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HvMCB1, a R1MYB transcription factor from barley with antagonistic regulatory functions during seed development and germination.

The functional analysis of hydrolase gene promoters induced by gibberellin (GA) in barley aleurone cells upon germination has identified a tripartite GA-response complex (GARC) containing a 5'-TATCCAC-3' box as well as the GA-responsive element (GARE) recognized by GAMYB and the pyrimidine box interacting with the DOF transcription factors BPBF and SAD. We show here that the MCB1 gene encoding a R1MYB protein binds to the 5'-TATCCAC-3' (GATA core) box in vitro and is a transcriptional repressor of a GA-induced amylase (Amy6.4) promoter in bombarded aleurone layers. Northern blot and mRNA in situ hybridization analyses showed that the MCB1 transcripts accumulate in the aleurone cells upon germination, as well as in endosperm tissues during seed development. The HvMCB1 protein expressed in bacteria binds in a specific manner to a 27-mer oligonucleotide containing the 5'-TATCCAC-3' sequence, derived from the promoter region of the Amy6.4 gene. Accumulation of the MCB1 transcript diminished in response to external GA incubation in aleurone cells, and in transient expression experiments HvMCB1 repressed transcription of the Amy6.4 promoter in GA-treated aleurone layers and reversed the GAMYB-mediated activation of this amylase promoter. In contrast, during endosperm maturation HvMCB1 acted as a transcription activator of the seed-specific Itr1 gene promoter through binding to a 5'-GATAAGATA-3' box.

Amino Acid Sequence↗

Expression of the PmSUC1 sucrose carrier gene from Plantago major L. is induced during seed development.

A cDNA clone of the plasma membrane sucrose-H+ symporter PmSUC1 from Plantago major L. has been isolated and expressed in Saccharomyces cerevisiae. The PmSUC1 protein was characterized in transgenic yeast and in proteoliposomes with an artificial proton-motive-force (pmf) generator. PmSUC1 catalyzes the active uptake of sucrose or maltose in the presence of pmf and is sensitive to uncouplers. Unlike the extremely pH-dependent PmSUC2 sucrose-H+ symporter, PmSUC1 is relatively insensitive to changes of the extracellular pH. In leaves and petioles of P. major, expression of PmSUC1 mRNA is restricted to the vascular system. The important new feature about PmSUC1 is that the highest mRNA levels are found in non-vascular tissue of P. major flowers where the gene is transiently expressed during the early stages of seed development. In situ hybridization experiments show that PmSUC1 is expressed only in young ovules; the putative physiological role of PmSUC1 is discussed.

Amino Acid Sequence↗

Antisense expression of the peptide transport gene AtPTR2-B delays flowering and arrests seed development in transgenic Arabidopsis plants.

Previously, we identified a peptide transport gene, AtPTR2-B, from Arabidopsis thaliana that was constitutively expressed in all plant organs, suggesting an important physiological role in plant growth and development. To evaluate the function of this transporter, transgenic Arabidopsis plants were constructed expressing antisense or sense AtPTR2-B. Genomic Southern analysis indicated that four independent antisense and three independent sense AtPTR2-B transgenic lines were obtained, which was confirmed by analysis of the segregation of the kanamycin resistance gene carried on the T-DNA. RNA blot data showed that the endogenous AtPTR2-B mRNA levels were significantly reduced in transgenic leaves and flowers, but not in transgenic roots. Consistent with this reduction in endogenous AtPTR2-B mRNA levels, all four antisense lines and one sense line exhibited significant phenotypic changes, including late flowering and arrested seed development. These phenotypic changes could be explained by a defect in nitrogen nutrition due to the reduced peptide transport activity conferred by AtPTR2-B. These results suggest that AtPTR2-B may play a general role in plant nutrition. The AtPTR2-B gene was mapped to chromosome 2, which is closely linked to the restriction fragment length polymorphism marker m246.

Agrobacterium tumefaciens↗

The plasma membrane H(+)-ATPase gene family in Arabidopsis: genomic sequence of AHA10 which is expressed primarily in developing seeds.

