Expression and functions of myo-inositol monophosphatase family genes in seed development of Arabidopsis

Myo-inositol monophosphatase (IMP) catalyzes the dephosphorylation of myo-inositol 3-phosphate in the last step of myo-inositol biosynthesis. IMP is also important in phosphate metabolism and is required for the biosynthesis of cell wall polysaccharides, phytic acid, and phosphatidylinositol. In Arabidopsis, IMP is encoded by VTC4. There are, however, two additional IMP candidate genes, IMPL1 and IMPL2, which have not yet been elucidated. In our genetic studies of Arabidopsis IMP genes, only the loss-of-function mutant impl2 showed embryonic lethality at the globular stage. All IMP genes were expressed in a similar manner both in the vegetative and reproductive organs. In developing seeds, expression of IMP genes was not coupled with the expression of the genes encoding myo-inositol phosphate synthases, which supply the substrate for IMPs in the de novo synthesis pathway. Instead, expression of IMP genes was correlated with expression of the gene for myo-inositol polyphosphate 1-phosphatase (SAL1), which is involved in the myo-inositol salvage pathway, suggesting a possible salvage pathway role in seed development. Moreover, the partial rescue of the impl2 phenotype by histidine application implies that IMPL2 is also involved in histidine biosynthesis during embryo development.     

Non-redundant functions of sHSP-CIs in acquired thermotolerance and their role in early seed development in Arabidopsis

Heat-shock proteins (HSPs) are a group of evolutionarily conserved polypeptides whose expression is induced in all organisms in response to environmental stresses and during various developmental processes. In this work, we show that the rose (Rosa hybrida) cytoplasmic 17.5-kDa Class I small HSP (sHSP17.5-CI, accession number: BQ103946) increases dramatically during flower development, and accumulates in closed bud petals and leaves only in response to heat stress. mRNA for a putative ortholog of this protein is also found in petals, but not leaves, of Arabidopsis (Arabidopsis thaliana) plants grown under optimal conditions, and it accumulates in leaves in response to heat stress. Analysis of Arabidopsis T-DNA insertion lines affected at three homologous genes revealed that their acquired thermotolerance, as measured by hypocotyl-elongation assay, is impaired. The correlation between sHSP-CI accumulation and expansion of rose petal cells, impairment of acquired thermotolerance, and defects in early embryogenesis of the double mutants (hsp17.4/hsp17.6A), all suggest that sHSP-CI proteins play a role in protecting cell proteins at various developmental stages, whereas in hypocotyl elongation they have a non-redundant function in acquired thermotolerance but have a redundant function in early embryogenesis.     

Genetic analysis of seed coat development in Arabidopsis

In the angiosperms, fertilization initiates the formation of the seed from the ovule, including the differentiation of the seed coat from the ovule integuments. Seed coat differentiation includes some of the most dramatic cellular changes of seed development and culminates in the death of the seed coat cells. Recently, genetic analyses in Arabidopsis have contributed substantially to our understanding of many aspects of seed coat biology and it might not be long before the entire differentiation pathway is understood. Such an advance would contribute substantially to our understanding of many important cellular events, including secondary cell wall synthesis, cell morphogenesis, vacuolar targeting and cell death, and would provide tools for the manipulation of seed dormancy and germination.     

Insertional mutagenesis of genes required for seed development in Arabidopsis thaliana.

The purpose of this project was to identify large numbers of Arabidopsis genes with essential functions during seed development. More than 120,000 T-DNA insertion lines were generated following Agrobacterium-mediated transformation. Transgenic plants were screened for defective seeds and putative mutants were subjected to detailed analysis in subsequent generations. Plasmid rescue and TAIL-PCR were used to recover plant sequences flanking insertion sites in tagged mutants. More than 4200 mutants with a wide range of seed phenotypes were identified. Over 1700 of these mutants were analyzed in detail. The 350 tagged embryo-defective (emb) mutants identified to date represent a significant advance toward saturation mutagenesis of EMB genes in Arabidopsis. Plant sequences adjacent to T-DNA borders in mutants with confirmed insertion sites were used to map genome locations and establish tentative identities for 167 EMB genes with diverse biological functions. The frequency of duplicate mutant alleles recovered is consistent with a relatively small number of essential (EMB) genes with nonredundant functions during seed development. Other functions critical to seed development in Arabidopsis may be protected from deleterious mutations by extensive genome duplications.     

From seed to seed: the role of photoreceptors in Arabidopsis development

As sessile organisms, plants have evolved a multitude of developmental responses to cope with the ever-changing environmental conditions that challenge the plant throughout its life cycle. Of the many environmental cues that regulate plant development, light is probably the most important. From determining the developmental pattern of the emerging seedling, to influencing the organization of organelles to best maximize energy available for photosynthesis, light has dramatic effects on development during all stages of plant life. In plants, three classes of photoreceptors that mediate light perception have been characterized at the molecular level. The phytochromes recognize light in the red portion of the spectrum, while cryptochromes and phototropins perceive blue and UVA light. In this review, we discuss the different aspects of development that are regulated by these photoreceptors in the model plant species Arabidopsis thaliana and how the phytochromes, cryptochromes, and phototropins bring about changes in development seen in the growing plant.     

