Vegetative Storage Protein Accumulation Research Articles

Evaluation of the susceptibility to green stem disorder in soybeans [Glycine max (L.) Merr.] with vegetative storage protein accumulation

ABSTRACT 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.

Analysis of vegetative storage protein accumulation in soybean cultivars expressing different green stem disorder severity affected by soil moisture change

ABSTRACT 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.

Development and metabolism of the fruit and seed of the Japanese plum Ozark premier (Rosaceae)

The growth characteristics of some plums and their component parts have been previously studied, as have some aspects of their developmental anatomy and composition. However, little is known about either their metabolism or about the interactions between the metabolism of their component parts. In this study we investigated these aspects in the Japanese plum Ozark Premier. Throughout fruit and seed development, changes in sugar and organic acid contents, protein composition and abundance of selected enzymes were determined. In the stone, there was a transient accumulation of vegetative storage proteins. These were subsequently mobilized and this coincided with the onset of the lignification of the stone and the start of storage protein accumulation in the seed. Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) was present in the seeds, even though they lacked chlorophyll, and its presence may be related to limited gas exchange. In the flesh of some fruits, phosphoenolpyruvate carboxykinase (PEPCK) and NADP malic enzyme (NADP-ME) are thought to function in the dissimilation of malate and/or citrate during ripening. However, PEPCK and NADP-ME were present in plum flesh for most of its development, although there was no net dissimilation of malate until the latter stages of ripening. There is an interaction between the developing seed and endocarp with respect to the utilization of imported sugars and amino acids. An hypothesis is presented to account for the presence of PEPCK and NADP-ME enzyme in plum flesh when there was no net dissimilation of organic acids.

Metabolism of the seed and endocarp of cherry (Prunus avium L.) during development

In this study some aspects of organic and amino acid metabolism in cherry endocarp and seed were investigated during their development. The abundance and location of a number of enzymes involved in these processes were investigated. These enzymes were aspartate aminotransferase (AspAT; EC:2.6.1.1), glutamine synthetase (GS; EC:6.3.1.2), phosphoenolpyruvate carboxylase (PEPC; EC:4.1.1.31), phosphoenolpyruvate carboxykinase (PEPCK; EC:4.1.1.49), and ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco; EC:4.1.1.39). There was a transient and massive accumulation of vegetative storage proteins in the endocarp. These proteins were remobilised as the endocarp lignified and at the same time that proteins were accumulated in the seed. This raised the possibility that a proportion of imported amino acids were temporarily stored in the endocarp as protein, and that these were later utilised by the seed when it started to accumulate storage proteins. Rubisco was present in the embryo and integuments of the seed although no chlorophyll was present. This is the first time that Rubisco has been detected in non-green seeds. The maximum abundance of Rubisco in the seed coincided with the deposition of seed storage proteins. A possible function for Rubisco in cherry seed is discussed. PEPCK was located in the integuments and appeared when seed storage proteins were being accumulated. In the integuments and embryo AspAT, GS, PEPC and Rubisco also appeared, or greatly increased in abundance, when seed storage proteins were being deposited.

VSP accumulation and cold-inducible gene expression during autumn hardening and overwintering of alfalfa

This study describes the time-course of the accumulation of total soluble proteins (TSPs) and vegetative storage proteins (VSPs) and of the transcripts of cold-inducible (CI) and VSP-encoding genes in taproots of two alfalfa cultivars (AC Caribou and Europe) during their acclimation to natural autumn hardening and overwintering conditions in eastern Canada. The impact of a defoliation in September on these winter hardening-related changes was also assessed. Both concentrations and pools of VSPs increased significantly between early September and mid-October and remained unchanged thereafter, concomitantly with the disappearance of VSP-encoding transcripts. Other soluble protein constituents continued to increase later in the autumn and early winter and accounted for nearly 60% of taproot TSP pools in winter. As a result, VSP abundance relative to TSPs decreased markedly during the winter. The increase in the levels of CI transcripts was induced by lowering temperatures, and distinct patterns suggest differences in the regulation of their accumulation. RNA analyses revealed that the accumulation of VSP transcripts during the autumn precedes the accumulation of CI transcripts. Autumn defoliation interrupted the accumulation of both TSPs and VSPs during autumn hardening and repressed the transcript levels of two CI genes differentially between cultivars. The well-documented impact of autumn defoliation on the vigour of spring regrowth and long-term persistence of alfalfa could be related to its negative impact on the accumulation of VSPs and TSPs and on the expression of genes encoding CI proteins potentially involved in cold tolerance and pathogen resistance.