The plasma membrane H(+)-ATPases in Arabidopsis thaliana represent the largest family of cation translocating P-type ATPases identified in plants or animals. We report here seven new isoforms, which were identified by polymerase chain reaction (PCR) amplification of genomic DNA. Amplifications were performed with degenerate primers corresponding to two short conserved sequence motifs ("CSDK" and "GDGV") found in most P-type ATPases. A comparison was made of three CSDK-side primers, which were used either as totally degenerate mixtures or rendered less degenerate by substitution with deoxyinosine or fluorodeoxyuridine. Amplified genomic fragments were cloned, partially sequenced and shown to correspond to Arabidopsis genes by Southern blot analysis with gene-specific probes. One newly identified isoform, AHA10, was isolated as a cosmid clone and sequenced. The 5' and 3' ends of the gene were determined by comparison with the AHA10 cDNA sequence. AHA10 is the most divergent isoform characterized in the Arabidopsis family. AHA10 appears to be expressed primarily in developing seeds, as indicated by Northern blot analysis of AHA10 mRNA and by the analysis of transgenic plants expressing a beta-glucuronidase (GUS) reporter gene fused to an AHA10 promoter. Our results indicate that one function of this unusually large H(+)-ATPase gene family is to allow for expression of different isoforms in different cell types.

Amino Acid Sequence↗

The in vivo synthesis and accumulation of lectin in developing seeds of black gram (Vigna mungo L. Hepper).

Black gram (Vigna mungo L. Hepper) seed contains two D-galactose-specific lectin species, BGL-I and BGL-II, identified on the basis of elution from ion exchange column and immunochemical cross-reactivity. BGL-I consisted of two monomeric lectins, BGL-I-1 and BGL-1-2, of relative molecular weights 94 and 89 kDa, respectively. BGL-II is another monomeric lectin with a molecular weight of 83 kDa. The in vivo synthesis studies using pulse-chase experiment showed that BGL-II lectin was synthesized as early as 14 days after flowering (DAF). The 94-kDa BGL-I-1 lectin was synthesized around 17 DAF. There was no cotranslational or posttranslational modification of the lectin proteins. The amount of lectin in developing seeds was determined by radial immunodiffusion assay technique. The maximum amount of lectin per seed was found at 28 DAF.

Food Analysis↗

Two tomato expansin genes show divergent expression and localization in embryos during seed development and germination.

Expansins are plant proteins that can induce extension of isolated cell walls and are proposed to mediate cell expansion. Three expansin genes were expressed in germinating tomato (Lycopersicon esculentum Mill.) seeds, one of which (LeEXP4) was expressed specifically in the endosperm cap tissue enclosing the radicle tip. The other two genes (LeEXP8 and LeEXP10) were expressed in the embryo and are further characterized here. LeEXP8 mRNA was not detected in developing or mature seeds but accumulated specifically in the radicle cortex during and after germination. In contrast, LeEXP10 mRNA was abundant at an early stage of seed development corresponding to the period of rapid embryo expansion; it then decreased during seed maturation and increased again during germination. When gibberellin-deficient (gib-1) mutant seeds were imbibed in water, LeEXP8 mRNA was not detected, but a low level of LeEXP10 mRNA was present. Expression of both genes increased when gib-1 seeds were imbibed in gibberellin. Abscisic acid did not prevent the initial expression of LeEXP8 and LeEXP10, but mRNA abundance of both genes subsequently decreased during extended incubation. The initial increase in LeEXP8, but not LeEXP10, mRNA accumulation was blocked by low water potential, but LeEXP10 mRNA amounts fell after longer incubation. When seeds were transferred from abscisic acid or low water potential solutions to water, abundance of both LeEXP8 and LeEXP10 mRNAs increased in association with germination. The tissue localization and expression patterns of both LeEXP8 and LeEXP10 suggest developmentally specific roles during embryo and seedling growth.

Cell Wall↗

Changes in the Ascorbate System during Seed Development of Vicia faba L.

Large changes occur in the ascorbate system during the development of Vicia faba seed and these appear closely related to what are generally considered to be the three stages of embryogenesis. During the first stage, characterized by embryonic cells with high mitotic activity, the ascorbic acid/dehydroascorbic acid ratio is about 7, whereas in the following stage, characterized by rapid cell elongation (stage 2), it is lower than 1. The different ascorbic/dehydroascorbic ratio may be correlated with the level of ascorbate free radical reductase activity, which is high in stage 1 and lower in stage 2. Ascorbate peroxidase activity is high and remains constant throughout stages 1 and 2, but it decreases when the water content of the seed begins to decline (stage 3). In the dry seed, the enzyme disappears together with ascorbic acid. Ascorbate peroxidase activity is observed to be 10 times higher than that of catalase, suggesting that ascorbate peroxidase, rather than catalase, is utilized in scavenging the H(2)O(2) produced in the cell metabolism. There is no ascorbate oxidase in the seed of V. faba. V. faba seeds acquire the capability to synthesize ascorbic acid only after 30 days from anthesis, i.e. shortly before the onset of seed desiccation. This suggests that (a) the young seed is furnished with ascorbic acid by the parent plant throughout the period of intense growth, and (b) it is necessary for the seed to be endowed with the ascorbic acid biosynthetic system before entering the resting state so that the seed can promptly synthesize the ascorbic acid needed to reestablish metabolic activity when germination starts.

