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PubMed2000

The expression of small heat shock proteins in seeds responds to discrete developmental signals and suggests a general protective role in desiccation tolerance.

To learn more about the function and regulation of small heat shock proteins (sHSPs) during seed development, we studied sHSP expression in wild-type and seed maturation mutants of Arabidopsis by western analysis and using an HSP17.4 promoter-driven beta-glucuronidase (GUS) reporter gene in transgenic plants. In the absence of stress, GUS activity increases during development until the entire embryo is stained before desiccation. Heat-stressed embryos stained for GUS at all stages, including early stages that showed no detectable HSP17. 4::GUS activity without heat. Examination of HSP17.4 expression in seeds of the transcriptional activator mutants abi3-6, fus3-3 (AIMS no. CS8014/N8014), and lec1-2 (AIMS no. CS2922/N2922) showed that protein and HSP17.4::GUS activity were highly reduced in fus3-3 and lec1-2 and undetectable in abi3-6 seeds. In contrast, heat-stressed abi3-6, fus3-3, and lec1-2 seeds stained for GUS activity throughout the embryo. These data indicate that there is distinct developmental and stress regulation of HSP17.4, and imply that ABI3 activates HSP17.4 transcription during development. Quantitation of sHSP protein in desiccation-intolerant seeds of abi3-6, fus3-3, lec1-2, and line24 showed that all had <2% of wild-type HSP17.4 levels. In contrast, the desiccation-tolerant but embryo-defective mutants emb266 (AIMS no. CS3049/N3049) and lec2-1 (AIMS no. CS2728/N2728) had wild-type levels of HSP17.4. These data correlate a reduction in sHSPs with desiccation intolerance and suggest that sHSPs have a general protective role throughout the seed.

Adaptation, Physiological↗
PubMed2000

Role of ethylene in cotyledon development of microspore-derived embryos of Brassica napus.

Ethylene production during seed development in Brassica napus occurs first at 20 d after pollination (DAP), while a second greater peak occurs at 35 DAP. Because of the inaccessible location of the embryo within the maternal tissue, microspore-derived embryos (MDEs) of B. napus were used as a model for studying the role of ethylene during embryo development. The MDEs also produced a peak in ethylene evolution at 20 DAC (i.e. the early cotyledonary stage), dropping to minimal levels by 25-30 DAC. At 20 DAC the excised cotyledon evolved 85% of the ethylene found in the whole MDE. To determine the role of ethylene, MDEs were treated with aminoethoxyvinylglycine (AVG, an inhibitor of ethylene biosynthesis), CoCl(2) (an inhibitor of 1-aminocyclopropane-1-carboxylic acid (ACC) oxidase), and silver thiosulphate (STS, an inhibitor of ethylene action). An inhibition in ethylene production or action at 20 DAC resulted in diminished lateral cotyledon expansion, due to a reduction in the lateral expansion of cells within the cotyledon. Recovery to 'control-type' levels of cotyledon cell expansion was achieved by application of ACC (the metabolic precursor of ethylene) to AVG-treated MDEs. Thus, ethylene production at 20 DAP likely controls cotyledon expansion during embryo development.

Brassica↗
PubMed2003

Seed 1-cysteine peroxiredoxin antioxidants are not involved in dormancy, but contribute to inhibition of germination during stress.