Poplar Trees (Populus canadensisMoench) Initiate Vegetative Storage Protein Accumulation During New Shoot Development in Spring

: It was generally assumed that the accumulation of vegetative storage protein (VSP) in poplar trees and/or temperate hardwoods did not occur in spring. To test this assumption, the accumulation of the 32-kDa VSP and the differential expression of a gene encoding for the protein in poplars were investigated using light and electron microscopy, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and reverse transcription-polymerase chain reaction (RT-PCR). We report, for the first time, that poplar trees initiated VSP accumulation in new shoots during the development of new shoots in spring under conditions of high temperature and long days. The amount of 32-kDa VSP increased gradually in the stem of new shoots and in two-year-old branches, but there were no detectable changes in its abundance in the bark tissues of trunks during new shoot development. Based on the presence of a 286-bp DNA fragment that is identical to the VSP gene bspA, encoding for the 32-kDa VSP in Populus deltoids Bartr. ex Marsh., the differential expression of the 32-kDa VSP gene in P. canadensis Moench was revealed by RT-PCR. The results indicated that the 32-kDa VSP gene was expressed strongly in new shoots, relative weakly in two-year-old branches and was not expressed in the trunk during new shoot development. This pattern of VSP accumulation and VSP gene space-time differential expression may be an important mechanism by which stored nitrogen compounds are used preferentially to exogenously available nitrogen and, in addition, the dynamic pattern may also have a role in the regulation of nitrogen metabolism, especially nitrogen uptake by the roots. (Managing editor: Wei WANG)

The functional status of paraveinal mesophyll vacuoles changes in response to altered metabolic conditions in soybean leaves.

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.

Effects of altered source–sink relationships on N allocation and vegetative storage protein accumulation in Brassica napus L.

Growth, N allocation and involvement of vegetative storage protein (VSP) in the temporary N accumulation were evaluated in field conditions of oilseed rape grown with altered source–sink relationships (by leaf halving or removal of stem, flowers or pods). As compared to control plants, removal of half the leaves early in the vegetative period-induced delayed flowering, a lower accumulation of the taproot 23 kDa VSP and finally a decreased pod production. Stem removal, when flower buds were still covered by leaves, increased taproot biomass and its N concentration but prevented taproot VSP accumulation. Continuous removal of flowers or pods increased the shoot growth and N uptake duration (for deflowered plants). When foliar senescence finally occurred in plants continuously subjected to flower or pod removal, the N was allocated in a large proportion to buffering organs (taproot and stem) and it led to an increased and late VSP accumulation without subsequent mobilization. In control plants, N stored as VSP was remobilized during late pod filling concomitant with stem and taproot sink to source transition. A summarized scheme of N source–sink relationships during the growth cycle is presented where the 23 kDa VSP may act as an N storage buffer in response to an asynchronism between mobilization of foliar N and N requirements for seed formation.

Vegetative storage proteins in overwintering storage organs of forage legumes: roles and regulation

In perennial forage legumes such as alfalfa (Medicago sativa L.) and white clover (Trifolium repens L.), vegetative storage proteins are extensively mobilized to meet the nitrogen requirements of new shoot growth in spring or after cutting in summer. The 32-kDa alfalfa storage protein possesses high homology with class III chitinases, belonging to a group of pathogenesis-related proteins that possess antifreeze protein properties in some species and exhibit chitinolytic activity in vitro. This protein and the corresponding mRNA accumulate in taproots of cold-hardy culti vars during acclimation for winter, and in response to short-day conditions in controlled environments. The 17.3-kDa storage protein of white clover possesses high homology with pathogenesis-related proteins and abscisic- acid-responsive proteins from several legume species and has characteristics common to stress-responsive proteins. Low temperature enhances accumulation of this 17.3-kDa protein and its corresponding transcript. Exogenous abscisic acid stimulates the accumulation of vegetative storage proteins and their transcripts in both legume species. These observations suggest that vegetative storage proteins do not exclusively serve as nitrogen reserves during specific phases of legume development, but may play important adaptive roles in plant protection against abiotic (low temperature) and biotic (pathogen attack) stresses.Key words: nitrogen reserves, vegetative storage proteins, regulation, cold tolerance, chitinase, pathogenesis-related proteins.

