Mobilization of nitrogen reserves during regrowth of defoliated Trifolium repens L. and identification of potential vegetative storage proteins

Although it is well established that carbon reserves contributeto shoot regrowth of leguminous forage species, little informationis available on nitrogen reserves except in Medicaqo sativaL. and Trifolium subterraneum L. In this study, reserves werelabelled with ¹⁵N to demonstrate the mobilization of endogenousnitrogen from roots and stolons to regrowing leaves and newstolons during 24 d of regrowth in white clover (Thfolium repensL.). About 55% and 70%, respectively, of the nitrogen contentsof these organs were mobilized to support the regrowth of leaves.During the first 6 d, nitrogen in regrowing leaves came mainlyfrom N reserves of organs remaining after defoliation. Afterthese first 6 d of regrowth, most of the shoot nitrogen wasderived from exogenous nitrogen taken up while the contributionof nitrogen reserves decreased. After defoliation, the buffer-solubleprotein content of roots and stolons decreased by 32% duringthe first 6 d of regrowth. To identify putative vegetative storageproteins, soluble proteins were separated using SDS-PAGE ortwo-dimensional electrophoresis. One protein of 17.3 kDa instolons and two proteins of 15 kDa in roots seemed to behaveas vegetative storage proteins. These three polypeptides, initiallyfound at high concentrations, decreased in relative abundanceto a large extent during early regrowth and then were accumulatedagain in roots and stolons once normal growth was re-established. Key words: White clover, regrowth, ¹⁵N-labelled, vegetative storage proteins, electrophoresis     

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.     

Nitrogen Reserve Mobilization during Regrowth of Medicago sativa L. (Relationships between Availability and Regrowth Yield)

An experiment was designed to study the role of N and C reserves on regrowth of the shoots following defoliation of forage species. Starch and N accumulation in root and crown tissue of nonnodulated Medicago sativa L. were modified during regrowth by applying different levels of N and different cutting heights. Plants were obtained with similar crown and root dry weights, but having either low starch and high tissue N or high starch and low tissue N. The plants were then submitted to a second defoliation and supplied with optimal N nutrition, and N flow from reserve was quantified using pulse-chase 15N labeling. Maximum yields following the second regrowth were obtained from those plants having a high tissue N, despite their low level of nonstructural carbohydrate. When N in the roots and crown exceeded 5 mg N plant-1 at the beginning of regrowth, about 68% was translocated to regrowing shoots. Highly significant correlations were also found between the amounts of N available in roots and crown at the beginning of regrowth and (a) the amount of N that was mobilized to new tissues, (b) the amount of N taken up during the regrowth period, and (c) the final shoot yield after 24 d of regrowth. No similar correlations were found for plants that varied in their initial starch content of roots and crown. It is suggested that N reserves were used mainly during the first 10 d after defoliation, and that the resulting aerial growth during this period should be sufficient to restore N2 fixation and/or N uptake to levels equal to those prior to defoliation. These data emphasize (a) the importance of root N reserves in initiating and sustaining new shoot growth, and (b) the need for a re-evaluation of the contribution of C reserves to shoot regrowth.     

Effect of elevated CO2, temperature and drought on dry matter partitioning and photosynthesis before and after cutting of nodulated alfalfa

The rising atmospheric CO₂ concentration resulting from industrial development may enhance photosynthesis and plant growth. However, there is a lack of research concerning the effect of combined factors such as CO₂, temperature and water availability on plant regrowth following cutting or grazing, which represent the usual methods of managing forage legumes like alfalfa. Elevated CO₂, temperature and drought can interact with cutting factors (e.g. cutting frequency or height), and source-sink balance differences before and after defoliation can modify photosynthetic behaviour and dry matter accumulation, as well as dry matter partitioning between above- and belowground organs. The aim of our study was to determine the interactive effect of CO₂ (ambient, around 350 μmol mol⁻¹ versus 700 μmol mol⁻¹), temperature (ambient versus ambient + 4 °C) and water availability (well-irrigated versus partially irrigated) on dry matter partitioning and photosynthesis in nodulated alfalfa after vegetative normal growth and during regrowth. At the end of vegetative normal growth, CO₂ enhanced dry matter accumulation despite photosynthesis being down-regulated at the end of this period. Photosynthesis was stimulated by elevated CO₂ and resulted in greater dry matter accumulation during the regrowth period. Aboveground organs were affected more by drought than belowground organs during the entire experiment, particularly during vegetative normal growth. The higher drought tolerance (greater growth) observed during the regrowth period may be related to higher mass and greater reserves accumulated in the roots of plants.     