Peroxiredoxins (Prx) are thiol-dependent antioxidants containing one (1-cysteine [-Cys]) or two (2-Cys) conserved Cys residues that protect lipids, enzymes, and DNA against reactive oxygen species. In plants, the 1-Cys Prxs are highly expressed during late seed development, and the expression pattern is dormancy related in mature seeds. We have expressed the Arabidopsis 1-Cys Prx AtPER1 in Escherichia coli and show that this protein has antioxidant activity in vitro and protects E. coli in vivo against the toxic oxidant cumene hydroperoxide. Although some 1-Cys Prxs are targeted to the nucleus, a green fluorescent protein-AtPER1 fusion protein was also localized to the cytoplasm in an onion epidermis subcellular localization assay. It has been proposed that seed Prxs are involved in maintenance of dormancy and/or protect the embryo and aleurone layer surviving desiccation against damage caused by reactive oxygen species. These hypotheses were tested using transgenic Arabidopsis lines overexpressing the barley (Hordeum vulgare) 1-Cys PER1 protein and lines with reduced levels of AtPER1 due to antisensing or RNA interference. We found no correlation between Prx levels and the duration of the after-ripening period required before germination. Thus, Prxs are unlikely to contribute to maintenance of dormancy. RNA interference lines almost devoid of AtPER1 protein developed and germinated normally under standard growth room conditions. However, seeds from lines overexpressing PER1 were less inclined to germinate than wild-type seeds in the presence of NaCl, mannitol, and methyl viologen, suggesting that Prx can sense harsh environmental surroundings and play a part in the inhibition of germination under unfavorable conditions.

Arabidopsis↗
PubMed1985

Novel application of quantitative immunoassays for screening seed globulins of cowpea varieties.

Using antibodies raised in rabbits, radioimmunoassay (RIA) and enzyme-linked immunosorbent assay (ELISA) are standardized for cowpea (var. Pusa Barsati) seed globulins. The RIA, when used to screen three stages of seed development, reveals that maximum globulins are detected at 28 days after flowering. When three different varieties of cowpea are assayed for their globulin content by RIA and ELISA, it is observed that the Bold Grain cowpea has the highest amount of related globulin as compared to two other varieties, namely Pusa Phalgun and Asparagus Bean.

Electrophoresis, Polyacrylamide Gel↗
PubMed1999

Engineering seed dormancy by the modification of zeaxanthin epoxidase gene expression.

Abscisic acid (ABA) is a plant hormone synthesized during seed development that is involved in the induction of seed dormancy. Delayed germination due to seed dormancy allows long-term seed survival in soil but is generally undesirable in crop species. Freshly harvested seeds of wild-type Nicotiana plumbaginifolia plants exhibit a clear primary dormancy that results in delayed germination, the degree of primary dormancy being influenced by environmental culture conditions of the mother plant. In contrast, seeds, obtained either from ABA-deficient mutant aba2-s1 plants directly or aba2-s1 plants grafted onto wild-type plant stocks, exhibited rapid germination under all conditions irrespective of the mother plant culture conditions. The ABA biosynthesis gene ABA2 of N. plumbaginifolia, encoding zeaxanthin epoxidase, was placed under the control of the constitutive 35S promoter. Transgenic plants overexpressing ABA2 mRNA exhibited delayed germination and increased ABA levels in mature seeds. Expression of an antisense ABA2 mRNA, however, resulted in rapid seed germination and in a reduction of ABA abundance in transgenic seeds. It appears possible, therefore, that seed dormancy can be controlled in this Nicotiana model species by the manipulation of ABA levels.

Abscisic Acid↗
PubMed2001

Cloning and expression of a sorghum gene with homology to maize vp1. Its potential involvement in pre-harvest sprouting resistance.

Pre-harvest sprouting (PHS) in sorghum is related to the lack of a normal dormancy level during seed development and maturation. Based on previous evidence that seed dormancy in maize is controlled by the vp1 gene, we used a PCR-based approach to isolate two Sorghum bicolor genomic and cDNA clones from two genotypes exhibiting different PHS behaviour and sensitivity to abscisic acid (ABA). The two 699 amino acid predicted protein sequences differ in two residues at positions 341 (Gly or Cys within the repression domain) and 448 (Pro or Ser) and show over 80, 70 and 60% homology to maize, rice and oat VP1 proteins respectively. Expression analysis of the sorghum vp1 gene in the two lines shows a slightly higher level of vp1 mRNA in the embryos susceptible to PHS than in those resistant to PHS during embryogenesis. However, timing of expression was different between these genotypes during this developmental process. Whereas for the former the main peak of expression was observed at 20 days after pollination (DAP), the peak in the latter was found at later developmental stages when seed maturation was almost complete. Under favourable germination conditions and in the presence of fluridone (an inhibitor of ABA biosynthesis), sorghum vp1 mRNA showed to be consistently correlated with sensitivity to ABA but not with ABA content and dormancy.