Short-day photoperiod induces changes in N uptake, N partitioning and accumulation of vegetative storage proteins in two Medicago sativa cultivars.

Our objective was to study the effect of short-day photoperiod for 28, 42 and 56 d on growth, N uptake and N partitioning, particularly vegetative storage protein (VSP) accumulation in taproots of two alfalfa (Medicagosativa L.) cultivars (Lodi and Europe). For both varieties, the reduction of daylength from 16 h (long day,LD) to 8 h (short day, SD) for 28 d reduced total plant growth by decreasing shoot growth. Nitrogen uptake and N distribution within the plant was determined by 15N labeling. N uptake decreased with SD treatment duration, and was 2- and 3-fold lower for Europe and Lodi, respectively, for 56 d in SD conditions when compared with LD plants. The SD treatment resulted in preferential partitioning of N to taproots in comparison with LD conditions (19vs 9% for Lodi and 12 vs 5% for Europe after 28 d). For both cultivars, the SD-induced changes in N allocation to taproots did not significantly affect taproot soluble protein concentrations during 42 d of daylength treatment. In contrast, VSP accumulation occurred after only 28 d for plants grown in SD conditions (6.2 vs 4.8 mg g-1 DW for Lodi and 5.1 vs 1.4 mg g-1 DW for Europe). SD exposure also increased vsp 57 and vsp 32 mRNA transcript levels in Lodi and Europe (up to 2-fold higher) taproots in SD for 28 d compared with LD conditions. Overall results indicate that photoperiod modulates taproot N accumulation in alfalfa by enhancing both β-amylase (vsp 57) and vsp 32 gene expression and accumulation. The enhanced VSP accumulation by short-day photoperiod may result from altered VSP gene expression / transcript stability or occur indirectly through altered N source-sink relationships. Additionally, when SD treatment included a night break with 15 min illumination with sodium high pressure light or red light, our results suggest that the induction of vsp 57 and vsp 32 gene expressions by SD signal is mediated by the phytochrome system.

Nitrogen storage and remobilization in Brassica napus L. during the growth cycle: effects of methyl jasmonate on nitrate uptake, senescence, growth, and VSP accumulation.

The role of methyl jasmonate (MeJa) in promoting senescence has been described previously in many species, but it has been questioned in monocarpic species whether induced senescence is a result of a potential death hormone like MeJa, or a consequence of an increased metabolic drain resulting from the growth of reproductive tissue. In oilseed rape (Brassica napus L.), a polypeptide of 23 kDa has been recently identified as a putative vegetative storage protein (VSP). This polypeptide could be used as a storage buffer between N losses from senescing leaves putatively promoted by methyl jasmonate that might be produced by flowers, and grain filling which occurs later on, while N uptake is strongly reduced. In order to describe causal relationships during Brassica napus L. plant responses to MeJa treatment, a kinetic experiment was performed to determine the order and the amplitude with which general processes such as growth, photosynthesis, chlorophyll content, N uptake, and N storage under the form of the 23 kDa VSP are affected. One of the most immediate consequences of MeJa treatment was the strong reduction of nitrate uptake within 6 h, relative to control plants. However, this was not a specific effect as K(+) uptake was similarly affected. Photosynthesis was reduced later (after 24 h), while chlorophyll content as well as leaf growth also decreased in a similar way. Moreover, this was concomitant with a remobilization of endogenous unlabelled N from senescing leaves to roots. Accumulation of the 23 kDa VSP was induced in the taproot after 24 h of MeJa treatment and was increased 10-fold within 8 d. On the other hand, the reversible effect of a MeJa pretreatment was tested in the long term (i.e. along the growth cycle) using plants previously grown in field conditions induced for flowering. Results show that a MeJa pulse induced a reversible effect on N uptake inhibition. In parallel, protein immunologically related to the 23 kDa VSP was detected in stems with a similar molecular weight (23 kDa), and in flowers and leaves with a molecular weight of 24 kDa. This accumulation was concomitant with the remobilization of both subunits of Rubisco. During stem and pod development, this protein induced by MeJa is fully hydrolysed. The external and intermittent supply of MeJa mimic some of the plant physiological processes previously reported under natural conditions. This suggests that in oilseed rape, methyl jasmonate could be considered as a possible monocarpic senescence factor while accumulation/mobilization of the 23 kDa VSP in taproot could be a marker for the cessation of N uptake and the initiation of a massive leaf senescence.

