Mechanism of transport of vegetative storage proteins to the vacuole of the paraveinal mesophyll of soybean leaf

Summary Microscopy techniques were used to identify the pathway of transport of soybean leaf vegetative storage proteins (VSP/ and VSP94) to the vacuoles of a specialized cell type, the paraveinal mesophyll (PVM), where they accumulate. PVM cells are enriched in endoplasmic reticulum and Golgi bodies relative to surrounding mesophyll cells. The margins of medial and trans Golgi cisternae had attached or closely associated noncoated vesicles with densely staining membranes and lumenal contents of the same appearance as material that accumulated in the vacuole. These vesicles appeared to be transported preferentially to the tonoplast, where fusion with the membrane released the granular contents into the vacuole. Cytochemical staining with phosphotungstic acid and silver methenamine supported this interpretation as both the Golgi vesicles and the tonoplast stained intensely with these reagents, unlike the tonoplast of mesophyll cells which do not accumulate VSP. Immunocytochemical localization for VSP/ labeled the Golgi bodies and associated vesicles, and vacuolar material in PVM cells, but not in mesophyll. Similar labeling was seen in PVM of another legume species previously found to accumulate antigenically similar VSPs. Immunolocalization for VSP94, a lipoxygenase, labeled the PVM cytosol and material in the PVM vacuole, but not the Golgi or vesicles. The results of this study demonstrate that the Golgi pathway is utilized for transport of VSP/ in the PVM, which follows the mechanism of deposition demonstrated for certain seed storage proteins. VSP94 appeared to follow a separate path for accumulation in PVM vacuoles.Abbreviations LOX lipoxygenase - PVM paraveinal mesophyll - RER rough endoplasmic reticulum - TEM transmission electron     

The paraveinal mesophyll of soybean leaves in relation to assimilate transfer and compartmentation

Nitrogen and carbohydrate assimilates were temporally and spatially compartmented among various cell types in soybean (Glycine max L., Merr.) leaves during seed filling. The paraveinal mesophyll (PVM), a unique cell layer found in soybean, was demonstrated to function in the synthesis, compartmentation and remobilization of nitrogen reserves prior to and during the seed-filling stages. At anthesis, the PVM vacuoles contain substantial protein which completely disappears by two weeks into the seed filling. Distinct changes in the PVM cytoplasm, tonoplast and organelles were correlated with the presence or absence of the vacuolar material. Microautoradiography following the accumulation of several radiolabeled sugars and amino acids demonstrated the glycoprotein nature of the vacuolar material. Incorporation of methionine, leucine, glucose, and glucosamine resulted in heavy labelling of the PVM vacuole, in contrast to galactose, proline, and mannose which resulted in a much reduced labelling pattern. In addition, starch is unequally compartmented and degraded among the various leaf cells during seed filling. At the end of the photoperiod at the flowering stage, the highest starch accumulation was in the second palisade layer followed by the spongy mesophyll and the first (uppermost) palisade layer. Starch in the first palisade layer was completely degraded during the dark whereas the starch in the second palisade and spongy mesophyll was not remobilized to any appreciable extent. By mid-podfilling (approximately five weeks postanthesis) starch was absent in the first palisade layer at the end of the photoperiod while the second palisade and spongy mesophyll layers contained substantial starch. Starch was remobilized from these latter cells during the remainder of seed filling when current photosynthetic production is low. Structural changes associated with cell senescence first appear in the upper palisade layer and then progress (excluding the PVM) to the second palisade and spongy mesophyll layer. The PVM and phloem appear to retain their structural integrity into the leaf yellowing stage. Reducing sink capacity by pod removal resulted in a continued accumulation of vacuolar protein, an increase in cytoplasmic volume, and fragmentation of the vacuole in the PVM. Pod removal also resulted in an increased amount of accumulated starch (which did not turn over) in all mesophyll layers, and an increase in cell size and cell-wall thickness.     

