Soybean Lipoxygenase L-4, a Component of the 94-kilodalton Storage Protein in Vegetative Tissues: Expression and Accumulation in Leaves Induced by Pod Removal and by Methyl Jasmonate

A lipoxygenase L-4 gene was isolated from a soybean genomiclibrary. The amino acid sequence of lipoxygenase L-4 is highlyhomologous with the partial amino acid sequence of the 94-kDavegetative storage protein, vsp94, found in paraveinal mesophyllcells in the leaves of depodded soybean plants. No L-4 expressionwas observed in maturing seeds. The L-4 gene is highly expressedin the vegetative tissues of young seedlings, including cotyledons,hypocotyls, roots and primary leaves. L-4 expression followedthe same pattern as lipoxygenase activity in cotyledons peaking3 to 5 days after germination, and returning to a basal levelby 9 days after germination. L-4 gene expression was low inthe roots, stems and leaves of 10-week-old plants. Exposureof 4-week-old plants to atmospheric methyl jasmonate increasedL-4 mRNA in leaves. Continuous pod removal from 7-week-old plantsover a 2 week period resulted in dramatic accumulation of L-4mRNA in leaves. Accumulation of the L-4 protein and three otherlipoxygenase fractions in the leaves of depodded plants wasdemonstrated by ion exchange chromatography. These results indicatethat lipoxygenase L-4 is a component of vsp94. (Received May 31, 1993; Accepted August 9, 1993)     

Expression and Accumulation Patterns of Nitrogen-Responsive Lipoxygenase in Soybeans

Gene expression and protein accumulation patterns of nitrogen-responsive lipoxygenase (LOX-NR), as a representative vegetative storage protein, were investigated in nonnodulated soybeans (Glycine max [L.] Merr. cv Wye). The form of available nitrogen (supplied as NH4NO3, NH4+, NO3-, or urea) influenced the mRNA level and the amount of LOX protein, indicating that preferential accumulation of LOX may occur. Soybeans were grown with 0, 2, 5, and 16 mM total nitrogen to determine the extent to which LOX accumulation responded to soil nitrogen levels. Analysis of both mRNA and protein levels was conducted in shoot tips, stems, pod walls, and leaves over the entire life cycle of the plant. A general correlation between increasing available nitrogen level and LOX level was seen in the shoot tip and other organs throughout the soybean life cycle. However, appreciable amounts of LOX-NR mRNA and protein accumulated even when plants were grown under conditions of nitrogen deficiency. The results indicate that LOX may play an important role as a temporary storage site for amino acids in the developing shoot tip. The expression patterns of LOX-NR in plants grown under nitrogen deficiency suggest that these proteins, although responsive to nitrogen status, may not function solely as temporary storage pools for amino acids.     

Identification and localization of vegetative STORAGE PROTEINS IN LEGUME LEAVES

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.     

Protein compositions of mesophyll and paraveinal mesophyll of soybean leaves at various developmental stages

Mesophyll and paraveinal mesophyll protoplasts (PVMP) were isolated from leaves of soybean (Glycine max) at various stages of physiological development, and protein compositions of the two protoplast types were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting. Polypeptides of 27, 29 (previously shown to be storage proteins), and 94 kilodaltons were found to be PVMP-specific proteins and were present in both nodulated and nonnodulated plants. The 27 and 94 kilodalton polypeptides were major PVMP constituents. All three polypeptides accumulate as early as one-quarter leaf expansion. Immunoblotting and immunocytochemical studies using antibodies against the 27/29 kilodalton proteins confirmed that they are specific to the paraveinal mesophyll (PVM) and that they are localized in the PVM vacuole. The 27 kilodalton polypeptide increased significantly by two weeks depodding, and this accumulation was restricted to the PVM vacuole. Radiolabeling experiments showed that the difference in relative amounts of the 27 and 29 kilodalton polypeptides was due to a greater rate of synthesis of the 27 kilodalton polypeptide. The 94 kilodalton polypeptide accumulated to a maximum at anthesis, but was absent at 2 weeks postanthesis in both depodded and podded nodulated plants, probably because they were nitrogen limited. In nonnodulated plants, it was present through 2 weeks postanthesis. The results confirm that the 27 and 29 kilodalton proteins of soybean leaf are stored in the PVM vacuole and show that they are accumulated early during leaf development while they are still strong sinks for nitrogen. The 94 kilodalton protein, previously found to accumulate in leaves after depodding, is also a PVM protein and is likely a third vegetative storage protein, although its accumulation appears to be more dependent on excess nitrogen availability. The results further support the hypothesis that the PVM is a specialized leaf tissue that functions in synthesis and compartmentation of storage proteins.     

