carbon clearance test. Adult Albinos Wistar mice randomly divided into four groups, were the first was served as a control, while the remaining groups respectively treated with extracted lectins from Illicium Verum at dose of: 10, 30 and 50 mg/kg by intra-peritoneal injection (IP). Change in phagocytic activity was determined after 48 h injection of carbon ink suspension. In carbone clearance test, extracted lectins from Illicium Verum exhibited significantly phagocytic index dose-dependent against control group, indicating stimulation of the reticulo-endothelial system. Present study thus reveals that extracted lectins from Illicium Verum holds promise as immunomodulatory agent, which act by stimulating dose dependent phagocytic function.
The most abundant protein of resting rhizomes of Calystegia sepium (L.) R.Br. (hedge bindweed) has been isolated and its corresponding cDNA cloned. The native protein consists of a single polypeptide of 212 amino acid residues and occurs as a mixture of glycosylated and unglycosylated isoforms. Both forms are derived from the same preproprotein containing a signal peptide and a C-terminal propeptide. Analysis of the deduced amino acid sequence indicated that the C. sepium protein shows high sequence identity and structural similarity with plant RNases. However, no RNase activity could be detected in highly purified preparations of the protein. This apparent lack of activity results most probably from the replacement of a conserved His residue, which is essential for the catalytic activity of plant RNases. Our findings not only demonstrate the occurrence of a catalytically inactive variant of an S-like RNase, but also provide further evidence that genes encoding storage proteins may have evolved from genes encoding enzymes or other biologically active proteins.
The ST (ShooT Specific) proteins are a new family of proteins characterized by a signal peptide, tandem repeats of 25/26 amino acids, and a domain of unknown function (DUF2775), whose presence is limited to a few families of dicotyledonous plants, mainly Fabaceae and Asteraceae. Their function remains unknown, although involvement in plant growth, fruit morphogenesis or in biotic and abiotic interactions have been suggested. This work is focused on ST1, a Cicer arietinum ST protein. We established the protein accumulation in different tissues and organs of chickpea seedlings and plants and its subcellular localization, which could indicate the possible function of ST1. The raising of specific antibodies against ST1 protein revealed that its accumulation in epicotyls and radicles was related to their elongation rate. Its pattern of tissue location in cotyledons during seed formation and early seed germination, as well as its localization in the perivascular fibres of epicotyls and radicles, indicated a possible involvement in seed germination and seedling growth. ST1 protein appears both inside the cell and in the cell wall. This double subcellular localization was found in every organ in which the ST1 protein was detected: seeds, cotyledons and seedling epicotyls and radicles.
Soybean vegetative storage proteins (VSPs) were purified and characterized. Anion exchange HPLC resolved partially purified VSPs into fractions containing 27-kD/27-kD and 29-kD/29-kD homodimers and 27-kD/29-kD heterodimers. Reversed-phase HPLC resolved partially purified VSPs into three fractions. One fraction contained only 27-kD VSP and the other two contained 29-kD VSP. The two 29-kD VSP fractions differed with respect to their cyanogen bromide cleavage patterns, an observation that indicated the 29-kD VSPs were heterogeneous. Genomic clones that contained 29-kD VSP genes were also isolated and characterized. One genomic clone contained a complete 29-kD VSP gene and was sequenced. The coding region in the clone contained two introns whose borders had regulatory sequences typical of other eukaryotic genes. Putative polyadenlyation signals were present in the 3'-flanking region of the gene, while putative TATA, CAAT, and enhancer core sequences were found in the 5'-flanking regions. A second genomic clone that was studied contained the 5' regions of two partial 29-kD VSP genes in an inverted linkage. Genomic DNA gel blots showed that the two genes were organized in the same arrangement in the soybean genome.
The inner bark tissues of three temperate hardwoods contain specific proteins which undergo seasonal fluctuations. Increases in particular proteins, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, occur within the bark of several Acer, Populus and Salix spp. during late summer and early autumn. These proteins are abundant in the bark throughout the winter and their levels decline the following spring. Light and electron microscopy showed that the parenchyma cells of the inner bark are packed with spherical organelles throughout the overwintering period. These organelles are rich in protein and analogous to protein bodies found in cells of mature seeds. The protein bodies of the parenchyma cells are replaced by large central vacuoles during spring and summer, presumably as a result of the mobilization of the storage protein and fusion of the protein bodies. The high levels of specific proteins in inner bark tissues and the presence of protein bodies within the parenchyma cells indicate that the living cells of the bark act as a nitrogen reserve in overwintering temperate hardwoods.