Two O-linked N-acetylglucosamine transferase genes of Arabidopsis thaliana L. Heynh. have overlapping functions necessary for gamete and seed development

The Arabidopsis SECRET AGENT (SEC) and SPINDLY (SPY) proteins are similar to animal O-linked N-acetylglucosamine transferases (OGTs). OGTs catalyze the transfer of N-acetylglucosamine (GlcNAc) from UDP-GlcNAc to Ser/Thr residues of proteins. In animals, O-GlcNAcylation has been shown to affect protein activity, stability, and/or localization. SEC protein expressed in Escherichia coli had autocatalytic OGT activity. To determine the function of SEC in plants, two tDNA insertional mutants were identified and analyzed. Although sec mutant plants did not exhibit obvious phenotypes, sec and spy mutations had a synthetic lethal interaction. This lethality was incompletely penetrant in gametes and completely penetrant postfertilization. The rate of both female and male sec spy gamete transmission was higher in plants heterozygous for both mutations than in plants heterozygous for sec and homozygous for spy. Double-mutant embryos aborted at various stages of development and no double-mutant seedlings were obtained. These results indicate that OGT activity is required during gametogenesis and embryogenesis with lethality occurring when parentally derived SEC, SPY, and/or O-GlcNAcylated proteins become limiting.     

Control of seed development in Arabidopsis thaliana by atmospheric oxygen

Seed development is known to be inhibited completely when plants are grown in oxygen concentrations below 5·1 kPa, but apart from reports of decreased seed weight little is known about embryogenesis at subambient oxygen concentrations above this critical level. Arabidopsis thaliana (L.) Heynh. plants were grown full term under continuous light in premixed atmospheres with oxygen partial pressures of 2·5, 5·1, 10·1, 16·2 and 21·3 kPa O₂, 0·035 kPa CO₂ and the balance nitrogen. Seeds were harvested for germination tests and microscopy when siliques had yellowed. Seed germination was depressed in O₂ treatments below 16·2 kPa, and seeds from plants grown in 2·5 kPa O₂ did not germinate at all. Fewer than 25% of the seeds from plants grown in 5·1 kPa oxygen germinated and most of the seedlings appeared abnormal. Light and scanning electron microscopic observation of non-germinated seeds showed that these embryos had stopped growing at different developmental stages depending upon the prevailing oxygen level. Embryos stopped growing at the heart-shaped to linear cotyledon stage in 5·1 kPa O₂, at around the curled cotyledon stage in 10·1 kPa O₂, and at the premature stage in 16·2 kPa O₂. Globular and heart-shaped embryos were observed in sectioned seeds from plants grown in 2·5 kPa O₂. Tissue degeneration caused by cell autolysis and changes in cell structure were observed in cotyledons and radicles. Transmission electron microscopy of mature seeds showed that storage substances, such as protein bodies, were reduced in subambient oxygen treatments. The results demonstrate control of embryo development by oxygen in Arabidopsis .     

LEUNIG has multiple functions in gynoecium development in Arabidopsis

The Arabidopsis gene LEUNIG was previously found to regulate floral organ identity. In this work we describe gynoecial phenotypes of newly isolated strong leunig alleles, leunig-101, leunig-102, and leunig-103. Gynoecia of these strong leunig mutants are united only at the basal part, leaving four unfused parts at the apex. Among them two medial ones are styles capped with stigmas, and two lateral ones are protrusions from valves. The gynoecium with unfused apex in leunig arises as a unit from a basal meristematic zone, suggesting that LEUNIG is required for normal congenital gynoecium fusion. The epidermal cells on growing inner surfaces of leunig gynoecium failed to fuse after they contact each other, indicating that LEUNIG is essential for the proper postgenital fusion. The epidermal cells at the very distal portion of protruded valves mimic those on wild-type styles, and those valves occasionally also have stigma-like tissues, indicating that LEUNIG function is required for the valve identity determination. We have also analyzed clavata1-4 leunig-101, clavata2-1 lug-101, fruitfull-1 leunig-101, and pinoid-1 leunig-101 double mutants. clavata1-4 leunig-101 and clavata2-1 leunig-101 exhibited additive phenotypes of single mutants, suggesting that LEUNIG and CLAVATA genes function in different pathways. In contrast, FRUITFULL and PINOID genes interact with LEUNIG to regulate gynoecium development. genesis 26:42-54, 2000.     

