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Current advances in abscisic acid action and signalling.

Abscisic acid (ABA) participates in the control of diverse physiological processes. The characterization of deficient mutants has clarified the ABA biosynthetic pathway in higher plants. Deficient mutants also lead to a revaluation of the extent of ABA action during seed development and in the response of vegetative tissues to environmental stress. Although ABA receptor(s) have not yet been identified, considerable progress has been recently made in the characterization of more downstream elements of the ABA regulatory network. ABA controls stomatal aperture by rapidly regulating identified ion transporters in guard cells, and the details of the underlying signalling pathways start to emerge. ABA actions in other cell types involve modifications of gene expression. The promoter analysis of ABA-responsive genes has revealed a diversity of cis-acting elements and a few associated trans-acting factors have been isolated. Finally, characterization of mutants defective in ABA responsiveness, and molecular cloning of the corresponding loci, has proven to be a powerful approach to dissect the molecular nature of ABA signalling cascades.

Abscisic Acid↗

Fagopyritol B1, O-alpha-D-galactopyranosyl-(1-->2)-D-chiro-inositol, a galactosyl cyclitol in maturing buckwheat seeds associated with desiccation tolerance.

O-alpha-D-Galactopyranosyl-(1-->2)-D-chiro-inositol, herein named fagopyritol B1, was identified as a major soluble carbohydrate (40% of total) in buckwheat (Fagopyrum esculentum Moench, Polygonaceae) embryos. Analysis of hydrolysis products of purified compounds and of the crude extract led to the conclusion that buckwheat embryos have five alpha-galactosyl D-chiro-inositols: fagopyritol A1 and fagopyritol B1 (mono-galactosyl D-chiro-inositol isomers), fagopyritol A2 and fagopyritol B2 (di-galactosyl D-chiro-inositol isomers), and fagopyritol B3 (tri-galactosyl D-chiro-inositol). Other soluble carbohydrates analyzed by high-resolution gas chromatography included sucrose (42% of total), D-chiro-inositol, myo-inositol, galactinol, raffinose and stachyose (1% of total), but no reducing sugars. All fagopyritols were readily hydrolyzed by alpha-galactosidase (EC 3.2.1.22) from green coffee bean, demonstrating alpha-galactosyl linkage. Retention time of fagopyritol B1 was identical to the retention time of O-alpha-D-galactopyranosyl-(1-->2)-D-chiro-inositol from soybean (Glycine max (L.) Merrill, Leguminosae), suggesting that the alpha-galactosyl linkage is to the 2-position of D-chiro-inositol. Accumulation of fagopyritol B1 was associated with acquisition of desiccation tolerance during seed development and maturation in planta, and loss of fagopyritol B1 correlated with loss of desiccation tolerance during germination. Embryos of seeds grown at 18 degrees C, a condition that favors enhanced seed vigor and storability, had a sucrose-to-fagopyritol B1 ratio of 0.8 compared to a ratio of 2.46 for seeds grown at 25 degrees C. We propose that fagopyritol B1 facilitates desiccation tolerance and storability of buckwheat seeds.

Adaptation, Physiological↗

Inhibition of UDP-glucose: protein transglucosylase by a maize endosperm protein factor.

UDP-glucose: protein transglucosylase (UPTG, EC 2.4.1.112) catalyzes the first step of protein-bound alpha-glucan synthesis in potato tuber and developing maize endosperm. The presence of a non-dialyzable, heat labile protein responsible for low levels of UPTG activity in developing maize endosperm was investigated. UPTG activity in 5-day old maize seedlings and potato tuber solubilized preparations was also reduced by the endosperm preparation. FPLC-Mono Q column chromatography of developing maize endosperm was effective in separating the inhibitor protein (IP) from UPTG. After gel filtration on Superose 12, IP yielded a major polypeptide of about 80 kDa on SDS-PAGE. IP was purified by gel filtration on Superose 12 and preparative SDS-PAGE, and specific antibodies were prepared. Polyclonal antibodies reacted specifically with an 80 kDa polypeptide of developing maize endosperm on Western blot. They also recognized a similar band in 5-day old maize seedlings, but not in potato tubers. The identification of a factor that regulates the level of UPTG activity in developing maize endosperm may help to elucidate the functional role of the enzyme in the initiation of starch synthesis during seed development.

Enzyme Inhibitors↗

Ontogenic changes in enzymes of carbon metabolism in relation to carbohydrate status in developing mungbean reproductive structures.