Influence of summer sowing dates, N fertilization and irrigation on autumn VSP accumulation and dynamics of spring regrowth in alfalfa (Medicago sativa L.)

Herbage yield of alfalfa (Medicago sativa L.) depends on forage management or environmental conditions that change C and N resource acquisition, and endogenous plants factors such as root organic reserves and number of active meristems. The aim of this work is to study the influence of two sowing dates in summer (12 July or 9 August), N fertilization (0 or 100 kg ha(-1)) and/or irrigation applied during the first year of alfalfa establishment on (i) the accumulation of N organic reserves (soluble proteins and more specifically vegetative storage protein) in taproots during autumn, (ii) the number of crown axillary meristems present at the end of winter and (iii) the dynamics of spring shoot growth. Delaying the sowing date for one month reduced root growth and root N storage, especially vegetative storage proteins (VSP) during autumn. Irrespective of sowing dates, N fertilization did not affect root biomass, number of crown buds, total root N, root soluble protein or VSP concentrations. By contrast, water deficiency during alfalfa establishment in the early summer reduced both root growth and N reserve accumulation. When spring growth resumed, there is a significant linear relationship between leaf area development and soluble protein and VSP concentrations in taproots, and also the number of crown buds. The results showed that an early sowing date and adequate water status during the summer allowed alfalfa plants to accumulate N reserves by increasing taproot mass and soluble protein concentrations, especially VSPs. This resulted in rapid shoot regrowth rates the following spring.

Influence of summer sowing dates, N fertilization and irrigation on autumn VSP accumulation and dynamics of spring regrowth in alfalfa ( Medicago sativa L.)

Herbage yield of alfalfa ( Medicago sativa L.) depends on forage management or environmental conditions that change C and N resource acquisition, and endogenous plants factors such as root organic reserves and number of active meristems. The aim of this work is to study the influence of two sowing dates in summer (12 July or 9 August), N fertilization (0 or 100 kg ha −1 ) and/or irrigation applied during the first year of alfalfa establishment on (i) the accumulation of N organic reserves (soluble proteins and more specifically vegetative storage protein) in taproots during autumn, (ii) the number of crown axillary meristems present at the end of winter and (iii) the dynamics of spring shoot growth. Delaying the sowing date for one month reduced root growth and root N storage, especially vegetative storage proteins (VSP) during autumn. Irrespective of sowing dates, N fertilization did not affect root biomass, number of crown buds, total root N, root soluble protein or VSP concentrations. By contrast, water deficiency during alfalfa establishment in the early summer reduced both root growth and N reserve accumulation. When spring growth resumed, there is a significant linear relationship between leaf area development and soluble protein and VSP concentrations in taproots, and also the number of crown buds. The results showed that an early sowing date and adequate water status during the summer allowed alfalfa plants to accumulate N reserves by increasing taproot mass and soluble protein concentrations, especially VSPs. This resulted in rapid shoot regrowth rates the following spring.

