Vegetative storage protein in Litchi chinensis, a subtropical evergreen fruit tree, possesses trypsin inhibitor activity

BACKGROUND AND AIMS: Vegetative storage proteins (VSPs) are commonly bioactive in herbaceous plants but few VSPs with bioactivity have been identified in trees. In addition, information on the characterization of VSPs in evergreen trees is limited. The objective of this study was to characterize the VSPs with bioactivity in evergreen trees. Methods The VSP in lychee (Litchi chinensis), an evergreen fruit tree, was characterized by a combination of cytological, biochemical and molecular biological techniques. KEY RESULTS: The VSP in lychee was a 22-kDa protein. It accumulated in the large central vacuoles of protein-storing cells (PSCs) in two distinguishable forms, granular and floccular. The PSCs were of a novel type. The 22-kDa protein is distributed in mature leaves, bark tissues of branches, trunk and large roots, paralleling the distribution of PSCs. Its homologues were present in mature seed. During young shoot development and fruiting, the 22-kDa protein decreased apparently, suggesting a nitrogen-storage function. The 22-kDa protein had several isoforms encoded by a small multigene family. One gene member, LcVSP1, was cloned. The LcVSP1 had no intron and contained a 675 bp open reading frame encoding a putative protein of 225 amino acids. LcVSP1 was homologous to Kunitz trypsin inhibitors. The 22-kDa protein inhibited trypsin and chymotrypsin, but had no inhibitory effect on subtilisin. CONCLUSIONS: Lychee is rich in a 22-kDa VSP with trypsin inhibitor activity. The VSP plays an important role in nitrogen storage while its possible defensive function remains to be elucidated.     

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.     

Gene expression profiling in the stem of young maritime pine trees: detection of ammonium stress-responsive genes in the apex

The shoots of young conifer trees represent an interesting model to study the development and growth of conifers from meristematic cells in the shoot apex to differentiated tissues at the shoot base. In this work, microarray analysis was used to monitor contrasting patterns of gene expression between the apex and the base of maritime pine shoots. A group of differentially expressed genes were selected and validated by examining their relative expression levels in different sections along the stem, from the top to the bottom. After validation of the microarray data, additional gene expression analyses were also performed in the shoots of young maritime pine trees exposed to different levels of ammonium nutrition. Our results show that the apex of maritime pine trees is extremely sensitive to conditions of ammonium excess or deficiency, as revealed by the observed changes in the expression of stress-responsive genes. This new knowledge may be used to precocious detection of early symptoms of nitrogen nutritional stresses, thereby increasing survival and growth rates of young trees in managed forests.     

生长素介导OsRGP1和0sSuS参与水稻地上部基部向重性弯曲生长

在水稻幼苗被放平后,地上部重力响应部位（地上部基部）的上半部生长比下半部生长慢,从而导致地上部基部发生向上弯曲．发生不对称生长的向重性反应。通过抑制差减杂交（SSH）实验发现．水稻地上部基部向重性弯曲生长过程中,反复糖基化多肽OsRGP1和蔗糖合成酶OsSuS在基部上下部有不对称表达。我们还采用Realtime PCR开展进一步研究。实验结果显示OsRGP1和0sSMS的表达受到IAA的调控。通过生物信息学分析也发现OsRGPl和OsSuS的启动子上存在着生长素的调控元件．生长素极性运输抑制剂TIBA处理能够消除OsRGP1和OsSuS在向重性过程中的不对称表达．这进一步说明向重性过程中不对称分布的生长素介导了OsRGP1和OsSuS基因的不对称表达。另外．向重性反应过程中,检测到基部下半部的己糖浓度增加。因此,OsRGP1的不对称表达可能有助于下半部细胞壁多糖的积累,进而促进了下半部细胞壁的扩张。OsSuS不对称表达也有可能会引起己糖在上下半部的不对称分布。己糖和细胞壁多糖在下半部的积累可能在下部细胞扩张从而导致向重性弯曲生长中起一定的作用。     

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.     