Roots are the overwintering structures of herbaceous perennial weeds growing in temperate climates. During the fall they accumulated reserves which are remobilized when growth resumes in the spring. An 18kDa (kilodalton) protein increases in both chicory and dandelion roots during the fall months. The proteins in both species are antigenically similar, and are recognized also by an antibody to a storage-protein deposited in Jerusalem artichoke (Helianthus tuberosus) tubers. In chicory, the protein is root-specific, but in dandelion it is detectable in the flowers, vestigial stem and the seed. Electrophoretic characterization of the 18-kDa protein shows that it is a single polypeptide, without subunits, with charge isomers of pI values close to pH 6.5. The major protein present in chicory and dandelion roots is unlike the vegetative storage proteins recently found in soybean or the storage proteins in the bark of trees.
The paraveinal mesophyll (PVM) layer of soybean leaves, which contains cells with various unique ultrastructural properties, has been studied for decades, and several hypotheses regarding its functional role have been developed. Here, we describe a method for obtaining PVM cells using laser capture microdissection and pressure catapulting, subsequent isolation of RNA from these cells, and downstream microarray analysis. A cell type-specific transcriptome analysis was used to compare the gene expression patterns in PVM cells with those of a mesophyll cell type (palisade parenchyma) as a reference. Transcripts related to vegetative storage protein (Vsp) and certain vegetative lipoxygenase (Vlx) isoforms were significantly enriched in PVM cells, which is in accordance with prior work that demonstrated an accumulation of the corresponding proteins. Potential roles of Vsp and Vlx in phosphate mobilization and defense responses, respectively, are discussed. In addition, we found an enrichment of several transport-related genes in PVM cells. Building on our transcriptome data, we provide a fresh discussion of the hypothesis that the PVM plays a role in photoassimilate mobilization and translocation.
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.
Leaves from 12 legume species representing two subtribes were examined by various techniques for the presence of vegetative storage proteins (VSPs) similar to the 27, 29, and 94 kD VSPs of soybean. Polyacrylamide gel electrophoresis (PAGE) of leaf protein followed by western immunoblotting using antibody that recognizes soybean VSP94, a lipoxygenase, demonstrated that this protein is present in six of the nine species tested. Blotting with antibody to soybean VSP27/29, which are glycoproteins, gave labelling in seven species and glycoprotein affino-blots showed that glycosylated proteins ranging around 27 to 29 kD were present in all nine species examined. Immunocytochemical localization studies of eight species demonstrated that proteins antigenically similar to VSP94 and VSP27/29 are specifically accumulated in the vacuole of paraveinal mesophyll (PVM) cells. They were not detectable at significant levels in other mesophyll cells using this technique. Comparisons of protein compositions of isolated PVM and mesophyll protoplasts from seven species further confirmed the specialized nature of the PVM. VSP94 and proteins ranging from 25 to 35 kD molecular mass were the major proteins of PVM of all but one species while Rubisco was quite low in amount compared to mesophyll protoplasts. The results show that VSP synthesis and accumulation is a general feature of legume leaves containing a PVM layer and indicate that the PVM plays a specialized role in nitrogen metabolism and partitioning in these species.
Antibodies raised against tonoplast intrinsic proteins (TIPs) were used to probe the functional status of the soybean [Glycine max (L.) Merr.] paraveinal mesophyll (PVM) vacuole during changes in nitrogen metabolism within the leaf. Young plants grown under standard conditions had PVM vacuoles characterised by the presence of γ-TIP, which is indicative of a lytic function. When plants were then subjected to shoot tip removal for a period of 15 d, forcing a sink-limited physiological condition, the γ-TIP marker diminished while the δ-TIP marker became present in the PVM vacuole, indicating the conversion of the PVM vacuole to a storage function. When the shoot tips were allowed to regrow, the γ-TIP marker again became dominant demonstrating the reversion of these PVM vacuoles back to a lytic compartment. The changes in TIP markers correlated with the accumulation of vegetative storage proteins and vegetative lipoxygenases, proteins implicated in nitrogen storage and assimilate partitioning. This research suggests that the PVM vacuole is able to undergo dynamic conversion between lytic and storage functions and further implicates this cell layer in assimilate storage and mobilisation in soybeans.
In young poplar (Populus nigra Muench × Populus maximowiczii A. Henry) plants, vegetative storage proteins (VSPs), the bark storage protein (BSP), and (or) wound-inducible 4 protein (WIN4) mRNAs were present in the apical and basal leaves and in the basal leaves, respectively. VSPs accumulated in the apical leaves and to a lesser extent in the stem. The response of the plants to 20 µM airborne methyl jasmonate (MJ) was examined in four parts ( apical and basal leaves, stem, and roots) in both short-term (within 72 h) and long-term (1, 2, 3, and 4 weeks) experiments. In the short-term, MJ-treated plants either induced or increased the part-specific expression of win4 and bsp, and accumulation of BSP and (or) WIN4. In the long-term, MJ treatment resulted in part-specific alterations in protein and nitrogen concentration and further altered BSP and WIN4 accumulation. The MJ-treated plants increased both the biomass allocation to the stem, without a change in the relative growth rate, and the tolerance low temperature (-2°C). Together, these results suggest the BSP and WIN4 are both involved in short-term N cycling and that exogenous MJ treatment promotes changes in nitrogen metabolism in poplar.