High-Pressure Effects on Vacuolar H+-ATPase from Etiolated Mung Bean Seedlings

A high-hydrostatic-pressure technique was employed to study the structure-function relationship of plant vacuolar H⁺-ATPase from etiolated mung bean seedlings (Vigna radiata L.). When isolated vacuolar H⁺-ATPase was subjected to hydrostatic pressure, the activity of ATP hydrolysis was markedly inhibited in a time-, protein concentration- and pressure-dependent manner. The pressure treatment decreased both V _max and K _m of solubilized vacuolar H⁺-ATPase, implying an increase in ATP binding affinity, but a decrease in the ATP hydrolysis activity. Physiological substrate, Mg²⁺-ATP, augmented the loss of enzymatic activity upon pressure treatment. However, ADP, AMP, and Pi exerted substantial protective effects against pressurization. Steady-state ATP hydrolysis was more sensitive to pressurization than single-site ATPase activity. The inactivation of solubilized vacuolar H⁺-ATPase by pressure may result from changes in protein–protein interaction. The conformational change of solubilized vacuolar H⁺-ATPase induced by hydrostatic pressure was further determined by spectroscopic techniques. The inhibition of vacuolar H⁺-ATPase under pressurization involved at least two steps. Taken together, our work indicates that subunit–subunit interaction is crucial for the integrity and the function of plant vacuolar H⁺-ATPase. It is also suggested that the assembly of the vacuolar H⁺-ATPase complex is probably not random, but follows a sequestered pathway.     

Ultrastructural features of chilling injury: injured cells and the early events during chilling of suspension-cultured mung bean cells

The ultrastructure of cells of mung bean (Vigna radiata L. var. Wilczek) in suspension culture was studied during chilling. During such treatment, three kinds of injured cells were observed: swollen cells, cells with broken vacuolar membranes, and cells with shrunken plasma membranes. Swelling was observed from the early stages of chilling, and in most cells during chilling. The other two types of cells were observed at the late stages of chilling. At the early stage of chilling, whorls of rough endoplasmic reticulum that surrounded clear regions of cytoplasm were observed. At the same time, markedly rough vacuolar membranes, plastids and mitochondria with vacuoles, enlargement of Golgi vesicles, and dilation of the ER were seen. These changes preceded the swelling of cells. These ultrastructural features of chilling injury are discussed in terms of biochemical observations. The disruption of the vacuolar membrane and the shrinking of the plasma membrane are discussed in terms of destruction of the cytoskeleton.     

Associative effect of inoculation of different strains of Azotobacter and homologous rhizobium on the yield of moong (Vigna radiata), soybean (Glycine max) and pea (Pisum sativum)

Summary It has been observed that in the case ofVigna radiata andGlycine max incorporation of suitable strain of Azotobacter gave higher yield than obtained by the use of Rhizobium as inoculant. In the case ofVigna radiata even a strain of Azotobacter isolated from the rhizosphere of berseem gave similar yields as Rhizobium. In the case ofPisum sativum association of Rhizobium with a strain ofAzotobacter chroococcum isolated from the rhizosphere of pea gave numerically higher yield than Rhizobium alone. It may be possible that statistically higher yield may be obtained when a suitable strain of Azotobacter is used after screening a large number of strains of Azotobacter from the rhizosphere of pea.     

Idioblasts and other unusual internal foliar secretory structures in Scrophulariaceae

 Leaf samples of mostly herbarium specimens (237 species of 172 genera) were cleared. Internal secretory structures of large size or unusual shape were detected and observed. Selected samples were processed into resin and sectioned for light microscopy or prepared for scanning electron microscopy. Adding results from two earlier publications, our survey includes 365 species from 174 genera. Five types of internal secretory structure, mostly unknown previously in the traditional Scrophulariaceae, occur in only nine genera: 1) single-celled subepidermal idioblasts, empty at maturity, in Scrophularia and Verbascum (Lersten and Curtis 1997), 2) 2–16 (or more)-celled nodules, with fibrous contents, in mid-mesophyll strata of Graderia and Radamaea, 3) epithelium-lined oil cavities in Leucophyllum (Lersten and Beaman 1998) and Capraria, 4) Kranz-type enlarged bundle sheath in Anticharis, and 5) paraveinal mesophyll (PVM) in Picria and Bonnaya. Received April 24, 2000 Accepted October 27, 2000     

Response to Salinity in Legume Species: An Insight on the Effects of Salt Stress during Seed Germination and Seedling Growth