A jasmonate-responsive element within the A. thaliana vsp1 promoter

The vsp1 gene of Arabidopsis thaliana encodes a storage protein that accumulates in vegetative organs. Transgenic plants expressing a vsp1 promoter-gus (beta-glucuronidase) gene fusion were found to contain high GUS activity when challenged with jasmonate, a volatile plant hormone. The induction of vsp1-gus expression by jasmonate could be measured in tobacco mesophyll protoplasts, after transient expression. A number of deletions were operated in the vsp1 promoter in order to locate its jasmonate-responsive element. A 41 bp sequence taken approximately 150 bp upstream of the vsp1 TATA box could confer jasmonate responsiveness upon a short CaMV 35S promoter. Whereas the deletion of a CAAT box-like element within the 41 bp sequence did not affect promoter activity, mutation of a short palindromic motif completely abolished jasmonate responsiveness. This motif shows no homology with the jasmonate-responsive elements of other promoters.     

木本植物营养贮藏蛋白质研究进展

本文根据最新的国内外研究资料对木本植物营养贮藏蛋白质的分类、定位、生化特性和生理功能等方面进行了全面的综述;着重论述了林木营养贮藏蛋白质的合成、转移、降解机理及基因表达与调控等方面的最新研究进展;对有待进一步研究的领域也进行了分析和讨论。     

木本植物营养贮藏蛋白质研究进展

本文根据最新的国内外研究资料对木本植物营养贮藏蛋白质的分类,定位,生化牧场生和生理功能等方面进行了全面的综述,着重论述了林木营养贮藏蛋白南的合成,转移,降解机理及其因表达与调控等方面的最新研究进展,对有待进一步研究的领域也进行了分析和讨论。     

Local and differential control of vegetative storage protein expression in response to herbivore damage in Arabidopsis thaliana

Vegetative storage proteins (VSPs) are thought to fulfil important nutritional roles during plant development and stress adaptation. Plant responses to mechanical wounding and herbivore damage include an activation of VSP expression. It was recently suggested that vsp is part of the systemic response of Arabidopsis to wounding. To test this proposal, we monitored the spatial regulation of vsp mRNAs and VSP proteins. Arabidopsis contains two vsp genes and real-time quantitative PCR allowed us to characterize their differential expression. The ratio of vsp1 to vsp2 mRNA abundance increased when plants were challenged with diamondback moth larvae or Egyptian cotton worms, but not when they were mechanically wounded. We observed a dramatic increase of vsp1 and vsp2 mRNA as well as VSP protein levels in leaves that experienced herbivore damage. By contrast, there was a relatively minor increase of vsp mRNA and VSP protein levels in undamaged leaves of infested plants. These results clearly demonstrate that VSPs are part of the local plant response to herbivore attack. To obtain additional information on vsp regulation, we analysed a fusion of a soybean vspB promoter fragment to the β-glucuronidase gene in transgenic Arabidopsis plants. The vspB promoter responded to both jasmonate and herbivore treatments, suggesting that similar signals regulate its expression in both plant species.     