Effects of environmental factors and endogenous signals on N uptake, N partitioning and taproot vegetative storage protein accumulation in Medicago sativa

Our objectives were to study the regulation of N partitioning within tissues of non-nodulated alfalfa (Medicago sativa L.) and N storage in taproots as vegetative storage proteins (VSP) of 15, 19, and 32 kDa and β-amylase (57 kDa) by environmental (photoperiod, temperature, N availability) and endogenous factors (methyl jasmonate). When compared to long-day conditions (LD, 16 h day/8 h night), short-day (SD, 8 h day/16 h night), exposure to low temperature (5˚C) or application of methyl jasmonate (MeJA, 100 M ) for 35 d reduced the biomass shoot/ root ratio and modified the source–sink relationships for N. SD and MeJA treatments resulted in partitioning of N to taproots and a concomitant accumulation of VSPs. In comparison with LD, SD treatment also stimulated β-amylase gene expression 2.5-fold. Although low temperature increased the N partitioning to root tissues and the accumulation of soluble proteins in taproot, VSP concentration and β-amylase mRNA levels remained low. Increasing N concentration from 1 to 5 mM KNO3 doubled the total dry matter but did not affect the N partitioning within the plant, VSP accumulation, or ‚ β-amylase expression. These results suggested that short photoperiod can result in preferential N allocation toward taproots with a concomitant induction of VSP accumulation.

Non-destructive Limits to Seed Growth and Leaf Protease Activities in Nodulating and Non-nodulating Soybean Isolines

Leaf senescence leads to a progressive decline in the photosynthetic competence of the leaf. This paper describes some effects of source:sink imbalance on leaf protein catabolism and senescence in soybean. We manipulated pod growth by restricting 100 or 50 % (PR-100 or PR-50, respectively) of young pods at the R4 stage in plastic drinking straws. This effectively reduces final seed mass without interrupting the vascular connections of pods. Nodulating (NOD+) and non-nodulating (NOD−) isolines of the ‘Clay’ soybean were grown in drainage lysimeters and three pod-restriction (PR) treatments were compared. Pod restriction decreased seed biomass per plant as a result of lower individual seed mass, which was only partially balanced by the increase in seed number. The nitrogen concentration in seeds remained unchanged in NOD+ plants, while it increased with the degree of sink restriction in seeds of NOD− plants. Leaf soluble protein, CO2 exchange rate and seed nitrogen content were consistently lower in NOD− plants; the leaf protein level remained stable with time in PR-100 plants, decreased for PR-50 and dropped for controls. Endoprotease (HBase) and carboxypeptidase (CPase) activities were significantly lower in leaves from PR-100 plants, while aminopeptidase activity was enhanced, indicating a de novo synthesis of leaf protein. This is consistent with the reported accumulation of vegetative storage proteins (VSPs) in soybean and other legumes after moderate or severe sink reduction. Thus, small modifications of the source:sink ratio such as those obtained by the non-destructive PR technique have an impact on leaf protein catabolism. Nodulating and non-nodulating soybean isolines showed similar responses to PR in terms of leaf senescence initiation and progression, but the rate of the processes appear to be largely influenced by plant N status.

Regrowth of White Clover after Chilling: Assimilate Partitioning and Vegetative Storage Proteins

ABSTRACTUnder temperate climates, grassland species are subjected to overwintering which may significantly influence their early spring growth capacity. In white clover (Trifolium repens L.), it is known that overwintering capacity can differ among cultivars. Ability of this forage legume to recover from winter damage will, therefore, have a great influence on its persistence in grass–clover associations. Experiments were undertaken with two different white clover cultivars (Huia and AberHerald). Leaf appearance rate, dry matter distribution, 14C assimilate partitioning, and vegetative storage protein accumulation were determined in plants subjected to a 4‐wk chilling period (5/0°C, day/night) and subsequent warmer temperatures (15/10°C), and compared with control plants (20/15°C). Chilling treatment decreased leaf appearance rate, with AberHerald producing more leaves than Huia. This can be considered as a major aspect of cold adaptation strategy because leaf appearance rate controlled carbon acquisition. Low temperature increased dry matter partitioning to below‐ground tissues. AberHerald allocated more assimilates to stolons than Huia. Accumulation of a 17.3‐kDa protein, believed to act as a vegetative storage protein, also increased after the chilling treatment. Regrowth was characterized by rapid mobilization of the 17.3‐kDa protein in stolons and by preferential carbon allocation to stolon apices. AberHerald showed a higher regrowth potential than Huia in view of its morphological and physiological characters which include carbon acquisition and assimilate partitioning patterns, favoring shoot regrowth and acquisition of stolon reserves.