An integrated overview of seed development in Arabidopsis thaliana ecotype WS

This work is part of a research program aiming at identifying and studying genes involved in Arabidopsis thaliana seed maturation. We focused here on the Wassilewskija ecotype seed development and linked physiological and biochemical data, including protein, oil, soluble sugars, starch and free amino acid measurements, to embryo development, to obtain a complete and thorough reference data set. A. thaliana seed development can be divided into three stages. During early embryogenesis (i.e. morphogenesis), seed weight and lipid content were low whereas important amounts of starch were transiently accumulated. In the second stage, or maturation phase, a rapid increase in seed dry weight was observed and storage oils and proteins were accumulated in large quantities, accounting for approximately 40% of dry matter each at the end of this stage. During the third and last stage (late maturation including acquisition of desiccation tolerance), seed dry weight remained constant while an acute loss of water took place in the seed. Storage compound synthesis ended concomitantly with sucrose, stachyose and raffinose accumulation. This study revealed the occurrence of metabolic activities such as protein synthesis, in the final phase of embryo desiccation. A striking correlation between peaks in hexose to sucrose ratio and transition phases during embryogenesis was observed.     

Disruptions in valine degradation affect seed development and germination in Arabidopsis

We have functionally characterized the role of two putative mitochondrial enzymes in valine degradation using insertional mutants. Prior to this study, the relationship between branched‐chain amino acid degradation (named for leucine, valine and isoleucine) and seed development was limited to leucine catabolism. Using a reverse genetics approach, we show that disruptions in the mitochondrial valine degradation pathway affect seed development and germination in Arabidopsis thaliana. A null mutant of 3‐hydroxyisobutyryl‐CoA hydrolase (CHY4, At4g31810) resulted in an embryo lethal phenotype, while a null mutant of methylmalonate semialdehyde dehydrogenase (MMSD, At2g14170) resulted in seeds with wrinkled coats, decreased storage reserves, elevated valine and leucine, and reduced germination rates. These data highlight the unique contributions CHY4 and MMSD make to the overall growth and viability of plants. It also increases our knowledge of the role branched‐chain amino acid catabolism plays in seed development and amino acid homeostasis.     

Parent-of-origin effects on seed development in Arabidopsis thaliana require DNA methylation

Some genes in mammals and flowering plants are subject to parental imprinting, a process by which differential epigenetic marks are imposed on male and female gametes so that one set of alleles is silenced on chromosomes contributed by the mother while another is silenced on paternal chromosomes. Therefore, each genome contributes a different set of active alleles to the offspring, which develop abnormally if the parental genome balance is disturbed. In Arabidopsis, seeds inheriting extra maternal genomes show distinctive phenotypes such as low weight and inhibition of mitosis in the endosperm, while extra paternal genomes result in reciprocal phenotypes such as high weight and endosperm overproliferation. DNA methylation is known to be an essential component of the parental imprinting mechanism in mammals, but there is less evidence for this in plants. For the present study, seed development was examined in crosses using a transgenic Arabidopsis line with reduced DNA methylation. Crosses between hypomethylated and wild-type diploid plants produced similar seed phenotypes to crosses between plants with normal methylation but different ploidies. This is consistent with a model in which hypomethylation of one parental genome prevents silencing of alleles that would normally be active only when inherited from the other parent - thus phenocopying the effects of extra genomes. These results suggest an important role for methylation in parent-of-origin effects, and by inference parental imprinting, in plants. The phenotype of biparentally hypomethylated seeds is less extreme than the reciprocal phenotypes of uniparentally hypomethylated seeds. The observation that development is less severely affected if gametes of both sexes (rather than just one) are 'neutralized' with respect to parent-of-origin effects supports the hypothesis that parental imprinting is not necessary to regulate development.     

The dioxygenase GIM2 functions in seed germination by altering gibberellin production in Arabidopsis

The phytohormones gibberellic acid (GA) and abscisic acid (ABA) antagonistically control seed germination. High levels of GA favor seed germination, whereas high levels of ABA hinder this process. The direct relationship between GA biosynthesis and seed germination ability need further investigation. Here, we identified the ABA‐insensitive gain‐of‐function mutant germination insensitive to ABA mutant 2 (gim2) by screening a population of XVE T‐DNA‐tagged mutant lines. Based on two loss‐of‐function gim2‐ko mutant lines, the disruption of GIM2 function caused a delay in seed germination. By contrast, upregulation of GIM2 accelerated seed germination, as observed in transgenic lines overexpressing GIM2 (OE). We detected a reduction in endogenous bioactive GA levels and an increase in endogenous ABA levels in the gim2‐ko mutants compared to wild type. Conversely, the OE lines had increased endogenous bioactive GA levels and decreased endogenous ABA levels. The expression levels of a set of GA‐ and/or ABA‐related genes were altered in both the gim2‐ko mutants and the OE lines. We confirmed that GIM2 has dioxygenase activity using an in vitro enzyme assay, observing that GIM2 can oxidize GA₁₂. Hence, our characterization of GIM2 demonstrates that it plays a role in seed germination by affecting the GA metabolic pathway in Arabidopsis.