The process of soil salinization and the preponderance of saline water sources all over the world represent one of the most harmful abiotic stress to plant growth. This pointed to the importance of obtaining plants which are tolerant or resistant to salt, considering that projection of climate change for the coming years indicate an increase in temperature and rain scarcity. In the current study, the effect of NaCl was investigated on germinating seeds of Lathyrus sativus L., Vicia sativa L., Vigna radiata L. R.Wilczek and Vigna unguiculata L. Walp., by combining physiological, biochemical, biostatistical and ultrastructural analyses. Our results revealed that germination was not influenced by high NaCl concentrations, while seedling growth was affected even at low NaCl concentrations, probably due to an alteration in water uptake and in organic matter biosynthesis. Nevertheless, the synthesis of antioxidant enzymes, phenolic acids and flavonoids was registered in all species, which tended to cope with the increasing salt stress, allowing a response mechanism such as cytoplasm detoxification and cellular turgor maintenance. Besides, the ultrastructural analysis evidenced plasmolyzed cells close to cells with a normal ultrastructure with no appreciable differences among the species. This research deeply investigates the mechanism of salt-stress response focusing on species never tested before for their possible tolerance to salinity.     

The Fine Structure of some Dry Seed Tissues Observed after Completely Anhydrous Chemical Fixation

Completely anhydrous fixation with acrolein vapour or osmiumtetroxide vapour was applied to tissues of air-dry seeds: thecoleoptile of wheat (Trilicum aestivum), and plumule and radicleof mung bean (Vigna radiata). Great shrinkage of cells and organelleswas noted, but all the usual organelles could be identified,except for Golgi bodies and (in most cases) ribosomes. The endoplasmicreticulum was very abundant and endoplasmic reticulum tubuleswere closely associated with the storage organelles, namelylipid bodies in the wheat coleoptile, and protein bodies inthe mung bean embryo axis. Aqueous fixation resulted in considerabledistortion of cellular structure. Triticum aestivum L., wheat, Vigna radiata L., mung bean, seed, fine structure, anhydrous fixation     

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.     

Distribution of low molecular weight carbohydrates in the subgenusCeratotropis of the genusVigna (Leguminosae)

The seeds of 9 members of the subgenusCeratotropis (Piper) Verdc., namelyVigna aconitifolia (Jacq.) Maréchal,V. angularis (Willd.) Ohwi et Ohashi,V. minima (Roxb.) Ohwi et Ohashi,V. nakashimae (Ohwi) Ohwi et Ohashi,V. reflexo-pilosa Hayata,V. umbellata (Thumb.) Ohwi et Ohashi,V. mungo (L.) Hepper,V. radiata (L.) Wilczek andV. sp., have been examined. On their low molecular weight carbohydrate compositions, this subgenus has been divided into 2 subgroups, mungo-radiata group and angularis group. Four other species referred to the subgeneraPlectotropis (Schumach.) Bak.,Lasiospron (Benth. emend Piper) Maréchal, Mascherpa et Stainier andVigna, V. vexillata (L.) A. Rich.,V. lasiocarpa (Benth.) Verdc.,V. marina (Burm.) Merr. andV. unguiculata (L.) Walp., were also analyzed and they had distinctive carbohydrate compositions. 1d-4-O-methyl-myo-inositol and 1d-5-O-(α-d-galactopyranosyl)-4-O-methyl-myo-inositol were detected in all species examined exceptV. vexillata, V. mungo andV. radiata.     

Nodulation and nitrogen fixation by fast- and slow-growing rhizobia strains of soybean on several temperate and tropical legumes

Three slow-growingBradyrhizobium japonicum (G₃, USDA-110 and KUL-150) of diverse origins and two fast-growing strains ofRhizobium fredii (USDA-192 and USDA-193) were tested with a cropped soybean (Glycine max L. Merrill) cultivar, two cowpeas (Vigna unguiculata), one mung-bean (Phaseolus radiata), one winged-bean (Psophocarpus tetragonolobus) and one field bean (Phaseolus vulgaris) varieties.TheR. fredii strains nodulated and fixed Nitrogen as effectively as the strains ofB. japonicum in a modern european soybean cultivar, namely Fiskeby V. The other western bred soybeans tested were not nodulated by theseR. fredii strains. All of the soybean rhizobia produced nodules in both cowpeas and in mung-bean; theR. fredii strains showed effective N₂-fixation in the cowpeas, particularly USDA-193, yielding shoot dry weights greater than those from theB. japonicum. The symbiotic performance of theR. fredii strains with soybean and other legumes indicated that they should be placed in an intermediate group between the slow-growingB. japonicum and cowpearhizobium sp.The hydrogen uptake activites suggested a possible host effect on the expression of such genes in one out of theB. japonicum strains tested. Furthermore, the slow-growing rhizobia showed significantly higher nitrate-reduction than theR. fredii in the nodules.     