The use of internal nitrogen stores in the rhizomatous grass Calamagrostis epigejos during regrowth after defoliation

BACKGROUND AND AIMS: The regrowth dynamics after defoliation of the invasive grass Calamagrostis epigejos were studied. As nitrogen (N) reserves have been shown to play an important role during plant regrowth, the identity, location and relative importance for regrowth of N stores were determined in this rhizomatous grass. METHODS: Plant growth, nitrate uptake and root respiration were followed during recovery from defoliation. Water soluble carbohydrates, nitrate, free amino acids and soluble proteins were analysed in the remaining organs. KEY RESULTS: Nitrate uptake and root respiration were severely reduced during the first days of regrowth. Roots were the main net source of mobilized N. The quantitatively dominant N storage compounds were free amino acids. Free amino acids and soluble proteins in the roots decreased by 55 and 50%, respectively, and a substantial (approximately 38%) decrease in stubble protein was also observed. Although the relative abundance of several soluble proteins in roots decreased during the initial recovery from defoliation, no evidence was found for vegetative storage protein (VSP). Furthermore, rhizomes did not act as a N storage compartment. CONCLUSIONS: Production of new leaf area was entirely reliant, during the first week after defoliation, on N stores present in the plant. Mobilized N originated mainly from free amino acids and soluble proteins located in roots, and less so from proteins in stubble. Presence of VSP in the roots was not confirmed. The data suggest that rhizomes played an important role in N transport but not in N storage.     

Regrowth dynamics of <Emphasis Type="Italic">Calamagrostis epigejos</Emphasis> after defoliation as affected by nitrogen availability

Young plants of a rhizomatous grass Calamagrostis epigejos (L.) Roth were grown from seed in nutrient solutions containing nitrogen in concentrations 0.1, 1.0, and 10 mM. After six weeks of cultivation the plants were defoliated and changes in growth parameters and in content of storage compounds were measured in the course of regrowth under highly reduced nitrogen availability. Plants grown at higher nitrogen supply before defoliation had higher amount of all types of nitrogen storage compounds (nitrates, free amino acids, soluble proteins), which was beneficial for their regrowth rate, in spite of lower content of storage saccharides. Amino acids and soluble proteins from roots and stubble bases were the most important sources of storage compounds for regrowth of the shoot. Faster growth of plants with higher N content was mediated by greater leaf area expansion and greater number of leaves. In plants with lower contents of N compounds number of green leaves decreased after defoliation significantly and senescing leaves presumably served as N source for other growing organs. Results suggest that internal N reserves can support regrowth of plants after defoliation even under fluctuating external N availability. Faster regrowth of C. epigejos with more reserves was mediated mainly by changes in plant morphogenesis.     