Abscisic Acid↗
PubMed1986

Uptake and processing of the precursor to the small subunit of ribulose 1,5-bisphosphate carboxylase by leucoplasts from the endosperm of developing castor oil seeds.

Intact leucoplasts from the endosperm of developing castor oil seed were isolated by Percoll density gradient centrifugation. The precursor to the small subunit of ribulose 1,5-bisphosphate carboxylase from pea was synthesized in vitro from hybrid-selected mRNA. Leucoplasts imported this precursor by an ATP-requiring mechanism similar to that described in chloroplasts (AR Grossman et al. 1980 Nature 285: 625-628). The small subunit precursor was processed to a molecular weight that was identical with that of the mature pea small subunit. These results show that leucoplasts, though specialized for fatty acid biosynthesis and not photosynthesis, have a mechanism of protein import similar to that of chloroplasts.

Journal Article↗
PubMed1997

Developmental regulation of a plant encoded inhibitor of eukaryotic initiation factor 2 alpha phosphorylation.

An inhibitor of eIF-2a phosphorylation was identified in various plant species. The plant protein (termed PKI) specifically cross-reacts with monoclonal antiserum that recognizes the glycosylated, active form of a M(r) 87 kD protein analog (p67) from reticulocytes. Northern blot analysis using a probe to the reticulocyte inhibitor cDNA further supports the presence of analogous transcripts in plant tissue. PKI specifically inhibits the phosphorylation of the plant encoded eIF-2 alpha kinase (pPKR) as well as plant and human eIF-2 alpha phosphorylation. The interaction between PKI and pPKR is indicated by their copurification on dsRNA agarose, despite evidence showing that PKI does not bind dsRNA. Further, wheat PKI inhibits human PKR phosphorylation but activity is recovered by immuno-depletion of PKI from wheat germ fractions. PKI is temporally regulated during plant growth and development. It is maximally present in extracts from dormant seeds, however, it is not detectable soon after leaf emergence at approximately 48 h post-imbibition. PKI levels are again detectable at the mid-milk stage in seed development. Protein levels of pPKR in ribosomal salt wash and cytosolic extracts from healthy plant tissue remain essentially constant throughout the life cycle. In contrast, pPKR activity levels based upon autophosphorylation vary significantly and are inversely correlated with PKI protein levels. Phosphorylation of eIF-2 alpha is a classical mechanism for the downregulation of protein synthesis suggesting that inhibition of pPKR activity by PKI may contribute to the dramatic and rapid increase in protein synthesis observed during seed germination.

Antibodies, Monoclonal↗
PubMed1984

C-Photosynthate Partitioning and Translocation in Soybeans during Reproductive Development.

Partitioning and translocation of (14)C-photosynthates were examined during flowering and seed maturation in soybean (Glycine max [L.]Merr.) plants to quantify allocation to sugars, amino acids, organic acids, and starch and to study transport of C and N from leaves to reproductive sinks. The trifoliolate leaf at the eighth node was exposed to steady state levels of (14)CO(2) for 2 hours, followed by immediate extraction and identification of radioactive assimilates in the fed leaf blade, tissues of the transport path (e.g. petiole and stem), and fruits if they were present. About one-third of the total (14)C recovered from the leaf blades was in starch until late pod-filling, after which the proportion dropped to 16%. Sugars comprised 70% to 86% of the recovered (14)C from soluble assimilates of the source leaf, with highest proportions occurring during late flowering and early pod-filling. Amino acids accounted for 8% to 17% of the (14)C recovered from the soluble fraction, and were most evident during early flowering and mid to late pod-filling. The (14)C-organic acids comprised from 3% to 14% of the soluble (14)C-assimilates in leaves. Petioles consistently contained a higher percentage of recovered radioactivity in sugars (87-97%) and a lower percentage in amino acids (3-12%) than did leaf blades. (14)C-Amino acids in petioles attained their highest levels during mid and late pod-filling, while (14)C-organic acids comprised 2% or less of the recovered radioactivity after pod initiation. The distribution of (14)C-assimilates in the internode below the source leaf was similar to that found in petioles. A comparison of the above data to calculated C and N requirements for seed development suggests that (14)C-amino acids derived from current photosynthesis and translocated from source leaves supply at least 12% to 48% of the seed N depending on the stage of pod-filling.