Characteristics of the enzymes involved in the assimilation of NO3− and NH4+, in particular the nitrate and nitrite reductases, glutamine synthetase, glutamate synthase, glutamate dehydrogenase, glutamate decarboxylase, and asparagine synthetase, are described. The cellular organization of these enzymes in root and leaf tissues are assessed in view of recent research developments that utilize various tissue blotting techniques. Regulation of nitrate assimilation is analyzed at the physiological, biochemical, and molecular levels. Key words: nitrate, ammonium, assimilation, regulation.
Nitrogen (N) nutrition enhances metabolic processes that influences the physicochemical environment at the soil-root interface, modifies rhizosphere conditions, interferes with the uptake of cations and anions, and enhances or represses the activity of several enzyme systems. Also, it affects growth patterns, protein content, and protein quality of seeds.Ammonium (NH4)-N nutrition increases anion uptake, free amino-N/protein ratios, and acidity of root free space; it reduces carbohydrate levels in plant tissues. NO3-N nutrition results in higher cation uptake, higher carbohydrate content in tissues, and alkalinization of root free space. N-Assimilation interferes with the allocation of dry matter and energy, which causes different growth rates of plant parts.In this article we review the effects of mineral-N nutrition on uptake of cations and anions, activity of enzymes, growth patterns of roots and shoots, and water use efficiency, protein content, and protein quality of seeds.
Accumulation and distribution of dry matter and nitrogen were examined by the sink cut treatment. The results obtained were as follows.1. In Gramineae except for maize, the amount of nitrogen in leaves decreased and nitrogen was translocated to stems regardless of the sink cut treatment, whereas the amount increased by the sink cut treatment in Leguminosae. Thus, it is assumed that leaf growth was autonomous in Gramineae, but was affected by the sink activity in Leguminosae.2. By the sink cut treatment, no specific protein bands were detected in leaves of both Gramineae and Leguminosae, as well as in stems of Gramineae. The vegetative storage proteins (VSP) were detected in stems of soybean and field bean, and its amount increased by the sink cut treatment. However, as the amount of soluble protein fraction in the stems of soybean and field bean was not affected by the sink cut treatment, the effect of VSP on nitrogen storage was negligible even when the sink was cut.
Chickpea (Cicer arietinum L., var. Pusa 256) plants raised under unirrigated and irrigated field conditions showed a decrease in leaf nitrogen and soluble protein content after flowering during pod development. This was found to be associated with a decrease in Rubisco content after flowering. Leaf nitrogen, soluble protein and Rubisco content, however, were higher in irrigated than in unirrigated plants. The Rubisco content at the flowering and post-flowering stages was 29.43 and 16.59 %, respectively, of leaf soluble protein in unirrigated plants. Under irrigated conditions, the Rubisco content was 49.91 and 37.99 %, respectively, at the flowering and post-flowering stages. These results therefore indicated a decrease in the mobilization of leaf nitrogen by irrigation in chickpea. The findings are discussed in relation to the decrease in seed yield and harvest index by irrigation commonly observed in this crop under north Indian conditions.
The seasonal pattern of concentrations of nitrogen, starch and vegetative storage protein (VSP) in stolons of Trifolium repens L. grown in the field were studied. Two different genotypes, cv. Aran and cv. Rivendel, differing in their morphology (stolon thickness and branching rate) but with similar growth rates, were used. Maximum concentrations of starch were found in summer whereas hydrolysis of starch took place throughout winter, suggesting that C storage is more important for winter survival than for promotion of early spring growth. On the other hand, VSP and nitrogen accumulated in autumn and early winter and then decreased when growth was resumed during early spring. For both cultivars, an inverse relationship was found between VSP concentration in stolons and mean air temperature, suggesting that VSP accumulation may be triggered by low temperature. Further experiments with plants grown under different regimes of temperature and daylength, suggested that VSP synthesis is stimulated by low root temperatures, with a slight synergistic effect of short daylength.
The effects of root temperature on growth, N 2 fixation, NH 4 ⁺ uptake and N allocation within Trifolium repens L., were studied under controlled conditions. The shoot growth rate was greatly reduced when root temperatures were lowered from 12 to 6°C, while the rate of stolon growth was less affected. Low root temperatures inhibited N2 fixation more than it did NH 4 ⁺ uptake, but the relative allocation of N to stolons was increased. Lowering root temperature also increased the accumulation of VSP in stolons. These results are discussed in terms of the mechanism associated with low temperature stimulation of VSP accumulation and its coupling with changes in the source/sink relations for allocation of N, between growth and storage.