Foraging dynamics of muskoxen in Peary Land, northern Greenland

Muskoxen Ovibos moschatus in northern Greenland (79-83°N) are at the northern limit of their distribution and exist under seasonal extremes dominated by nearly 10 months of winter, much of which is without sunlight. The period of summer vegetative growth is less than two months. In the Kap København area (82°30'N), diversity of plant species is low (76 species of vascular plants) and forage biomass in major vegetation types in summer varies from over 40 g m^-2 in sedge-dominated fens to ≤5 g m^-2 in polar barrens. Nonetheless, 90-95% of the ice-free area consists of barren ground or sparcely vegetated polar desert. During summer, muskoxen apparently foraged opportunistically to maximize intake, with sedges the major food item in fens while willows were the major dietary component when on willow-dominated slopes. Quality of summer forage was high during its early phenological stages, with 21-28% crude protein and 60-75% in vitro digestibility. Microhistological analysis of winter feces indicated dominance by graminoids. Muskoxen spent > 50% of their daily activity feeding, which fits a cline of increasing feeding time with increasing latitude in summer. Increased feeding times at high latitudes appears to be a function of both reduced forage biomass and need to maximize forage intake during the brief summer period when forage quality is high. Movement rates in summer while foraging were inversely related to available forage biomass. Seasonal activity of muskoxen peaks during the rutting period (July-September) and then declines gradually through early winter to a low in late winter (March-April).     

Partitioning of reserve and newly assimilated carbon in roots and leaf tissues of Lolium perenne during regrowth after defoliation: assessment by 13C steady-state labelling and carbohydrate analysis

The relative significance of the use of stored or currently absorbed C for the growth of leaves or roots of Lolium perenne L. after defoliation was assessed by steady-state labelling of atmospheric CO₂. Leaf growth for the first two days after defoliation was to a large extent dependent on the use of C reserves. The basal part of the elongating leaves was mainly new tissue and 91% of the C in this part of the leaf was derived from reserves assimilated prior to defoliation. However, half of the sucrose in the growth zone was produced from photosynthesis by the emerged leaves. Fructans that were initially present in elongating leaf bases were hydrolysed (loss of 93 to 100%) and the resulting fructose was found in the new leaf bases, suggesting that this pool may be used to support cell division and elongation. Despite a negative C balance at the whole-plant level, fructans were synthesized from sucrose that was translocated to the new leaf bases. After a regrowth period of 28 d, 45% of the C fixed before defoliation was still present in the root and leaf tissue and only 1% was incorporated in entirely new tissue.     

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.     

Fluctuation of Vegetative Storage Proteins in the Seedlings of Swietenia macrophylla, Analogous to the Seasonal Changes of Those in the Shoot of the Adult Tree

In order to Identify appropriate plant materials for studying the gene expression and biological function of vegetative storage proteins （VSPs） in woody plants, the VSPs in the seedlings of Swietenla rnacrophylla King were investigated by using light microscopy, sodium dodecyl sulfate-polyacrylamide gel electrophoresis （SDS-PAGE） and Western.blotting. The seed of S. macrophylla was rich in storage proteins that accumulated In the vacuoles of cotyledon parenchyma cells in appearance of compact spherical grains. The growth and development of S. macrophylla seedlings were characterized by an obvious growth rhythm. The storage proteins In seeds disappeared during seedling growth while VSPs appeared in the stem 2 weeks after seedling leaves matured. Thereafter, the VSPs In the seedling stem almost exhausted during new shoot growth, and when the leaves of new shoot Just matured, both the stem beneath the new shoot of seedlings and the stem of new shoot started to accumulate VSPs. Nitrogen application dramatically Increased the level of VSPs, but had little influence on the dynamics of VSP consumption and accumulation in seedling stem. Together with these data, the fluctuation of VSPs in seedlings was very similar to that in the branches of the adult trees. In addition, seedlings are easy to be treated due to their small size. Our results suggested that S. rnacrophylla seedlings were suitable for Investigating the biological roles of VSPs and the mechanism of nitrogen storage in trees.     