The content of free sugars and the activities of enzymes involved in carbon metabolism-sucrose synthase, acid and alkaline invertase, phosphoenol pyruvate carboxylase, malic enzyme and isocitrate dehydrogenase were determined during seed development in mungbean pods. A decrease in carbohydrate content of pod wall from 10 to 25 days after flowering (DAF) and a concomitant increase in the seed till 20 DAF was observed. Sucrose remained the dominant soluble sugar in the pod wall and seed. In the branch of inflorescence and pod wall, the activities of sucrose metabolizing enzymes, viz. acid and alkaline invertase, sucrose synthase (synthesis and cleavage) and sucrose phosphate synthase were higher at 5-10 DAF, whereas in seed the maximum activities of these enzymes were observed at the time of maximum seed filling stage (10-20 DAF). High activities of sucrose synthase at the time of rapid seed filling can be correlated to its sink strength. Higher activities of phosphoenol pyruvate carboxylase in the branch of inflorescence and pod wall than in seed may indicate the involvement of the fruiting structure for recapturing respired CO2. High activities of isocitrate dehydrogenase and malic enzyme in the seed at the time of rapid seed filling could provide NADPH and carbon skeletons required for the synthesis of various seed reserves.

Carbohydrate Metabolism↗

Patterns of Dwarf expression and brassinosteroid accumulation in tomato reveal the importance of brassinosteroid synthesis during fruit development.

Brassinosteroids (BRs) are essential for many physiological functions in plants, however little is known concerning where and when they are synthesized. This is especially true during flower and fruit production. To address this we have used a promoter-GUS reporter fusion and RT-PCR to determine the relative expression levels of the tomato Dwarf (D) gene that encodes a BR C-6 oxidase. In young seedlings GUS reporter activity was observed mainly in apical and root tissues undergoing expansion. In flowers GUS activity was observed in the pedicel joints and ovaries, whereas in fruits it was strongest during early seed development and was associated with the locular jelly and seeds. RT-PCR analysis showed that tissue-specific expression of Dwarf mRNA was consistent with that of the Dwarf:GUS fusion. In good correlation with the high local Dwarf activity, quantitative measurements of endogenous BRs indicated intense biosynthesis in developing tomato fruits, which were also found to contain high amounts of brassinolide. Grafting experiments showed the lack of BR transport indicating that BR action occurs at the site of synthesis.

Flowers↗

Molecular cloning and characterization of genes encoding two microsomal oleate desaturases (FAD2) from olive.

Two different cDNA sequences, designated OepFAD2-1 and OepFAD2-2, encoding two microsomal oleate desaturases (FAD2) have been isolated from olive (Olea europaea cv. Picual) using a PCR approach. Both deduced amino acid sequences showed the three histidine boxes characteristic of all membrane-bound desaturases, and possess a C-terminal endoplasmic reticulum retention signal. Phylogenetic analysis shows that OepFAD2-1 and OepFAD2-2 are grouped with other plant FAD2 sequences. Functional expression of the corresponding FAD2 cDNAs in yeast confirmed that they encode microsomal oleate desaturases. Genomic Southern blot analysis is consistent with the presence of at least two copies of each OepFAD2 gene in the olive genome. OepFAD2-1 transcript was strongly detected in very young seeds and in leaves, showing low levels in mesocarps, while the transcript of the OepFAD2-2 gene was moderately expressed in developing seeds, ripening mesocarp and leaves. These expression data suggest differential functions for the two olive microsomal oleate desaturase genes, with FAD2-1 possibly responsible for the desaturation of reserve lipids in the young seed, while FAD2-2 may be mainly involved in storage lipid desaturation in the mature seeds and the mesocarp.

Amino Acid Sequence↗

The ltk gene family encodes novel receptor-like kinases with temporal expression in developing maize endosperm.