Seasonal pattern of accumulation and effects of low temperatures on storage compounds in Trifolium repens

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, N2 fixation, NH4+ 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 NH4+ 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.

Seedling Development and Deposition of Starch and Storage Proteins in Alfalfa Roots

The physiological basis for defoliation tolerance in alfalfa (Medicago sativa L.) is associated with taproot organic reserves, including soluble sugars, starch, and vegetative storage protein (VSP). It is not known when organic reserve accumulation begins in developing seedlings or the factors that influence their deposition. The objectives of three greenhouse experiments were to determine (i) the onset starch and VSP accumulation in roots of alfalfa seedlings, (ii) if N fertilization of seedlings stimulated root VSP deposition, and (iii) seedling defoliation tolerance is associated with the presence of root organic reserves. In Exp. 1, roots and shoots of ‘Vernal’, ‘Resistar’, and ‘WL 605’ were sampled periodically beginning 19 d after planting (DAP) to determine the developmental paltern of starch and VSP deposition in roots. In Exp. 2, normal and ineffective nodulating ‘Saranac’ was grown with or without 10 mM N to determine if applied N stimulates early VSP deposition. In Exp. 3, alfalfa seedlings were completely defoliated (including cotyledons) 14, 28, 42, and 56 DAP, and shoot regrowth and seedling survival were used to measure defoliation tolerance. In all experiments, starch, soluble sugars, protein, and VSP concentrations in roots were determined. Root starch and VSP concentrations were low up to 30 DAP. Starch and VSP accumulation began by 39 DAP and coincided with declining root protein and soluble sugar concentrations. Addition of N increased herbage growth but did not stimulate early VSP deposition. In Exp. 3, ≈80% of seedlings survived complete defoliation 14, 42, and 56 DAP, while only 52% survived defoliation 28 DAP. Roots contained low starch and VSP concentrations 14 and 28 DAP. Although seedling survival 42 and 56 DAP was associated with presence of starch and VSP in seedling roots, defoliation tolerance of alfalfa seedlings 14 DAP appears to be influenced by factors other than root starch and VSP concentrations.

Characterization of seed storage proteins in Populus and their homology with Populus vegetative storage proteins.

We investigated the synthesis and accumulation of vegetative storage proteins (VSPs) in poplar plantlets and the homology between poplar seed storage proteins (SSPs) and VSPs. One-dimensional SDS polyacrylamide gel electrophoresis confirmed that both seed and vegetative storage proteins contained two predominant polypeptides of MW 32 and 36 kDa, but the subunit composition of the polypeptides differed. The 32- and 36-kDa polypeptides were highly abundant in basal leaves, stems, and roots of poplar plantlets. The 36-kDa subunit was synthesized in all plantlet tissues examined, but the 32-kDa subunit was not, suggesting that the 36-kDa polypeptide is a precursor of the 32-kDa polypeptide. The 36- and 32-kDa polypeptides of both SSPs and VSPs were glycosylated and both were found to be albumins. In addition, both polypeptides cross-reacted with a VSP antibody. Protein fingerprint patterns generated with two different proteolytic enzymes were identical for the 36-kDa polypeptide isolated from seeds or from stem tissue. Our study provides evidence that poplar SSPs and VSPs exhibit homology, and that expression is neither tissue-specific nor regulated solely by photoperiod.

Ringing induces the accumulation of vegetative storage proteins in poplar bark

Poplar branches were ringed in late spring to determine whether the interruption of the phloem flow could induce the accumulation of vegetative storage proteins (VSPs) in the bark of adult trees. Eight days after ringing, an increased deposition of starch as well as a premature rise in the soluble-protein level occurred in the bark tissues located 1 cm above the ring. Changes in the SDS-PAGE pattern of bark proteins were characterized by the accumulation of three polypeptides (32, 36 and 38 kDa), which exhibited the same molecular weight as VSPs described in poplar bark during winter, cross-reacted to antibodies raised against a poplar VSP, and bound to several lectins in the same way as poplar bark VSPs. These results indicate that during the vegetative period, ringing induces the accumulation of VSPs in the bark of poplar.