Periods of organogenesis in mono- and bicyclic annual shoots of Juglans regia L. (Juglandaceae)

The organogenetic cycle of shoots on main branches of 4-year-old Juglans regia trees was studied. Mono- and bicyclic floriferous and vegetative annual shoots were analysed. Five parent annual shoot types were sampled between October 1992 and August 1993. Organogenesis of summer growth units was monitored between 16 Jun. and 3 Aug. 1993. Variations over time in the number of nodes, cataphylls and embryonic green leaves of terminal buds were studied. The number of nodes of parent shoot buds was compared with the number of nodes of shoots derived from parent shoot buds. The spring growth units of mono- and bicyclic shoots consist exclusively of preformed leaves which were differentiated, respectively, during the spring flush of growth (mid-April until mid-May) or the summer flush of growth (mid-June until early August) in the previous growing season. Thus, winter buds may consist of flower and leaf primordia differentiated in two different periods during annual shoot extension. The summer growth units of bicyclic shoots consist of preformed leaves that were differentiated in spring buds during the spring flush of growth in the current growing season. Bud morphology is compared between spring and summer shoots.     

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.     

Relationship between reproductive behavior and new shoot development in 5-year-old branches of olive trees (Olea europaea L.)

The development of new shoots plays a central role in the complex interactions determining vegetative and reproductive growth in woody plants. To explore this role we evaluated the new shoots in the olive tree, Olea europaea L., and the effect of fruiting on new shoot growth and subsequent flowering. Five-year-old branches served as canopy subunits in order to obtain a global, whole-tree view of new shoot number, size and morphological origin. The non-bearing trees had many more shoots than the fruit-bearing trees, and a greater number of longer shoots. In both bearing conditions, however, the majority of shoots were less than 4 cm long, with shoots of progressively longer lengths present in successively decreasing frequencies. Six major shoot types were defined on the basis of apical or lateral bud origin and of parent shoot age. On fruit-bearing trees, the new shoots originated predominantly from the shoot apex, while on non-fruiting trees, they formed mainly from axillary buds, but in both cases, they tended to develop on younger parent shoots. The previous bearing condition of the tree was the main determinant for subsequent inflorescence development, which was independent of both shoot type and length. Thus, reproductive behavior strongly affected both the amount and type of new branching, but subsequent flowering level was more influenced by previous bearing than by the potential flowering sites on new shoots.     

Size structure of current-year shoots in mature crowns

Characteristics of current-year shoot populations were examined for three mature trees of each of three deciduous broad-leaved species. For first-order branches (branches emerging from the vertical trunk) of the trees examined, lengths or diameters of all current-year shoots were measured. Total leaf mass and total current-year stem mass of first-order branches were estimated using an allometric relationship between leaf or stem mass and length or diameter of current-year stems. For each tree, the number of current-year shoots on a first-order branch was proportional to the basal stem cross-sectional area of the branch. On the other hand, first-order branches had shoot populations with size structures similar to each other. As a result, the leaf mass of a first-order branch was proportional to the basal stem cross-sectional area of the branch, being compatible with the pipe-model relationship. All current-year shoot populations had positively skewed size structures. Because small shoots have a larger ratio of leaf mass to stem mass than large shoots, first-order branches had an extremely large ratio of leaf mass to current-year stem mass. This biased mass allocation will reduce costs for current stem production, respiration and future radial growth, and is beneficial to mature trees with a huge accumulation of non- photosynthetic organs. The allometric relationships between leaf mass and basal stem diameter and that between leaf mass and current-year stem mass of first-order branches were each similar across the trees examined. Characteristics of shoot populations tended to offset inter-species diversity of shoot allometry so that branch allometry shows inter-species convergence.     

Vegetative storage protein with trypsin inhibitor activity occurs in Sapindus mukorassi, a sapindaceae deciduous tree