Journal Article↗
PubMed1999

Analyses of isoamylase gene activity in wild-type barley indicate its involvement in starch synthesis.

The notion of debranching enzyme activity as a participant in starch synthesis is gaining acceptance. Inconsistent reports from mutant analyses implicate either isoamylase or pullulanase as a determinant in amylopectin formation and whether wild-type plants utilize one or the other, or both, of these debranching enzymes in starch synthesis is unclear. Recent results on the sul mutant in maize suggest that both forms of debranching enzymes might be involved in amylopectin formation. We wished to find out if isoamylase takes part in starch synthesis by comparing isoamylase gene activity under three conditions: (1) during starch accumulation in developing sink tissues; (2) during starch degradation in germinating seeds; (3) in ectopic expression after applying sucrose, a starch precursor. We isolated the gene for barley isoamylase, isol, and analysed its expression and regulation in germinating seeds, developing endosperm and vegetative tissues, and compared the isoamylase gene expression in sink tissues from three different species. Our results indicate that isoamylase gene activity is involved in starch synthesis in wild-type plants and is modulated by sucrose.

Amino Acid Sequence↗
PubMed2002

Cinnamomin, a type II ribosome-inactivating protein, is a storage protein in the seed of the camphor tree (Cinnamomum camphora).

Cinnamomin is a novel type II ribosome-inactivating protein (RIP) isolated in our laboratory from the seed of the camphor tree (Cinnamomum camphora). In this paper the physiological role it plays in the plant cell was studied. Northern and Western blotting revealed that cinnamomin was expressed specifically in cotyledons. It accumulated in large amounts simultaneously with other proteins at the post-stages of seed development. Cinnamomin degraded rapidly during the early stages of seed germination. Endopeptidase was proved to play an important role in the degradation of cinnamomin. Western blotting of total proteins from the protein body with antibodies against cinnamomin demonstrated that it only existed in this specific cellular organelle as a storage protein. The similar properties of cinnamomin and other seed storage proteins of dicotyledons were compared. We conclude that cinnamomin is a special storage protein in the seed of C. camphora.

Algal Proteins↗
PubMed1999

Stachyose synthesis in seeds of adzuki bean (Vigna angularis): molecular cloning and functional expression of stachyose synthase.

Stachyose is the major soluble carbohydrate in seeds of a number of important crop species. It is synthesized from raffinose and galactinol by the action of stachyose synthase (EC 2.4.1.67). We report here on the identification of a cDNA encoding stachyose synthase from seeds of adzuki bean (Vigna angularis Ohwi et Ohashi). Based on internal amino acid sequences of the enzyme purified from adzuki bean, oligonucleotides were designed and used to amplify corresponding sequences from adzuki bean cDNA by RT-PCR, followed by rapid amplification of cDNA ends (RACE-PCR). The complete cDNA sequence comprised 3046 nucleotides and included an open reading frame which encoded a polypeptide of 857 amino acid residues. The entire coding region was amplified by PCR, engineered into the baculovirus expression vector pVL1393 and introduced into Spodoptera frugiperda (Sf21) insect cells for heterologous expression. The recombinant protein was immunologically reactive with polyclonal antibodies raised against stachyose synthase purified from adzuki bean and was shown to be a functional stachyose synthase with the same catalytic properties as its native counterpart. High levels of stachyose synthase mRNA were transiently accumulated midway through seed development, and the enzyme was also present in mature seeds and during germination.

Amino Acid Sequence↗
PubMed2006

Timing of reproductive and vegetative development in Saxifraga oppositifolia in an alpine and a subnival climate.