We describe the isolation and characterization of maize cDNAs that are transcribed from a small gene family and encode a novel group of receptor-like kinases (RLKs). The distinctive extracellular domain of these novel RLKs includes a unique number and arrangement of leucine-rich repeats (LRRs), a proline-rich region (PRR), a putative protein degradation target sequence (PEST), and a serine-rich region (SRR). The intracellular domain contains a putative serine/threonine protein kinase. To distinguish them from other reported RLKs, these novel RLKs were termed leucine-rich repeat transmembrane protein kinases (LTKs). Based on analysis of available deduced protein sequences, LTK1 and LTK2 were predicted to be 92.1% identical, while LTK2 and LTK3 were predicted to be 97.5% identical. Though the three LTK proteins showed high homology, the region that most distinguished LTK1 from LTK2 and LTK3 was found in the extracellular domain, in the SRR. To differentiate between expression of the individual ltk genes, we used the reverse transcriptase polymerase chain reaction (RT-PCR) in combination with restriction enzyme analysis. While ltk1 transcripts were constantly present in all tissues tested, ltk2 and ltk3 transcripts were only detected in the endosperm. Furthermore, transcript levels for both ltk1 and ltk2 showed modulation during endosperm development, peaking at 20 days after pollination. These results suggest that members of the ltk gene family mediate signals associated with seed development and maturation.

Amino Acid Sequence↗

[A comparative study on cytochemistry between spring and fall peanut seeds].

A comparative study was conducted on cytochemistry of spring and fall crop seeds in peanut cultivars Quanhua No. 10 and Shanyou 71 respectively. Lipids, protein, and polysaccharides in cells of axis and coteledon were simultaneously shown in the Epon812 buried section by means of cytochemistry, and their morphology, quantity and distribution were compared. Embryo cells of spring crop seed develop fully with big cell more vivid contrasting texture and more regularly disposed organelle, but the counterpart cells in fall crop seeds were not as much mature and their organelle arrangement appeared somewhat irregular. In cotyledon storage cells, there were also some difference between spring crop seed and fall crop seed. Cells of spring crop seeds were full of reserves, with more lipid and protein bodies that were closely ranged and extruded with each other. However, the cell structure in fall crop seeds was more loosely arranged, vacuoles had not been filled with protein, but starch grains accumulated more. Therefore, it was shown clearly that spring crop seeds have some advantages over fall crop seeds on production application. Moreover, some cytochemical techniques for demonstration of lipid, polysaccharide and protein in thick resin section and the stain protection were discussed in the paper.

Arachis↗

Identification of gametophytic mutations affecting female gametophyte development in Arabidopsis.

The female gametophyte (embryo sac or megagametophyte) plays a critical role in sexual reproduction of angiosperms. It is the structure that produces the egg cell and central cell which, following fertilization, give rise to the seed's embryo and endosperm, respectively. In addition, the female gametophyte mediates a host of reproductive processes including pollen tube guidance, fertilization, and the induction of seed development. Several major events occur during megagametogenesis, including syncitial nuclear divisions, cellularization, nuclear migration and fusion, and cell death. While these events have been described morphologically, the molecules regulating them in the female gametophyte are largely unknown. We discuss a genetic screen based on reduced seed set and segregation distortion to identify mutations affecting megagametogenesis and female gametophyte function. We report on the isolation of four mutants (fem1, fem2, fem3, and fem4) and show that the four mutations map to different locations within the genome. Additionally, we show that the fem1 and fem2 mutations affect only the female gametophyte, while the fem3 and fem4 mutations affect both the female and male gametophyte. We analyzed female gametophyte development in these four mutants as well as in the gfa2, gfa3, gfa4, gfa5, and gfa7 mutants. We found that the fem2, fem3, gfa4, and gfa5 mutants abort development at the one-nucleate stage, while the fem1, fem4, gfa2, gfa3, and gfa7 mutants are affected in processes later in development such as polar nuclei fusion and cellularization. The establishment of a genetic screen to identify mutants and the development of a rapid procedure for analyzing mutant phenotypes represent a first step in the isolation of molecules that regulate female gametophyte development and function.

Arabidopsis↗

Towards normalization of soybean somatic embryo maturation.

Soybean (Glycine max L. Merrill) somatic embryos have been useful for assaying seed-specific traits prior to plant recovery. Such traits could be assessed more accurately if somatic embryos more closely mimicked seed development. Amino acid supplements, carbon source, and abscisic acid and basal salt formulations were tested in an effort to modify existing soybean embryogenesis histodifferentiation/maturation media to further normalize the development of soybean somatic embryos. The resultant liquid medium, referred to as soybean histodifferentiation and maturation medium (SHaM), consists of FNL basal salts, 3% sucrose, 3% sorbitol, filter-sterilized 30 mM glutamine and 1 mM methionine. SHaM-derived somatic embryos are more similar to seed in terms of protein and fatty acid/lipid composition, and conversion ability, than somatic embryos obtained from traditional soybean histodifferentiation and maturation media.