A vegetative storage protein (VSP) with trypsin inhibitor activity in a deciduous tree,Sapindus mukorassi,was characterized by means of sodium dodecyl sulfate-polyacrylamide gel electrophoresis,Western-blot,immuno-histochemical localization,light- and electro-microscopy,together with analysis of proteinase inhibitor activity of the purified VSP in vitro.There were two proteins with molecular masses of about 23 and 27 kDa in a relatively high content in the bark tissues of terminal branches of S.mukorassi in leafless periods.The proteins decreased markedly during young shoot development,indicating their role in seasonal nitrogen storage.Immuno-histochemical localization with the polyclonal antibodies raised against the 23 kDa protein demonstrated that the 23 kDa protein was the major component of protein inclusions in protein-storing cells.The protein inclusions were identified by protein-specific staining and should correspond to the electron-dense materials in different forms in the vacuoles of phloem parenchyma cells and phloem ray parenchyma cells under an electron microscope.So,the 23 kDa protein was a typical VSP in S.mukorassi.The 23 and 27 kDa proteins shared no immuno-relatedness,whereas the 23 kDa protein was immuno-related with the 22 kDa VSP in lychee and possessed trypsin inhibitor activity.The 23 kDa protein may confer dual functions:nitrogen storage and defense.     

结果初期黄冠梨对春施15N-尿素的吸收、分配与利用

以3年生黄冠梨为材料,探讨了早春施用¹⁵N尿素后,树体在萌芽期-新梢缓慢生长期和新梢缓慢生长期-果实成熟期对氮素的吸收、分配与利用特性。结果表明: 梨树在萌芽期-新梢缓慢生长期主要以新梢和叶片等营养器官生长为核心;在新梢缓慢生长期-果实成熟期则以根系等贮藏器官生长为主,果实产量品质形成为辅,且树体尤其是贮藏器官的生物量成倍增加。由于各器官尤其是新梢和叶片生长旺盛、新梢缓慢生长期吸收的标记氮量相对较多,各器官吸收的肥料氮(Ndff)值相对较高;果实成熟期除粗根外各器官的Ndff值均低于新梢缓慢生长期。萌芽期到新梢缓慢生长期吸收的标记氮主要分配在新梢和叶片营养器官中,新梢缓慢生长期到果实成熟期吸收的标记氮则主要分配在贮藏器官中;整个生育期间,植株吸收的标记氮在贮藏器官中分配率最高,营养器官次之,生殖器官中分配率最低。3年生梨树从萌芽期-新梢缓慢生长期、新梢缓慢生长期-果实成熟期吸收的肥料氮分别占当年总吸氮量的31.1%和21.0%,而两个时期内吸收的土壤氮占比分别达68.9%和79.0%。     

Within-tree and among-tree variation in growth of Epirrita autumnata on mountain birch leaves

Abstract.

• 1 We studied among-tree and within-tree variation in the growth of larvae of a geometrid, Epirrita autumnata, on mountain birch (Betula pubescens ssp. tortuosa) leaves at different hierarchical levels: among trees (genets), among ramets within trees, among branches within ramets, among shoots within branches and among leaves within shoots. We used only short shoot leaves, which burst simultaneously in spring.
• 2 Trees accounted for most of the variation in larval growth rate, but there was substantial variation also among ramets within trees, among branches within ramets, and among short shoots within branches. Variation among leaves within short shoots was negligible. When the probabilities from different experiments were combined, the differences were statistically significant at the tree, ramet and branch levels, and approached significance at the shoot level.
• 3 In different experiments, larval growth was from 9% to 54% lower on the worst tree than on the best tree of the experiment. On average, larval growth rate was in different experiments from 11% to 32% lower on the worst ramet than on the best ramet within tree, from 8% to 18% lower on the worst than on the best branch within ramet, and from 12% to 30% lower on the worst than on the best shoot within branch.
• 4 The amount of among-and within-tree variation shown by our results may have ecological and evolutionary implications: among-tree variation should select for discrimination by ovipositing females and dispersing larvae, within-tree variation should select also for optimal foraging behaviour of larvae.

     

Aspen shoots are carbon autonomous during bud break

Current thinking holds that carbon autonomy of branches in trees is unlikely, particularly during bud break, when the new developing shoots require significant influx of carbon resources from more distant sources. Results from recent studies indicate that the impact of bud break on overall tree reserves might be small. In two studies the independence of flushing shoots from stored carbon reserves and the photosynthesis in developing new leaves and shoots of Populus tremuloides were explored. New developing shoots quickly became a positive carbon source and only a few days into flush, the photosynthetic system of the newly developing shoots was efficient enough to achieve positive carbon gain even at low light levels. Only 14% of the stored shoot reserves, without any mobilization from more distant reserves, were used during bud break and early shoot expansion. Without any underlying stress, shoots of deciduous trees appear to be carbon autonomous during bud break when demand on stored carbon should be the highest. The development of an efficient photosynthetic system in new shoots is critical in the recovery of carbon reserves in aspen. It minimizes the cost of bud break to the overall stored carbon reserves by optimizing the assimilation of carbon in the newly developed leaves, while eliminating the cost for mobilizing carbon reserves from more distant sources. This carbon autonomy of shoots has important implications for the whole tree carbon balance particularly to the non-photosynthetic tissues which functions solely depending on carbon export from the newly developing leaves and shoots.     