Specific lipoxygenase isoforms accumulate in distinct regions of soybean pod walls and mark a unique cell layer

Developing seeds constitute a strong sink for the plant and rely on the turnover and mobilization of carbon and nitrogen assimilates to supply the nutrients needed for their maturation. In large part these nutrients emanate from the vegetative organs including leaves and pod walls. Vegetative lipoxygenases (VLXs) accumulate in the paraveinal mesophyll cell layer of soybean (Glycine max L.) leaves where individual isoforms are proposed to play a role(s) as active enzymes or as transient storage proteins. VLXs also are prominent proteins in soybean pod walls, representing approximately 12% of the total soluble protein. Examining the temporal, tissue, and subcellular patterns of individual VLX isoform accumulation and of lipoxygenase activity through pod wall development indicates that VLXD is the principal VLX isoform playing a role in storage in this organ. The major accumulation of VLXD occurs just prior to seed fill within the endocarp middle zone, and protein extracted from this region shows relatively low levels of lipoxygenase activity, suggesting the middle zone may act as a storage tissue. Three other VLX isoforms, VLXA, VLXB, and VLXC colocalize to the cytoplasm of a single discrete cell layer in the mesocarp. Thus, the patterns of VLX cellular and subcellular localization in pod walls suggest independent functions for these different isoforms while also serving as specific markers for a novel cell layer in the pod wall.     

杨树新梢积累营养贮藏蛋白质的细胞学研究

采用光学显微镜和电子显微镜技术,对杨树新梢中的营养贮藏蛋白质进行了细胞学鉴定。在用戊二醛固定的标本中,营养贮藏蛋白质呈颗粒状,积累在中央大液泡里。在新梢伸长生长时期,新梢茎的基部已积累了营养贮藏蛋白质,在伸长生长刚停止,中上部的叶片近成熟时,整个新梢的茎都有营养贮藏蛋白质的积累,其中,以新梢基部的茎最为丰富。营养贮藏蛋白质优先在次生韧皮部的韧皮薄壁细胞和韧皮射线薄壁细胞中积累,在新梢伸长生长停止后,新梢基部茎的木质部中也积累了相当数量的营养贮藏蛋白质,主要分布在初生木质部和内侧次生木质部的各种生活的薄壁细胞中。新梢较早地积累营养贮藏蛋白质是热带树木和温带树木的一个共同特点,对于树木的氮代谢和树木当年的生长发育可能具有重要的调控作用。     

Wheat vegetative nitrogen compositional changes in response to reduced reproductive sink strength

N redistribution patterns and the N composition of vegetative tissues above the peduncle node of wheat (Triticum aestivum L.) plants with altered reproductive sink strength were evaluated to determine the role of vegetative storage proteins in the temporary storage of excess N destined for export. The degree of leaf senescence symptoms (loss of chlorophyll, total N, and ribulose-1,5-bisphosphate carboxylase/oxygenase) were initially reduced, but the complete senescence of vegetative tissues proceeded even for plants completely lacking reproductive sinks. Plants with 50% less sink strength than control plants with intact spikes redistributed vegetative N to the spike almost as effectively as the control plants. Plants without reproductive sinks exported less N from the flag leaf and had flag leaf blades and peduncle tissues with higher soluble protein and α-NH₂ amino acid levels than control plants. An abundant accumulation of polypeptides in the soluble protein profiles of vegetative tissues was not evident in plants with reduced sink strength. Storage of amino acids apparently accommodates any excess N accumulated by vegetative tissues during tissue reproductive growth. Any significant role of vegetative storage proteins in the N economy of wheat is unlikely.     

Soybean vegetative storage protein structure and gene expression

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.     