Abscisic Acid↗

Coffee and tomato share common gene repertoires as revealed by deep sequencing of seed and cherry transcripts.

An EST database has been generated for coffee based on sequences from approximately 47,000 cDNA clones derived from five different stages/tissues, with a special focus on developing seeds. When computationally assembled, these sequences correspond to 13,175 unigenes, which were analyzed with respect to functional annotation, expression profile and evolution. Compared with Arabidopsis, the coffee unigenes encode a higher proportion of proteins related to protein modification/turnover and metabolism-an observation that may explain the high diversity of metabolites found in coffee and related species. Several gene families were found to be either expanded or unique to coffee when compared with Arabidopsis. A high proportion of these families encode proteins assigned to functions related to disease resistance. Such families may have expanded and evolved rapidly under the intense pathogen pressure experienced by a tropical, perennial species like coffee. Finally, the coffee gene repertoire was compared with that of Arabidopsis and Solanaceous species (e.g. tomato). Unlike Arabidopsis, tomato has a nearly perfect gene-for-gene match with coffee. These results are consistent with the facts that coffee and tomato have a similar genome size, chromosome karyotype (tomato, n=12; coffee n=11) and chromosome architecture. Moreover, both belong to the Asterid I clade of dicot plant families. Thus, the biology of coffee (family Rubiacaeae) and tomato (family Solanaceae) may be united into one common network of shared discoveries, resources and information.

Arabidopsis↗

Genetic engineering of grain and pasture legumes for improved nutritive value.

This review describes work aimed at the improvement of the nutritive value of grain and forage legumes using gene transfer techniques. Two traits which are amenable to manipulation by genetic engineering have been identified. These are plant protein quality and lignin content. In order to increase the quality of protein provided by the legume grains peas and lupins, we are attempting to introduce into these species chimeric genes encoding a sunflower seed protein rich in the sulphur-containing amino acids methionine and cysteine. These genes are designed to be expressed only in developing seeds of transgenic host plants. Chimeric genes incorporating a similar protein-coding region, but different transcriptional controls, are being introduced into the forage legumes lucerne and subterranean clover. In this case the genes are highly expressed in the leaves of transformed plants, and modifications have been made to the sunflower seed protein-coding sequences in order to increase the stability of the resultant protein in leaf tissue. Another approach to increasing plant nutritive value is represented by attempts to reduce the content of indigestible lignin in lucerne.

Amino Acid Sequence↗

Subcellular localization of the 2S globulin narbonin in seeds of Vicia narbonensis.

Narbonin is a 2S protein from the globulin fraction of narbon bean (Vicia narbonensis L.) cotyledons. Its amino acid composition and the pattern of its regulated accumulation in developing seeds led to the suggestion that narbonin could be a storage protein. Therefore, it was expected to be present in protein bodies of the storage tissue cells. Comparison of the cDNA-derived amino acid sequence with a directly determined partial N-terminal sequence revealed that the primary translation product of narbonin mRNA lacks a transient N-terminal signal peptide (V.H. Nong et al., 1995, Plant Mol Biol 28: 61 - 72). Narbonin polypeptides that had been synthesized in a cell-free translation system supplemented with dog pancreas microsomes were not protected against degradation by posttranslationally added proteases (protease protection assay). In accordance with the lack of a signal peptide this indicates that the polypeptide was not cotranslationally sequestered into the microsomes. The protein-body fraction that had been isolated from mature narbon bean cotyledons by a non-aqueous gradient centrifugation procedure was free of narbonin; this was found in the soluble cell fraction. In electron micrographs, narbonin could be localized in the cytoplasm using the immuno gold-labelling technique. Previously, it had already been shown that narbonin is too slowly degraded during narbon bean germination to act as a storage protein. From all these results it has to be concluded that narbonin is a cytoplasmic protein which does not belong to the storage proteins in the restricted sense. Other possible functions are discussed.

Animals↗

T-DNA tagging of a seed coat-specific cryptic promoter in tobacco.

T-DNA tagging with a promoterless beta-glucuronidase (GUS) gene generated a transgenic Nicotiana tabacum plant that expressed GUS activity only in developing seed coats. Cloning and deletion analysis of the GUS fusion revealed that the promoter responsible for seed coat specificity was located in the plant DNA proximal to the GUS gene. A 3.3 kb fragment corresponding to the insertion site was isolated from untransformed plants. No long open reading frames were detected in this region. Northern blots and RNase protection assays failed to detect transcripts from this region in untransformed plants. Furthermore, the insertion site was situated within the N. tomentosiformis genome of the allotetraploid species N. tabacum, in a region which is not conserved within the genus Nicotiana. It is concluded that seed coat-specific GUS expression in this transgenic plant resulted from T-DNA insertion next to a cryptic promoter. These results suggest that at least some of the fusions generated to marker genes in promoter trapping studies are not associated with conventional gene promoters. The possibility that similar insertion events play a role in gene evolution is discussed.