Journal Article↗

A fiberless seed mutation in cotton is associated with lack of fiber cell initiation in ovule epidermis and alterations in sucrose synthase expression and carbon partitioning in developing seeds

Fiber cell initiation in the epidermal cells of cotton (Gossypium hirsutum L.) ovules represents a unique example of trichome development in higher plants. Little is known about the molecular and metabolic mechanisms controlling this process. Here we report a comparative analysis of a fiberless seed (fls) mutant (lacking fibers) and a normal (FLS) mutant to better understand the initial cytological events in fiber development and to analyze the metabolic changes that are associated with the loss of a major sink for sucrose during cellulose biosynthesis in the mutant seeds. On the day of anthesis (0 DAA), the mutant ovular epidermal cells lacked the typical bud-like projections that are seen in FLS ovules and are required for commitment to the fiber development pathway. Cell-specific gene expression analyses at 0 DAA showed that sucrose synthase (SuSy) RNA and protein were undetectable in fls ovules but were in abundant, steady-state levels in initiating fiber cells of the FLS ovules. Tissue-level analyses of developing seeds 15 to 35 DAA revealed an altered temporal pattern of SuSy expression in the mutant relative to the normal genotype. Whether the altered programming of SuSy expression is the cause or the result of the mutation is unknown. The developing seeds of the fls mutant have also shown several correlated changes that represent altered carbon partitioning in seed coats and cotyledons as compared with the FLS genotype.

Journal Article↗

Mutations in the FIE and MEA genes that encode interacting polycomb proteins cause parent-of-origin effects on seed development by distinct mechanisms.

In flowering plants, two cells are fertilized in the haploid female gametophyte. Egg and sperm nuclei fuse to form the embryo. A second sperm nucleus fuses with the central cell nucleus, which replicates to generate the endosperm, a tissue that supports embryo development. The FERTILIZATION-INDEPENDENT ENDOSPERM (FIE) and MEDEA (MEA) genes encode WD and SET domain polycomb proteins, respectively. In the absence of fertilization, a female gametophyte with a loss-of-function fie or mea allele initiates endosperm development without fertilization. fie and mea mutations also cause parent-of-origin effects, in which the wild-type maternal allele is essential and the paternal allele is dispensable for seed viability. Here, we show that FIE and MEA polycomb proteins interact physically, suggesting that the molecular partnership of WD and SET domain polycomb proteins has been conserved during the evolution of flowering plants. The overlapping expression patterns of FIE and MEA are consistent with their suppression of gene transcription and endosperm development in the central cell as well as their control of seed development after fertilization. Although FIE and MEA interact, differences in maternal versus paternal patterns of expression, as well as the effect of a recessive mutation in the DECREASE IN DNA METHYLATION1 (DDM1) gene on mutant allele transmission, indicate that fie and mea mutations cause parent-of-origin effects on seed development by distinct mechanisms.

Arabidopsis↗

Cloning and characterization of a cowpea seed lipid transfer protein cDNA: expression analysis during seed development and under fungal and cold stresses in seedlings' tissues.