Chromosome Banding Patterns in Some Indian Pulses

By using the Giemsa C-banding technique, chromosome bandingpatterns on the somatic chromosomes of eight important pulsecrops, pea, lentil, guar (cluster bean), chick pea, pigeon pea,mung bean (green gram), urd (black gram) and cowpea have beenstudied. Each species has a characteristic C-banding pattern.The significance of such banding patterns which correlate withthe position of pachytene knobs, in chromosome identification,and in assigning relationships at the cytological level in thepulses of genus Vigna is stressed. Chromosome banding, Giemsa C-banding, pulse crops, Pisum sativum L., garden pea, Lens culinaris Medik, lentil, Cyamopsis tetragonoloba (L.) Taub., guar, Cicer arietinum L., chick pea, gram, Cajanus cajan (L.) Millsp., pigeon pea, Vigna radiata (L.) Wilczek, mung bean, Vigna mungo (L.) Hepper, urd, Vigna unguiculata (L.) Walp, cowpea     

Effect of Ethanol, Methanol and Acetone on Rooting Etiolated Cuttings of Vigna radiata in Presence of Sucrose and Auxin

Root formation on etiolated hypocotyl cuttings of Vigna radiataL. is greatly enhanced in the presence of ethanol, methanoland acctone. Low concentrations of solvents plus sucrose slightlypromoted the formation of roots. Vigna radiata, alcohol, rooting, auxin     

Low Temperature-Induced Alkalization of Vacuoles in Suspension-Cultured Cells of Mung Bean (Vigna radiata [L.] Wilczek)

In mung bean (Vigna radiata [L.] Wilczek) suspension-culturedcells, which are extremely chilling-sensitive at the early stageof exponential growth, cold-induced acidification of the cytoplasmhas been shown to be closely correlated with the cold-inducedinactivation of the vacuolar H⁺-ATPase [Yoshida (1994) PlantPhysiol. 104: 1131]. To gain more insight into the mechanismof the cold-induced acidification of the cytoplasm in thesesuspension-cultured cells, we focused in the present study onchanges in the vacuolar pH during cold incubation. The vacuolarpH was measured by fluorescence-ratio imaging cryomicroscopy,with CDCF-DA [5-(and 6-)carboxy-2'-7'-dichlorofluorescein diacetate]as a pH-sensitive probe. During cold incubation of the CDCF-loadedprotoplasts from cells at the early stage of exponential growth,the vacuolar pH shifted toward alkaline values with time, increasingfrom 5.1 to 5.7 over the course of 8 hours. The increase invacuolar pH was closely correlated with the acidification ofcytoplasm. The cold-induced acidification of the cytoplasm appearedto be less dependent on the pH of the external medium. Fromthe results, it appears that the cold-induced acidificationof the cytoplasm probably reflects, to a considerable extent,the passive release of protons or proton equivalents from vacuolesas a consequence of the cold-induced inhibition of the ATP-driventransport of protons across vacuolar membranes. ¹Contribution no. 3814 from the Institute of Low TemperatureScience, Hokkaido University.     