Amino Acid Sequence↗

ABA and sugar interactions regulating development: cross-talk or voices in a crowd?

Plant growth and development are controlled by the concerted action of many signaling pathways that integrate information from environmental signals with that from developmental and metabolic cues. Physiological studies have demonstrated that abscisic acid and sugars have both similar and antagonistic effects on diverse processes, including seed development, germination, and seedling growth. Recent genetic studies have identified several loci that are involved in both sugar and hormonal responses. It is rarely clear whether these apparent linkages reflect direct or indirect interactions between sugar and hormone signaling pathways, but the identification of gene products that are encoded at these loci is allowing these possibilities to be tested.

Abscisic Acid↗

Mineral Nutrient Requirements of a Loblolly Pine (Pinus taeda) Cell Suspension Culture : Evaluation of a Medium Formulated from Seed Composition Data.

The mineral nutrient requirements of Pinus taeda cells were explored using quantitative cell culture growth measurements. An appraisal was thereby made of the critical features of a novel and successful medium which was developed specifically for this gymnosperm using chemical composition data for developing seeds, and characterized by generally high concentration of all micronutrients, high magnesium, and low calcium. The high magnesium concentration was found not to be detrimental and possibly beneficial whereas the calcium level bordered on a deficiency threshold. Within the microelements high iodide was found to be essential, as was a higher borate level than is present in media developed for angiosperms. High zinc concentrations were also beneficial, with normal levels permitting slower but nevertheless healthy growth. An improved medium was thereby formulated which was stress-free and exhibited broader genotype specificity. This new formulation has proved very successful in maintaining long-term growth of highly uniform and apparently meristematic suspension cultures of Pinus radiata.

Journal Article↗

Characterization of rapeseed myrosinase-binding protein.

Myrosinase-binding proteins (MBPs) were purified from seeds of Brassica napus L. (oilseed rape). The proteins were characterized with respect to amino-acid composition, peptide sequence and isoelectric points. Gel electrophoresis and Western blotting of protein extracts from mature seeds showed the existence of at least ten proteins reacting with a monoclonal anti-MBP antibody and ranging in molecular size from 110 to 30 kDa. Proteins other than MBP reacting with the anti-MBP antibody were assigned as myrosinase-binding protein-related proteins (MBPRPs). Two MBPRPs were purified by immunoaffinity chromatography and characterized with respect to partial amino-acid sequence. Sequence identities were found between MBP and MBPRP. Western blot analysis of protein extracts from different tissues of B. napus showed that MBPRP is present in the whole plant, whereas MBP mostly occurs in the mature seed. A double-antibody sandwich enzyme-linked immunosorbent assay (ELISA) was used to investigate the occurrence of MBP and MBPRP in developing seeds of some species in the Brassicaceae family.

Amino Acid Sequence↗

Structure and regulated expression of Kunitz chymotrypsin inhibitor genes in winged bean [Psophocarpus tetragonolobus (L.) DC].

We analyzed the structure and the expression of Kunitz chymotrypsin inhibitor (WCI) genes in winged bean. WCI was encoded by a multigene family which comprised at least seven members. From their primary structures, four genes (WCI-2, WCI-3a, WCI-3b, and WCI-x) were expected to be functional ones and the other three (WCI-P1, WCI-P2, and WCI-P3) to be pseudogenes. The nucleotide sequences of the WCI-3a and WCI-3b genes were nearly identical, and they encoded the WCI-3 protein, the major chymotrypsin inhibitor in seeds. The WCI-2 gene also encoded the chymotrypsin inhibitor found in seeds and the WCI-x gene was expected to encode an unidentified chymotrypsin inhibitor. WCI messenger RNA and protein accumulated mainly in developing seeds and tuberous roots, small amounts of WCI mRNA being present in stems and pericarps. In seeds, transient accumulation of WCI mRNA was observed during the seed maturation period. These results suggest that the expression of WCI genes is regulated organ-specifically and developmentally in winged bean.

Amino Acid Sequence↗