Lipid transfer proteins (LTPs) are antimicrobial peptides (AMPs) involved in the defense of plants against pathogens. Our group has previously characterized and purified a LTP from cowpea (Vigna unguiculata (L.) Walp.) seeds which caused the inhibition of growth of fungal pathogens in vitro. The aim of this work was to obtain the cDNA encoding the cowpea LTP and after cloning, to use the cDNA as a probe for studying its expression profile during the development of cowpea seeds. In this work, the N-terminal sequence of the mature LTP peptide from cowpea was used to produce a degenerated oligonucleotide. This primer allowed the amplification of the LTP cDNA by RT-PCR from mRNA of cowpea seeds. The sequence analysis of the cloned cDNA, named VULTP, showed 494 bp which encoded a polypeptide of 91 amino acids. The deduced peptide presented high homology of similarity to plant LTPs of Vigna radiata var. radiate (94%), Prunus domestica (82%) and Zea mays (72%). The expression profile of the VULTP gene in cowpea was analyzed by Northern blot and revealed that the transcript is not accumulated in adult tissues. Conversely, VULTP mRNA is early and strongly accumulated during seed development. The results obtained to seedling of cowpea demonstrate that the VULTP gene presents differential expression in response to different stress. Further studies will be conducted to try to gain better understanding about the physiological role of this gene in cowpea.

Adaptation, Physiological↗

A RING-H2 zinc-finger protein gene RIE1 is essential for seed development in Arabidopsis.

RING zinc-finger proteins play important roles in the regulation of development in a variety of organisms. In the plant kingdom, few genes encoding RING zinc-finger proteins have been documented with visible effects on plant growth and development. A novel gene, RIE1, encoding a RING-H2 zinc-finger protein was identified in Arabidopsis thaliana and is characterized in this paper. RIE1 encodes a predicted protein product of 359 amino acids residues with a molecular mass of 40 kDa, with a RING-H2 zinc-finger motif located at the extreme end of the C-terminus. Characterization of a Dissociation (Ds) insertion line (SGT4559) and a T-DNA insertion line (SRIE1) demonstrated that disruption of RIE1 is embryo-lethal. SGT4559 heterozygous plants produced seeds with embryo development arrested from globular to torpedo stages. Some mutant seeds were rescued by embryo culture, and the mutant (rie1) plants seemed to grow normally compared to wild-type plants, except that the mutants produced only abnormal seeds. However, RIE1 was expressed in different tissues throughout the whole plant as revealed by northern blot analysis and gene fusion assay of RIE1 promoter with the beta-glucuronidase (GUS) gene. Our results indicated that RIE1 plays an essential role in seed development.

Amino Acid Sequence↗

The DOF protein, SAD, interacts with GAMYB in plant nuclei and activates transcription of endosperm-specific genes during barley seed development.

The DOF protein, SAD, previously shown to be a transcriptional activator in barley aleurone cells upon seed germination, also has an important role in gene regulation during endosperm development. mRNA was detected in early (10 days after flowering) developing barley seeds where it accumulated in the starchy endosperm, aleurone cells, nucellar projection, vascular tissues and the immature embryo, as shown by RT-PCR and in situ hybridization analyses. The SAD protein, expressed in bacteria, binds to oligonucleotides containing the prolamine box, 5'-A/TAAAG-3'sequence, derived from the promoter regions of the endosperm-specific genes Hor2 and Itr1, encoding a B-hordein and trypsin-inhibitor BTI-CMe, respectively. SAD competed for the same binding sites with another endosperm-expressed DOF protein, BPBF. Transient expression experiments in co-bombarded developing endosperms demonstrated that SAD trans-activated transcription from Hor2 and Itr1 promoters through binding to the intact DOF motifs. When the two DOF factors are co-bombarded together an additive effect was observed upon the expression of the Itr1 gene. In-frame fusion of the Sad ORF to the reporter green fluorescent protein gene directs the fluorescence expression to the nucleus in transiently transformed onion epidermal layers. The visualization of fluorescence in the nucleus of onion cells, using the bimolecular fluorescent complex (BiFC) approach, has shown the in vivo interaction between SAD and the R2R3MYB protein GAMYB. The interaction in plant cells has also been documented for the DOF protein BPBF and GAMYB, but nuclear interaction could not be detected between BPBF and SAD by this procedure.

Cell Nucleus↗

Suppression of sucrose synthase gene expression represses cotton fiber cell initiation, elongation, and seed development.