Direct DNA delivery via microprojectile bombardment has become an established approach for gene transfer into peanut (Arachis hypogaea L.). To optimize our transformation protocol and to simultaneously explore the function of a heterologous promoter whose activity is developmentally regulated, embryogenic cultures from three peanut cultivars were bombarded with two plasmid constructs containing a uidA gene controlled by either a soybean vegetative storage protein gene promoter or a cauliflower mosaic virus 35S promoter. We found that GUS transient expression was useful to predict stable transformation and confirmed that image analysis could provide a quick and efficient method for semi-quantitation of transient expression. One hundred and sixty hygromycin-resistant cell lines were recovered from and maintained on selective medium, and those tested by Southern blot analysis showed integration of the foreign gene. Over 200 transgenic plants were regenerated from 38 cell lines. More than 100 plants from 32 cell lines flowered and 79 plants from 19 cell lines produced pods. Over 1000 R1 seeds were harvested. Analysis of expression in primary transgenic plants showed that GUS expression driven by the vspB promoter was modulated by chemical and positional information.
A new method for the chemical deglycosylation of glycoproteins is described. Treatment of fetuin with trifluoromethanesulfonic acid at 0 or 25°C results in rapid cleavage of peripheral sugars, slow loss of serine- and threonine-linked N-acetylgalactosamine, and retention of N-glycosidically linked N-acetylglucosamine. Amino acid analysis and sodium dodecyl sulfate-polyacrylamide gel electrophoresis reveal that the peptide backbone is left intact after reaction at 0°C for 2.5 h or at 25°C for 1 h. Treatment of ovine luteinizing hormone and human M,N-active erythrocyte sialoglycoproteins at 0°C for 3 h gives similar results. In the case of ovine luteinizing hormone, the recovered deglycosylated hormone has approximately 2% of the starting material's activity as measured in a receptor binding assay. In the case of bovine nasal septum proteoglycan, the deglycosylated core protein acts as an excellent acceptor for UDP-d-xylose:core protein β-O-xylosyltransferase. The reagent is far less toxic than HF and does not require special equipment for handling. The procedure should prove useful not only for generation of deglycosylated proteins, but also for assessment of the number of asparagine-linked saccharide chains in a glycoprotein.
The rapid, ultrasensitive silver stains that have been developed recently for detecting proteins in polyacrylamide gels show variation in staining from gel to gel and do not stain certain proteins at all. It was found that treatment of gels with dithiothreitol prior to impregnation with silver nitrate results in more reproducible staining patterns that are also qualitatively similar to those obtained with Coomassie blue. In addition, it obviates the need for treatment with intense light, and results in sensitivities at least as high as those obtained with previously published methods.
Pod removal or petiole girdling, which causes obstruction of translocation, was found in our previous study to cause reduced rates of photosynthesis in soybean leaves due to stomatal closure. The purpose of this research was to determine the involvement of photoassimilate accumulation and leaf abscisic acid (ABA) levels in the mechanism of stomatal closure induced by such treatments.Leaf glucose and sucrose levels increased during the initial 12-hour period after depodding or petiole girdling. Starch, which represents a much larger pool of leaf carbohydrate, was not perceptibly increased above control levels during the 12-hour posttreatment period.When leaflets were exposed to nonphotosynthetic environments (shading or CO(2)-free air) for a 24-hour period after the translocation-obstructing treatments were applied and then returned to normal light or CO(2) concentration, stomatal diffusive conductivity was reduced 65% and 85% with depodding and girdling, respectively. These reductions were comparable to those previously observed without an intervening nonphotosynthetic exposure, thus indicating that photosynthate accumulations were not necessary for the observed response.Free and bound ABA (released on alkaline hydrolysis) were determined by gas liquid chromatography with electron capture detection following preparative high performance liquid chromatography. Free ABA in monitored leaves increased almost 10-fold 48 hours after complete depodding and 25-fold 24 hours after petiole girdling of such leaves. By 3 hours after treatment, in a time course study, free ABA had increased 2-fold above control values in depodded and 5-fold in girdled leaves. Leaf concentrations of bound ABA did not appear to be related to the treatment effects on stomata.Thus, the translocation-obstructing treatments cause an increased level of ABA by a mechanism not involving accumulation of photoassimilate. Increased leaf ABA levels, which were independent of water stress or leaf water potential, appear to be involved in the stomatal closure response. It is suggested that the mechanism of increased leaf ABA levels following translocation-obstruction may be due to an interference with normal translocation of ABA out of leaves.