Morphogenesis of floral structures, dynamics of reproductive development from floral initiation until fruit maturation, and leaf turnover in vegetative short-stem shoots of Saxifraga oppositifolia were studied in three consecutive years at an alpine site (2300 m) and at an early- and late-thawing subnival site (2650 m) in the Austrian Alps. Marked differences in the timing and progression of reproductive and vegetative development occurred: individuals of the alpine population required a four-month growing season to complete reproductive development and initiate new flower buds, whereas later thawing individuals from the subnival sites attained the same structural and functional state within only two and a half months. Reproductive and vegetative development were not strictly correlated because timing of flowering, seed development, and shoot growth depended mainly on the date of snowmelt, whereas the initiation of flower primordia was evidently controlled by photoperiod. Floral induction occurred during June and July, from which a critical day length for primary floral induction of about 15 h could be inferred. Preformed flower buds overwinter in a pre-meiotic state and meiosis starts immediately after snowmelt in spring. Vegetative short-stem shoots performed a full leaf turnover within a growing season: 16 (+/-0.8 SE) new leaves per shoot developed in alpine and early-thawing subnival individuals and 12 (+/-1.2 SE) leaves in late-thawing subnival individuals. New leaf primordia emerged continuously from snowmelt until late autumn, even when plants were temporarily covered with snow. Differences in the developmental dynamics between the alpine and subnival population were independent of site temperatures, and are probably the result of ecotypic adaptation to differences in growing season length.

Adaptation, Physiological↗
PubMed1999

Expression of a polygalacturonase associated with tomato seed germination.

Radicle protrusion from tomato (Lycopersicon esculentum Mill.) seeds to complete germination requires weakening of the endosperm tissue opposite the radicle tip. In common with other cell wall disassembly processes in plants, polygalacturonases (PGs) may be involved. Only calcium-dependent exo-PG activity was detected in tomato seed protein extracts. Chromatographic profiles of a partially acid-hydrolyzed fraction of polygalacturonic acid further digested with seed extract were consistent with the presence of only calcium-dependent exo-PG activity. In addition, a transcript encoding a previously unknown PG was detected prior to the completion of germination. The mRNA, produced from a gene (LeXPG1) estimated by Southern analysis to be represented once in the genome, was also present in flowers (anthers) and in lower amounts in roots and stems. LeXPG1 mRNA abundance was low during seed development, increased during imbibition, and was even greater in seeds that had completed germination. Expression of LeXPG1 during germination predominates in the endosperm cap and radicle tip, and in the radicle appears as a distinct band possibly associated with vascular tissue differentiation. We suggest that PG is involved in cell wall loosening of the endosperm necessary for radicle protrusion from tomato seeds and in subsequent embryo and seedling growth.

Gene Expression Regulation, Enzymologic↗
PubMed1986

Remobilization patterns of C and N in soybeans with different sink-source ratios induced by various night temperatures.

The effects of increased sink-source ratios, induced by elevating night temperatures, on remobilization of (14)C-assimilates and N within field-grown soybeans (Glycine max [L.] Merr.) was investigated from preflowering to maturity. Raising the mean minimum night temperature for the entire growing season from 10 (check, uncontrolled) to 16 degrees C increased seed growth without appreciable effect on final leaf area. Increasing this temperature to 24 degrees C increased seed growth and reduced final leaf area. Leaves, stems, petioles, and pods acted as intermediate storage sites for (14)C assimilates. Only plants with higher night temperatures remobilized some of the stored assimilates during the period of rapid seed growth. Even the seeds in the 24 degrees C plants with the largest sink-source ratios did not utilize all the C-assimilates potentially available for remobilization. Nitrogen was readily remobilized from petioles, stems, and pods of all treatments as early as the beginning of seed development, but from the leaves only during late seed-filling. However, only plants with elevated night temperatures tended to remobilize all of the available N from vegetative tissues and pods. We concluded that a larger portion of stored assimilates may be remobilized to the seed if a strong seed sink can be sustained. It also appeared that with increasing sink-source ratios, N shortage might limit seed yield before a lack of C-assimilates would. A proposed model for soybean assimilate demand, distribution, partitioning, and remobilization is presented.