The simultaneous use of 14CO2 and 15N2 labelling techniques to study the carbon and nitrogen economy of legumes grown under natural conditions

A method based on simultaneous short-term exposure to ¹⁴CO₂ and ¹⁵N₂ is described for studying nitrogen fixation and distribution in legumes relative to carbon assimilation and use. Equipment designed to accomodate experiments under natural conditions with very little disturbance of the N₂ fixing association is used. It permits continuous measurement and regulation of variables such as air temperature, humidity and CO₂ concentration as well as soil aeration. Measurements of distribution and use of assimilates, respiration of nodulated roots, quantitative N₂ fixation and the distribution and fate of fixed N as a function of time lead to a precise estimation of C and N budgets for each labelling period. When experiments are done at several phenological stages they give a new insight into the complex C and N interrelations in legume symbiosis.
A series of trials throughout the growth period of Glycine max (L.) Merr. cv. Hodgson demonstrated the sensitivity of the method. The development of the plants from vegetative to reproductive stages was accompanied by a complete change in the distribution patterns of current assimilates and products of nitrogen fixation. Maximum sink strength moved from the leaves to the pods and seeds which ended up receiving 70% of the incoming C and 35% of the fixed N. The fact that up to 85% of fixed N in the plants was in the reproductive organs at maturity can be accounted for by remobilisation from vegetative parts.
The respiration of nodulated roots utilized 33% of carbon translocated to below-ground plant parts before nitrogen fixation started, but as much as 50% during the period of optimal fixation. The advantages and limitations of the isotopic method described are critically discussed as a prelude to future investigations.     

银杏营养贮藏蛋白质的亚细胞定位

在电子显微镜下,对银杏(Ginkgo biloba L.)枝条营养贮藏蛋白质的超微结构特征及在亚细胞水平的定位进行了系统研究.结果表明:银杏营养贮藏蛋白质主要存在于韧皮薄壁细胞的液泡内.银杏韧皮薄壁细胞内的营养贮藏蛋白质在细胞质内合成,由内质网膨大的槽库、质膜内折或高尔基体小泡发育形成贮藏蛋白质的液泡.液泡蛋白质主要以不定形块状、絮状或颗粒状形态存在.贮藏蛋白质在整个越冬期一直保持高含量,直到翌年春季萌芽时,贮藏蛋白质迅速转移再利用.随着新梢的生长,到了夏末秋初,又重新开始积累贮藏蛋白质.     

Morphological pattern of development affects the contribution of nitrogen reserves to regrowth of defoliated white clover (Trifolium repens L.)

The contribution of nitrogen reserves to regrowth following defoliation was studied in white clover plants (Trifolium repens cv. Huia). This was found to be closely linked to the morphological pattern of development of the aerial parts during the same period. Low temperature (6 degrees C) and short day exposure (8 h photoperiod) were used to induce dwarf development, i.e. to increase branching rate and to enhance new sites of leaf production during a period of regrowth. Treated plants exhibited a large reduction in leaf area and a large increase in leaf pool size for the first 10 d of a subsequent regrowth under standard culture conditions (16 h daylight; 22/18 degrees C day/night). The contribution of nitrogen from storage compounds in organs remaining after defoliation (sources) to regrowing tissues (sinks) was assessed by 15N pulse-chase labelling during regrowth following shoot removal. The mobilization of nitrogen reserves from storage tissues of regrowing clover was closely linked to the pattern of differentiation of the newly developed organs. It appeared that regrowth was supported less by endogenous N for the first 10 d after defoliation in treated plants, compared with control plants grown continuously in standard conditions. It is assumed that dwarf plants exhibit a lower dependence upon the mobilization of soluble proteins previously accumulated in roots and uncut stolons. The relationship between leaf development rate and N-uptake recovery following defoliation is discussed.     

Effect of elevated CO2, temperature and drought on photosynthesis of nodulated alfalfa during a cutting regrowth cycle