Collections of xylem exudate of root stumps or detached nodules, and of phloem bleeding sap from stems, petioles, and fruits were made from variously aged plants of Lupinus albus L. relying on nodules for their N supply. Sucrose was the major organic solute of phloem, asparagine, glutamine, serine, aspartic acid, valine, lysine, isoleucine, and leucine, the principal N solutes of both xylem and phloem. Xylem sap exhibited higher relative proportions of asparagine, glutamine and aspartic acid than phloem sap, but lower proportions of other amino acids. Phloem sap of petioles was less concentrated in asparagine and glutamine but richer in sucrose than was phloem sap of stem and fruit, suggesting that sucrose was unloaded from phloem and amides added to phloem as translocate passed through stems to sinks of the plant. Evidence was obtained of loading of histidine, lysine, threonine, serine, leucine and valine onto phloem of stems but the amounts involved were small compared with amides. Analyses of petiole phloem sap from different age groups of leaves indicated ontogenetic changes and effects of position on a shoot on relative rates of export of sucrose and N solutes. Diurnal fluctuations were demonstrated in relative rates of loading of sucrose and N solutes onto phloem of leaves. Daily variations in the ability of stem tissue to load N onto phloem streams were of lesser amplitude than, or out of phase with fluctuations in translocation of N from leaves. Data were related to recent information on C and N transport in the species.
Methods used to construct whole-plant models of nitrogen translocation are reviewed. Translocation of nitrogen in phloem and xylem are examined in relation to the overall nutrition of a plant. Partitioning of nitrogen within shoots is influenced by the extent to which nitrogenous solutes are transferred laterally (xylem-to-phloem; xylem-to-xylem) and by the extent of nitrogen cycling in leaves and roots. Considered in the context of the whole plant these processes contribute to the maintenance of an appropriate balance of carbon and nitrogen for supply to developing apices and seeds. Floral structures also play an important role in seed nutrition. It is argued that control of nitrogen partitioning occurs within the shoot and that transfer of amides between xylem and phloem, and cytokinins translocated from roots may play an important role in partitioning of nitrogen.
Cytyledons of the common bean, Phaseolus vulgaris L., were incubated with radioactive amino acids at different stages of seed development. The proteins were fractionated by ion-exchange chromatography, sucrose gradients, and sodium dodecylsulfate (SDS) polyacrylamide gel electrophoresis. From 16 to 28 d after flowering about 40% of the incorporated radioactivity was associated with the polypeptides of vicilin and 10% with those of phytohemagglutinin.
Polysomes were isolated from developing cotyledons 20–25 d after flowering and free polysomes were separated from membrane-bound polysomes. Aurintricarboxylic acid, an inhibitor of initiation in cell-free translation systems, did not inhibit the incorporation of amino acids into in-vitro synthesized proteins, indicating that synthesis was limited to the completion of already initiated polypeptides. Autofluorography of SDS-polyacrylamide gels showed that the two classes of polysomes made two different sets of polypeptides and that there was little overlap between these two sets.
Four polypeptides similar in size to the 4 polypeptides of vicilin were made by membrane-bound polysomes and not by free polysomes. Antibodies specific for vicilin bound to those 4 polypeptides. Free polysomes made only polypeptides which did not bind to antibodies specific for vicilin. Antibodies against phytohemagglutinin did not bind to any of the invitro synthesized polypeptides.
The membranes to which the polysomes were bound were characterized on sucrose gradients and by electron microscopy. Polysomes recovered from membranes which banded on top of 35 and 50% sucrose synthesized the vicilin polypeptides most rapidly. These membrane fractions were rich in vesicles of rough endoplasmic reticulum (ER). The ER marker-enzyme NADH-cytochrome-c reductase banded with an average density of 1.18 g/cm3 (40% w/w sucrose) on continuous gradients. These experiments demonstrate that the ER is the site of vicilin synthesis in developing bean cotyledons. Quantitative determinations of several ER parameters (RNA and lipid-phosphate content, NADH-cytochrome-c-reductase activity) show that expansion of the cotyledons is accompanied by a 4-6-fold increase in ER.