Methyl jasmonate treatment eliminates cell-specific expression of vegetative storage protein genes in soybean leaves

Soybean (Glycine max) plants accumulate a vacuolar glycoprotein in the parenchymal cells of leaves, petioles, stems, seed pods, and germinating cotyledons that acts in temporary nitrogen storage during vegetative growth. In situ immunolocalization of this vegetative storage protein (VSP) revealed that it accumulates in those parenchymal cells in close proximity to existing and developing vasculature, as well as in epidermal and cortical cells. The protein was more prevalent in younger, nitrogen-importing tissues before pod and seed development. Removal of actively growing seed pods greatly enhanced VSP accumulation, primarily in bundle sheath and paraveinal mesophyll cells. In situ hybridization of a VSP RNA probe to mRNA in leaf sections demonstrated that cell-specific mRNA accumulation corresponded with the pattern of protein localization. Treatment of leaf explants with 50 micromolar methyl jasmonate resulted in accumulation of VSP mRNA and protein in all cell types.     

Preferential Loss of an Abundant Storage Protein from Soybean Pods during Seed Development

A temporary vegetative storage protein, composed of similar 25 kilodalton and 27 kilodalton subunits, was found to be abundant in soybean (Glycine max (L.) Herr. var Hobbit) leaves, stems, pods, flower petals, germinated cotyledons, and less abundant in roots, nodules and seeds. Total pod protein was highest at 3 weeks after flowering and declined by 37% within 3 weeks during seed development. During this time the vegetative storage protein declined from 18% to 1.5% of the total pod protein and accounted for 45% of the protein lost from pods. This indicates that the vegetative storage protein makes a significant contribution to the pool of nutrients mobilized from pods for transport to developing seeds.     

Rubiscolytics: fate of Rubisco after its enzymatic function in a cell is terminated

Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) is the predominant protein in photosynthesizing plant parts and the most abundant protein on earth. Amino acids deriving from its net degradation during senescence are transported to sinks (e.g. developing leaves, fruits). Rubisco catabolism is not controlled only by the overall sink demand. An accumulation of carbohydrates may also accelerate senescence and Rubisco degradation under certain conditions. Amino acids produced by proteolysis are rapidly redistributed in plants with proper source-sink relationships. In leaves of wheat plants with reduced sink capacity (e.g. sink removal, phloem interruption by steam girdling at the leaf base), Rubisco is degraded and free amino acids accumulate. They may be washed out in the rain during late senescence. In leaves of depodded soybeans, Rubisco is degraded and amino acids can be reutilized in these leaves for the synthesis of special vacuolar proteins in the paraveinal mesophyll (vegetative storage proteins). Nitrogen deriving from Rubisco degradation in older (senescing) leaves of annual crops is integrated to some extent again in newly synthesized Rubisco in younger leaves or photosynthesizing tissues of fruits. Finally, a high percentage of this nitrogen is accumulated in protein bodies (storage proteins). At the subcellular level, Rubisco can be degraded in intact chloroplasts. Reactive oxygen species may directly cleave the large subunit or modify it to become more susceptible to proteolysis. A metalloendopeptidase may play an important role in Rubisco degradation within intact chloroplasts. Additionally, the involvement of vacuolar endopeptidase(s) in Rubisco catabolism (at least under certain conditions) was postulated by various laboratories.     

Developmental changes in abundance of the VSPβ protein following nuclear transformation of maize with the Soybean <Emphasis Type="Italic">vspβ</Emphasis> cDNA

Background  

Developing monocots that accumulate more vegetative tissue protein is one strategy for improving nitrogen-sequestration and nutritive value of forage and silage crops. In soybeans (a dicotyledonous legume), the vspA and B genes encode subunits of a dimeric vegetative storage protein that plays an important role in nitrogen storage in vegetative tissues. Similar genes are found in monocots; however, they do not accumulate in leaves as storage proteins, and the ability of monocot leaves to support accumulation of an ectopically expressed soybean VSP is in question. To test this, transgenic maize (Zea Mays L. Hi-II hybrid) lines were created expressing soybean vspB from a maize ubiquitin Ubi-1 promoter.