Impact of biocontrol agents in combination with Prosopis juliflora (Swartz) DC. in controlling the root-infecting fungi of leguminous crops

Biocontrol agents, viz., Rhizobium meliloti, Pseudomonas aeruginosa, Aspergillus niger and Trichoderma harzianum, are used as seed dressing and soil is amended with Prsosopis juliflora (Swartz) DC. plant parts like stem, leaves and flower at 1% w/w for the control of root-rot fungi. All antagonists suppressed the infection of root-rot fungi viz., Fusarium spp., Rhizoctonia solani and Macrophomina phaseolina whereas the infection of R. solani and M. phaseolina was controlled when cowpea (Vigna unguiculata L.) and mungbean (Vigna radiata L.) seeds were treated with P. aeruginosa and T. harzianum and the soil was amended with P. juliflora leaves’ powder at 1% w/w. However, germination of both the crops was observed in all treatments. Growth parameters like shoot and root length, shoot and root weight, and leaf area significantly increased in all the treatments as compared to the control parameters. P. aeruginosa and T. harzianum in combination with soil amendment with P. juliflora plant parts at 1% w/w were the most effective for the control of root-rot fungi of leguminous plants.     

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.     

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.     

D-Amino-acid-stimulated ethylene production in seed tissues

Ethylene production by axial and cotyledonary tissues excised from Xanthium pennsylvanicum Wallr. seeds was markedly (up to 5-fold) stimulated by the D-isomers of phenylalanine, valine, leucine, threonine, methionine and eithionine while the L-isomers caused no such effect. Responsiveness of these seed tissues to D-methionine appeared soon after the beginning of imbibition, reached a maximum after 6–12 and 12–24 h for the axial and cotyledonary tissues, respectively, and then decreased sharply. D-Phenylalanine and D-methionine also stimulated ethylene production in seed tissues of X. canadense Mill. and in cotyledonary segments from seeds of Helianthus annuus L., Cucurbita moschata Duch. and Vigna radiata (L.) Wilczek. The endogeneous ethylene production and the D-amino-acid-stimulated ethylene production by the seed segments was strongly inhibited by aminoethoxyvinyl glycine, a potent inhibitor of ethylene synthesis from L-methionine.     

Ontogeny of Somatic Embryos of Vigna aconitifolia, Vigna mungo and Vigna radiata

Callus cultures were initiated from immature cotyledons of Vignaaconitifolia, V. mungo and V. radiata on MS medium supplementedwith NAA, picloram or 2, 4-D. On transfer to L-6 liquid mediumsupplemented with low concentrations of picloram, GA₃ and cytokinins,large number of somatic embryos differentiated from the callus.The cells destined to become somatic embryos divided to formspherical or filamentous proembryos. From the filamentous proembryo,the embryo proper developed either at single or multiple sites.Development of somatic embryos from multiple sites resultedin several embryos connected by a common suspensor at the radicleend. Continued divisions of the proembryos led to globular,heart shaped and dicotyledonary stages of somatic embryogenesis.The somatic embryos of V. mungo and V. aconitifolia differentiatedinto tiny plantlets at low frequency (1%) in liquid suspensioncultures supplemented with zeatin, picloram and GA₃. Vigna aconitifolia Jacq, Marechal, mothbean, Vigna mungo L. Hepper, urdbean, Vigna radiata L. Wilczk, mungbean, somatic embryo     

Sequence related amplified polymorphism (SRAP) analysis for genetic diversity and micronutrient content among gene pools in mungbean [Vigna radiata (L.) Wilczek]

Vigna radiata (L.) Wilczek, commonly called mungbean is an important pulse crop. Commercial cultivars contain low levels of iron and zinc and it is important to assess genetic variability in the available germplasm for improving micronutrient content in commercial cultivars. The present study was undertaken to study molecular diversity using Sequence-related amplified polymorphism (SRAP) among 21 Vigna radiata genotypes. Twenty nine SRAP primer combinations produced a total of 121 amplified bands which were polymorphic with an average of 4.65 bands per primer. The size of amplified bands ranged from 70 bp to 3,000 bp and 6 out of 29 SRAP primers were most useful in fingerprinting Vigna radiata genotypes under study. The similarity coefficients between different genotypes ranged from 0.45 to 0.96 with an average similarity value of 0.71. At an arbitrary cut-off at 60 % similarity level on a dendrogram, the Vigna radiata accessions were categorized into two major clusters. ML1108 and 2KM115 were found to be genetically similar. SMH99-1A and ML776 showed high iron and zinc content while Satya was poor in iron as well as zinc content. Mapping population involving ML776 and Satya could be used for tagging gene(s) for micronutrient content. The results indicated that SRAP markers were efficient for identification of Vigna radiata genotypes and assessment of the genetic relationships among them.