Nitrogen and carbohydrate assimilates were temporally and spatially compartmented among various cell types in soybean (Glycine max L., Merr.) leaves during seed filling. The paraveinal mesophyll (PVM), a unique cell layer found in soybean, was demonstrated to function in the synthesis, compartmentation and remobilization of nitrogen reserves prior to and during the seed-filling stages. At anthesis, the PVM vacuoles contain substantial protein which completely disappears by two weeks into the seed filling. Distinct changes in the PVM cytoplasm, tonoplast and organelles were correlated with the presence or absence of the vacuolar material. Microautoradiography following the accumulation of several radiolabeled sugars and amino acids demonstrated the glycoprotein nature of the vacuolar material. Incorporation of methionine, leucine, glucose, and glucosamine resulted in heavy labelling of the PVM vacuole, in contrast to galactose, proline, and mannose which resulted in a much reduced labelling pattern. In addition, starch is unequally compartmented and degraded among the various leaf cells during seed filling. At the end of the photoperiod at the flowering stage, the highest starch accumulation was in the second palisade layer followed by the spongy mesophyll and the first (uppermost) palisade layer. Starch in the first palisade layer was completely degraded during the dark whereas the starch in the second palisade and spongy mesophyll was not remobilized to any appreciable extent. By mid-podfilling (approximately five weeks postanthesis) starch was absent in the first palisade layer at the end of the photoperiod while the second palisade and spongy mesophyll layers contained substantial starch. Starch was remobilized from these latter cells during the remainder of seed filling when current photosynthetic production is low. Structural changes associated with cell senescence first appear in the upper palisade layer and then progress (excluding the PVM) to the second palisade and spongy mesophyll layer. The PVM and phloem appear to retain their structural integrity into the leaf yellowing stage. Reducing sink capacity by pod removal resulted in a continued accumulation of vacuolar protein, an increase in cytoplasmic volume, and fragmentation of the vacuole in the PVM. Pod removal also resulted in an increased amount of accumulated starch (which did not turn over) in all mesophyll layers, and an increase in cell size and cell-wall thickness.
Due to the large nitrogen requirement by soybeans ( Glycine max L. Merr.), efficient N utilization might conserve plant energy for other metabolic processes. One method of improving efficiency of N utilization would be to have a high total N content in the seeds vs. that in the above‐ground portion of the plant (i.e. a high N index). The objective of this field study was to evaluate a diverse group of 32 nonnodulating lines and nodulating and non‐nodulating near isogenic lines of ‘Clark’ and ‘Harosoy’ for genetic variability in harvest nitrogen index when grown at three N fertilizer rates. In a second year's experiments lines representing extremes in harvest N index were sampled throughout reproductive development and quantity of N in leaflets, petioles, stems and pods was determined. Harvest indices for grain yield and N varied among the genotypes tested. Harvest indices and harvest nitrogen indices were positively correlated with seed yield. A significant positive correlation between harvest and harvest nitrogen indices suggested that certain genotypes are efficient in mobilizing both N and dry matter to the developing seed. Differences in harvest nitrogen index at maturity resulted from differential N mobilization from leaflets, petioles and stems of efficient (high harvest N index) as compared to inefficient (low harvest N index) genotypes. In general stem N characteristics were most diagnostic of harvest N index. At maturity harvest N index was statistically correlated with grams of N in stems, percent N in stems, and percent of maximum N accumulated that was translocated from the stems. Averaged across genotypes adding N fertilizer increased the quantity of whole plant N, grain yield and percent protein in the seed. However harvest N indices were not significantly affected by N fertilization. The ranking of genotypes for harvest and harvest N indices were consistent in different environments, but the inclusion of abscised leaflets and petioles caused some differences in ranking. Of the genotypes evaluated noduating Harosoy had the greatest grain yield and was among the highest in N index.
The redistribution of N from vegetative plant parts to the developing seed in soybeans [Glycine max (L.) Merrill] may influence the duration of seed filling and yield. The objective of this study was to investigate the N redistribution characteristics of soybean cultivars of varying maturities and growth habit. Eight cultivars ranging from Maturity Group II to V and including indeterminate, determinate, and semi-dwarf growth habits were grown in the field in 1977 and 1978 at Lexington, Ky. using conventional cultural practices. The soil type was a Lanton silt loam (Cumulic Haplaquolls) in 1977 and an Eagam silt loam (Cumulic Hapludolls) in 1978. Nitrogen redistribution was estimated by harvesting plants at beginning seed growth (R5) and at maturity. The abscised leaf blades and petioles were also collected and the dry weight and total N was measured in all plant parts. The vegetative dry weight at RS increased in cultivars of later maturity. There were no consistent culvar differences in N concentration at R5. The proportion of seed N that came from redistribution varied from 30 to essentially 100% and there were significant cultivar differences. The cultivar differences were positively correlated with the amount of N in the plant at R5 which was determined primarily by the vegetative dry weight at R5. Late maturing cultivars got more of their seed N from redistribution than early maturing cultivars. Although there were significant cultivar differences in yield and the duration of seed fill, they were not related to the amount of seed N that came from redistribution. Nitrogen redistribution does not appear to be an important factor determining the duration of seed filling or yield in soybeans.
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The accumulation of dry weight by the pod is an important process during reproductive growth in soybeans [Glycine max (L.) Merr.]. Thus, field experiments on Maury silt loam (Typic Paleudalfs) and Egam silt loam (Cumulic Hapludolls) with soybean cultivars differing in seed size were conducted for 2 years to investigate pod and seed development characteristics and associated changes in seed moisture content. Pods were tagged and random samples from different plants were taken at weekly intervals. Measurements included: pod length and width (1979 experiment only), pod wall and seed dry weight, seed moisture percentage, and water content (mg . seed⁻¹). Cultivars differed in maturity and final seed size which ranged from 92 to 262 mg . seed⁻¹.