Putative zeatin O‐glucosyltransferase OscZOG1 regulates root and shoot development and formation of agronomic traits in rice

As a ubiquitous reaction, glucosylation controls the bioactivity of cytokinins in plant growth and development.Here we show that genetic manipulation of zeatin-Oglucosylation regulates the formation of important agronomic traits in rice by manipulating the expression of OscZOG1 gene,encoding a putative zeatin O-glucosyltransferase. We found that OscZOG1 was preferentially expressed in shoot and root meristematic tissues and nascent organs. The growth of lateral roots was stimulated in the overexpression lines, but inhibited in RNA interference lines. In shoots, knockdown of OscZOG1 expression by RNA interference significantly improved tillering, panicle branching, grain number per panicle and seed size, which are important agronomic traits for grain yield. In contrast, constitutive expression of OscZOG1 leads to negative effects on the formation of the grain-yielding traits with a marked increase in the accumulation levels of cis-zeatin O-glucoside(c ZOG) in the transgenic rice plants. In this study,our findings demonstrate the feasibility of improving the critical yield-determinant agronomic traits, including tiller number, panicle branches, total grain number per panicle and grain weight by downregulating the expression level of OscZOG1. Our results suggest that modulating the levels of cytokinin glucosylation can function as a fine-tuning switch in regulating the formation of agronomic traits in rice.     

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.     

Trypanosoma cruzi: a gene family encoding chitin-binding-like proteins is posttranscriptionally regulated during metacyclogenesis.

The development of the representation of differential expression method has lead to the cloning of Trypanosoma cruzi stage-specific genes. We used this method to characterize a multicopy gene family differentially expressed during metacyclogenesis. The genomic and cDNA clones sequenced encoded three short cysteine-rich polypeptides, of two types, with predicted molecular masses of 7.1, 10.4, and 10.8 kDa. We searched GenBank for similar sequences and found that the sequences of these clones were similar to that encoding the wheat germ agglutinin protein. The region of similarity corresponds to the chitin-binding domain, with eight similarly positioned half-cysteines and conserved aromatic residues involved in chitin recognition. Multiple copies of the genes of this family are present on a high- molecular-mass chromosome. We studied the expression of genes of this family during metacyclogenesis by determining messenger RNA (mRNA) levels. The mRNAs for the members of this gene family were present in the total RNA fraction but were mobilized to the polysomal fraction of adhered (differentiating) epimastigotes during metacyclogenesis, with a peak of accumulation at 24 of differentiation. Polyclonal antisera were raised against a recombinant protein and a synthetic peptide. The specific sera obtained detected 7- and 11-kDa proteins in T. cruzi total protein extracts. The 11-kDa protein was present in similar amounts in the various cell populations, whereas the 7-kDa protein displayed differential synthesis during metacyclogenesis, with maximal levels in 24-h-adhered (differentiating) epimastigotes.     

Architectural analysis of 1-year-old apple seedlings according to main shoot growth and sylleptic branching characteristics

Main shoot and sylleptic shoot growth characteristics were measured during and after the first year of growth of 255 Telamon x Braeburn apple seedlings. Although mean main shoot growth characteristics between branched and non-branched trees were significantly different, many non-branched trees expressed similar main shoot growth to branched trees. The variables describing length, number and position of the sylleptic shoots were used to classify branched trees into architecturally different groups. A continuum from trees with few and short shoots to trees with many long shoots is observed. The release of axillary buds from apical dominance is not under complete control by the apical meristem. Genetic seedling difference at the level of roots presumably plays an important role in sylleptic branching. Genetic variation in terms of number, position, and subsequent elongation of sylleptic shoots is clearly observed.