Journal Article↗
PubMed2002

Hormone and seed-specific regulation of pea fruit growth.

Growth of young pea (Pisum sativum) fruit (pericarp) requires developing seeds or, in the absence of seeds, treatment with gibberellin (GA) or auxin (4-chloroindole-3-acetic acid). This study examined the role of seeds and hormones in the regulation of cell division and elongation in early pea fruit development. Profiling histone H2A and gamma-tonoplast intrinsic protein (TIP) gene expression during early fruit development identified the relative contributions of cell division and elongation to fruit growth, whereas histological studies identified specific zones of cell division and elongation in exocarp, mesocarp, and endocarp tissues. Molecular and histological studies showed that maximal cell division was from -2 to 2 d after anthesis (DAA) and elongation from 2 to 5 DAA in pea pericarp. Maximal increase in pericarp gamma-TIP message level preceded the maximal rate of fruit growth and, in general, gamma-TIP mRNA level was useful as a qualitative marker for expanding tissue, but not as a quantitative marker for cell expansion. Seed removal resulted in rapid decreases in pericarp growth and in gamma-TIP and histone H2A message levels. In general, GA and 4-chloroindole-3-acetic acid maintained these processes in deseeded pericarp similarly to pericarps with seeds, and both hormones were required to obtain mesocarp cell sizes equivalent to intact fruit. However, GA treatment to deseeded pericarps resulted in elevated levels of gamma-TIP mRNA (6 and 7 DAA) when pericarp growth and cell enlargement were minimal. Our data support the theory that cell division and elongation are developmentally regulated during early pea fruit growth and are maintained by the hormonal interaction of GA and auxin.

Aquaporins↗
PubMed1999

Differential ovule development following self- and cross-pollination: the basis of self-sterility in Narcissus triandrus (Amaryllidaceae).

Self-pollination results in significantly lower seed set than cross-pollination in tristylous Narcissus triandrus. We investigated structural and functional aspects of pollen-pistil interactions and ovule-seed development following cross- and self-pollination to assess the timing and mechanism of self-sterility. Ovule development within an ovary was asynchronous at anthesis. There were no significant differences in pollen tube behavior following cross- vs. self-pollination during the first 6 d of growth, regardless of style morph type. Double fertilization was significantly higher following cross- vs. self-pollination. Aborted embryo development was not detected following either pollination type up to seed maturity. Prior to pollen tube entry, a significantly greater number of ovules ceased to develop following self- vs. cross-pollination. These results indicate that self-sterility in N. triandrus operates prezygotically but does not involve differential pollen tube growth typical of many self-incompatibility (SI) systems. Instead, low seed set following self-pollination is caused by a reduction in ovule availability resulting from embryo sac degeneration. We hypothesize that this is due to the absence of a required stimulus for normal ovule development. If this is correct, current concepts of SI may need to be broadened to include a wider range of pollen-pistil interactions.

Journal Article↗
PubMed2003

Plant reproductive development during spaceflight.

Reproductive development in microgravity has now been studied in a variety of plants; Arabidopsis, Brassica, and Triticum have been especially well studied. Earlier indications that gravity might be required for some stage of reproductive development have now been refuted. Nevertheless, the spaceflight environment presents many unique challenges that have often compromised the ability of plants to reproduce. These include limitations in hardware design to compensate for the unique environmental characteristics of microgravity, especially absence of convective air movement. Pollen development has been shown to be sensitive to high concentrations of ethylene prevailing on various orbital platforms. Barring these gross environmental problems, androecium and gynoecium development occur normally in microgravity, in that functional propagules are produced. Nonetheless, qualitative changes in anther and pistil development have been shown, and significant qualitative changes occur in storage reserve deposition during seed development. Apart from the intrinsic biological importance of these results, consequences of diminished seed quality when plants are grown in the absence of gravity will detract from the utility of plant-based life support systems. By understanding gravity's role in determining the microenvironments that prevail during reproductive development, counter-measures to these obstacles can be found, while at the same time providing basic knowledge that will have broader agricultural significance.

Fruit↗