Pod length and width in all cultivars in 1979 were at a maximum when the seeds averaged 4% of their maximum dry weight. Length and width were positively correlated with final seed size. In most cultivars sampled in both 1975 and 1979 there appeared to be little redistribution of dry matter from pod walls prior to seed physiological maturity. However, some redistribution was apparent in ‘Emerald’ and ‘Essex’ in 1979 and ‘Cutler 71’ in 1975. Seed of all cultivars showed rapid water uptake during early seed development and water content (mg . seed⁻¹) reached a maximum before seed physiological maturity. Moisture percentage of the seed was greater than 80% when the seed started rapid accumulation of dry matter and declined progressively during seed development, reaching less than 60% as the seed approached physiological maturity. These changes in percentage moisture during seed development were consistent across cultivars in both years, regardless of seed size. Water uptake and moisture content seemed to he directly related to the stage of seed development and were not affected by genetic differences in seed size.
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The RNAs and proteins specified by five early genes of bacteriophage T7 have been identified by electrophoresis on sodium dodecyl sulfate, polyacrylamide gels. Extracts of cells infected by different deletion strains and point mutants of T7 are analyzed on a slab gel system in which 25 samples can be run simultaneously and then dried for autoradiography. The high capacity of this system makes it possible to do many types of experiment that would be extremely tedious by other means.The five early genes are designated 0.3,0.7, 1, 1.1 and 1.3, in order from left to right on the T7 genetic map. The stop signal that prevents host RNA polymerase from transcribing into the late region of T7 DNA is located to the right of gene 1.3 (ligase). Most deletions that affect gene 1.3 also delete the stop signal, and some of them affect at least one late protein, the 1.7 protein. Several small, early RNAs can be resolved that are not affected by any of the deletions. These small RNAs could not come from between the five early genes or from the right end of the early region, and other work (Dunn & Studier, 1973) indicates that at least some of them come from the region to the left of gene 0.3.Deletions have been found that enter either end of the gene 1 RNA or the right ends of the 0.3 or 1.1 RNAs without seeming to affect the proteins specified by these RNAs. Perhaps all of the early messenger RNAs of T7 have untranslated regions at both ends. Some deletions that enter the left end of the gene 1 RNA reduce the amount of gene 1 protein that is synthesized, presumably by interfering with initiation of protein synthesis.
Removing the pods from soybean (Glycine max [L.] Merr. cv Wye) plants induces a change in leaf function which is characterized by a change in the leaf soluble protein pattern. The synthesis of at least four polypeptides ( approximately 27, 29, 54, and 80 kilodaltons) is enhanced, and these polypeptides accumulate to levels comprising over 50% of the soluble protein. Heat girdling the petiole also causes the accumulation of these polypeptides, suggesting that the signal for changing leaf function may be associated with inhibition of phloem transport. The 27 and 29 kilodalton polypeptides are glycosylated and have been purified to greater than 90% by (NH(4))(2)SO(4) fractionation, concanavilin A affinity, and gel filtration chromatography. These peptides appear to comprise a single protein. Mouse antiserum has been prepared against this glycoprotein and has been used to check for cross-reactivity with seed proteins and to quantitate changes with leaf development. No cross-reactivity was observed with seed soluble proteins from several stages of development. Quantitation showed the highest content in podded plants at, and shortly following, flowering, with levels subsequently declining in conjunction with seed growth. In depodded plants, the level of glycoprotein continued to increase following flowering and accounted for 45% of the soluble leaf protein by 4 weeks after depodding.
Well nodulated, field-grown soybeans (Glycine max [L.] Merr. var Williams) were depodded just prior to seed development and near mid pod-fill. Both treatments caused a considerable increase in leaf dry weight, suggesting continued photosynthate production following pod removal. Moreover, depodding had a marked effect on leaf soluble protein without affecting total proteolytic activity. Early depodding caused a 50% increase in leaf protein, and both early and late depodding caused the retention of protein for several weeks following the decline in control leaves. But despite this retention of protein, leaves of depodded plants showed no difference in the onset of the irreversible decline in photosynthesis. Therefore, although depodding delayed the loss of leaf chlorophyll and protein, it did not delay the onset of functional leaf senescence and in fact, actually appeared to enhance the rate of decline in photosynthesis. There was a good correlation between the irreversible decline in ribulose bisphosphate carboxylase (activity and amount) and that of photosynthesis. In contrast, the correlation did not seem as good between stomatal closure and the onset of the irreversible decline in photosynthesis. The reason total soluble protein remained high following depodding while carboxylase, which normally comprised 40% of the soluble protein, declined was because several polypeptides increased in amounts sufficient to offset the loss of carboxylase. This change in leaf protein composition indicates a change in leaf function; this is discussed in terms of other recent findings.