Rising atmospheric CO₂ may increase potential net leaf photosynthesis under short-term exposure, but this response decreases under long-term exposure because plants acclimate to elevated CO₂ concentrations through a process known as downregulation. One of the main factors that may influence this phenomenon is the balance between sources and sinks in the plant. The usual method of managing a forage legume like alfalfa requires the cutting of shoots and subsequent regrowth, which alters the source/sink ratio and thus photosynthetic behaviour. The aim of this study was to determine the effect of CO₂ (ambient, around 350 vs. 700 µmol mol⁻¹), temperature (ambient vs. ambient + 4° C) and water availability (well-irrigated vs. partially irrigated) on photosynthetic behaviour in nodulated alfalfa before defoliation and after 1 month of regrowth. At the end of vegetative normal growth, plants grown under conditions of elevated CO₂ showed photosynthetic acclimation with lower photosynthetic rates, V_cmax and ribulose-1,5-bisphosphate carboxylase/oxygenase (rubisco) activity. This decay was probably a consequence of a specific rubisco protein reduction and/or inactivation. In contrast, high CO₂ during regrowth did not change net photosynthetic rates or yield differences in V_cmax or rubisco total activity. This absence of photosynthetic acclimation was directly associated with the new source-sink status of the plants during regrowth. After cutting, the higher root/shoot ratio in plants and remaining respiration can function as a strong sink for photosynthates, avoiding leaf sugar accumulation, the negative feed-back control of photosynthesis, and as a consequence, photosynthetic downregulation.     

Seasonal variation in nitrogen storage reserves in the roots of leafy spurge (Euphorbia esula) and responses to decapitation and defoliation

Seasonal fluctuations of carbohydrates and nitrogenous components in the roots of the noxious perennial leafy spurge ( Euphorbia esula L.) are strongly associated with overwintering strategy, Amino acids and distinct soluble proteins accumulate during fall and remain at elevated levels throughout winter. The formation of carbohydrate reserves in roots was not significantly affected by decapitation or selective defoliation; however, maximum amino acid and soluble protein contents were markedly reduced. In particular, the accumulation pattern of a 26 kDa protein was altered. This protein may play a role in plant conditioning and regenerative potential.     

Effects of competitive stress on vegetative growth, storage, and regrowth after defoliation in Phleum pratense

The capacity for resource storage is important to population persistence in nonforaging plants with a consolidation strategy. In a competitive environment, selection may favor genetic variants with a well-developed ability to store resources for future vegetative growth or to regrow following herbivory. To determine the evolutionary potential for changes in vegetative growth, storage and regrowth after defoliation in response to competitive stress, half-sib families of the caespitose grass Phleum pratense were grown in pots in a glasshouse either alone or in competition with four individuals of Lolium perenne . They were defoliated after 16 wk and permitted to regrow for 8 wk. Production of new tillers, leaf area and leaf dry mass were recorded before and after defoliation; resource storage was estimated by the dry mass of stem bases. Tiller numbers, leaf area and mass, regrowth, and stem base mass were significantly reduced by competition; however, there was significant variation among families for many variables. A significant proportion of the variation in clipped and regrowth leaf mass, and in stem base mass, was due to a competition by family interaction. Storage in stem bases was correlated with total accumulated leaf mass in both control and competition groups. Genetic variation for competitive ability and the ability to regrow rapidly after defoliation exist in this P. pratense population. A well-developed ability to regrow following release from competitive or grazing pressures is likely to be a critical adaptation of caespitose grasses.     

Availability of water and inorganic nutrients in the persistent leaf bases of the grasstree Kingia australis, and uptake and translocation of labelled phosphate by the embedded aerial roots

Numerous lateral branches of the concealed aerial roots of the grasstree Kingia australis ramify through the persistent leaf bases, suggesting a role in uptake of water and nutrients localed up to 8 m above ground. These leaf bases were shown to hold up to more than three times their weight in water. Water and nutrients (N, P and K) available in the leaf bases may greatly exceed that in an equal volume of surface soil, especially during the dry summer months. At this time, ³²P injected# among the leaf bases is strongly absorbed by the aerial roots and translocated to the meristematic regions of other laterals beneath the point of application, but mostly to the stern and leaves above it. Scattered vascular bundles in the pith are the major routes for the rapid distribution of "P up and down the trunk following its departure from the aerial roots. Water and nutrients stored in the leaf bases and their subsequent uptake by the associated aerial roots may therefore contribute significantly to this species' tolerance of long summer droughts and extremely impoverished soils.