Cotton is the most important textile crop as a result of its long cellulose-enriched mature fibers. These single-celled hairs initiate at anthesis from the ovule epidermis. To date, genes proven to be critical for fiber development have not been identified. Here, we examined the role of the sucrose synthase gene (Sus) in cotton fiber and seed by transforming cotton with Sus suppression constructs. We focused our analysis on 0 to 3 days after anthesis (DAA) for early fiber development and 25 DAA, when the fiber and seed are maximal in size. Suppression of Sus activity by 70% or more in the ovule epidermis led to a fiberless phenotype. The fiber initials in those ovules were fewer and shrunken or collapsed. The level of Sus suppression correlated strongly with the degree of inhibition of fiber initiation and elongation, probably as a result of the reduction of hexoses. By 25 DAA, a portion of the seeds in the fruit showed Sus suppression only in the seed coat fibers and transfer cells but not in the endosperm and embryo. These transgenic seeds were identical to wild-type seeds except for much reduced fiber growth. However, the remaining seeds in the fruit showed Sus suppression both in the seed coat and in the endosperm and embryo. These seeds were shrunken with loss of the transfer cells and were <5% of wild-type seed weight. These results demonstrate that Sus plays a rate-limiting role in the initiation and elongation of the single-celled fibers. These analyses also show that suppression of Sus only in the maternal seed tissue represses fiber development without affecting embryo development and seed size. Additional suppression in the endosperm and embryo inhibits their own development, which blocks the formation of adjacent seed coat transfer cells and arrests seed development entirely.

Cell Division↗

Triacylglycerol synthesis in developing seeds of groundnut (Arachis hypogaea): pathway and properties of enzymes of sn-glycerol 3-phosphate formation.

The enzymatic pathway for the synthesis of sn-glycerol 3-phosphate was investigated in developing groundnut seeds (Arachis hypogaea). Glycerol-3-phosphate dehydrogenase was not detected in this tissue but an active glycerokinase was demonstrated in the cytosolic fraction. It showed an optimum pH at 8.6 and positive cooperative interactions with both glycerol and ATP. Triosephosphate isomerase and glyceraldehyde-3-phosphate phosphatase were observed mainly in the cytosolic fraction while an active glyceraldehyde reductase was found mainly in the mitochondrial and microsomal fractions. The glyceraldehyde 3-phosphate phosphatase showed specificity and positive cooperativity with respect to glyceraldehyde 3-phosphate. The glyceraldehyde reductase was active toward glucose and fructose but not toward formaldehyde and showed absolute specificity toward NADPH. It is concluded that in the developing groundnut seed, sn-glycerol 3-phosphate is synthesized essentially by the pathway dihydroxyacetone phosphate----glyceraldehyde 3-phosphate Pi----glyceraldehyde NADPH----glycerol ATP----glycerol 3-phosphate. All the enzymes of this pathway showed activity profiles commensurate with their participation in triacylglycerol synthesis which is maximal during the period 15-35 days after fertilization. Glycerokinase appears to be the rate-limiting enzyme in this pathway.

Arachis↗

Endosperm: the crossroad of seed development.

The endosperm is an essential part of the seed that sustains embryo development and reserve storage. Several genes that are involved in endosperm differentiation and that have domains of expression arranged along a conserved antero-posterior axis have been isolated in Arabidopsis and in cereals. Endosperm polarity is controlled maternally by chromatin-remodeling complexes. Endosperm development appears to be predominantly under epigenetic controls that might be linked with its evolutionary origin.

Arabidopsis↗

Changes in lipid status and glass properties in cotyledons of developing sunflower seeds.

Biochemical events involved in the acquisition of germinability and storability during orthodox seed development are well documented; however, the roles played by the physical organization of lipids and water are poorly characterized. The aim of this work was to determine, using a thermodynamic approach, whether changes in thermal properties of lipid reserves, and intracellular glasses might play a role in sunflower (Helianthus annuus L.) seed development. Triacyglycerols (TAGs) accumulated in cotyledons until the end of seed filling, which occurred 42 days after anthesis (DAA). Further seed development, leading to mature seed at 58 DAA, was mainly associated with an enlargement of lipid bodies without significant changes either in the lipid content or in their composition. When cooled to -100 degrees C, lipid reserves from cotyledons of mature seeds displayed alpha and beta' polymorphic crystalline structures; however, the ability to form alpha crystals, which was an indicator of lipid purity, progressively appeared during seed development. Characteristics of lipid melting confirmed that seed maturation drying was associated with changes in TAG physical organization. Cotyledon development was associated with an increase in the temperature of glass to rubber transition (Tg), thus suggesting a decrease in molecular mobility during maturation drying. This phenomenon was concomitant with an increase in raffinose content. Our results demonstrate that physical characteristics of lipid reserves and glasses of sunflower cotyledons are developmentally regulated and might play a role in acquisition of seed germinability and storability.

Cold Temperature↗