Immunohistochemical staining was used to determine the cellular distribution of two glycosylated polypeptides (molecular weights of 27 and 29 kilodaltons) which are normally present at low levels in soybean (Glycine max var ;Wye') leaves but which markedly accumulate after depodding. These polypeptides, which comprise a substantial portion of the total leaf soluble protein of depodded plants, were exclusively located in the vacuoles of paraveinal mesophyll and associated bundle sheath cells. These results support the unique role of the soybean leaf paraveinal mesophyll in the transport and spatial compartmentation of nitrogen reserves in relation to seed filling.
Exchanges of CO(2) and changes in content of C and N were studied over the life of a leaf of Lupinus albus L. These data were combined with measurements of C:N weight ratios of xylem (upper stem tracheal) and phloem (petiole) sap to determine net fluxes of C and N between leaf and plant. Phase 1 of leaf development (first 11 days, leaf to one-third area) showed increasing net import of C and N, with phloem contributing 61% of the imported C and 18% of the N. (14)C feeding studies suggested the potential for simultaneous import and export through phloem over the period 9 to 12 days. Phase 2 (11-20 days, leaf attaining maximum area and net photosynthesis rate) exhibited net import through xylem and increasing export through phloem. Eighty-two% of xylem-delivered N was consumed in leaf growth, the remainder exported in phloem. Phase 3 (20-38 days) showed high but declining rates of photosynthesis, translocation, and net export of N. Phase 4 (38-66 days) exhibited substantial losses of N and declining photosynthesis and translocation of C. C:N ratio of xylem sap remained constant (2.3-2.6) during leaf life; petiole phloem sap C:N ratio varied from 25 to 135 over leaf development. The relationships between net photosynthesis and N import in xylem were: phase 1, 4.8 milligrams C per milligram N; phase 2, 24.7 milligrams C per milligram N; phase 3, 91.9 milligrams C per milligram N; and phase 4, 47.7 milligrams C per milligram N.
The effects of increased sink-source ratios, induced by elevating night temperatures, on remobilization of (14)C-assimilates and N within field-grown soybeans (Glycine max [L.] Merr.) was investigated from preflowering to maturity. Raising the mean minimum night temperature for the entire growing season from 10 (check, uncontrolled) to 16 degrees C increased seed growth without appreciable effect on final leaf area. Increasing this temperature to 24 degrees C increased seed growth and reduced final leaf area. Leaves, stems, petioles, and pods acted as intermediate storage sites for (14)C assimilates. Only plants with higher night temperatures remobilized some of the stored assimilates during the period of rapid seed growth. Even the seeds in the 24 degrees C plants with the largest sink-source ratios did not utilize all the C-assimilates potentially available for remobilization. Nitrogen was readily remobilized from petioles, stems, and pods of all treatments as early as the beginning of seed development, but from the leaves only during late seed-filling. However, only plants with elevated night temperatures tended to remobilize all of the available N from vegetative tissues and pods. We concluded that a larger portion of stored assimilates may be remobilized to the seed if a strong seed sink can be sustained. It also appeared that with increasing sink-source ratios, N shortage might limit seed yield before a lack of C-assimilates would. A proposed model for soybean assimilate demand, distribution, partitioning, and remobilization is presented.
Depodded soybean (Glycine max [L] Merr. cv Williams) plants accumulate high levels of a glycoprotein in their leaves that has many features of a storage protein. The protein is found in all vegetative tissues which have been examined but not in the seeds. Translation in vitro indicated that elevated mRNA levels were at least partially responsible for the specific increase in vegetative storage protein. cDNA clones were isolated and sequenced, and an amino acid sequence was predicted. Although the amino acid composition is similar to that of seed storage proteins, no sequence similarity could be detected. Northern blot hybridization confirmed a large increase in vegetative storage protein mRNA in leaves of depodded plants. The vegetative storage proteins are represented by about four gene copies in the haploid genome.
Jasmonic acid-dependent increase in paraveinal mesophyll proteins in soybean leavesand mixotrophic suspension cultures (abstract 687)
- Jm Anderson
- Sr Spilatro
- Sf Klauer
- Franceschi
Anderson JM, Spilatro SR, Klauer SF, Franceschi VR (1988) Jasmonic acid-dependent increase in paraveinal mesophyll proteins in soybean leavesand mixotrophic suspension cultures (abstract 687). Plant Physiol 86: S-114
Molecular cloning, a laboratory manual. Cold Spring Harbor
- T Maniatis
Maniatis T (1982) Molecular cloning, a laboratory manual. Cold
Spring Harbor, New York.