Expression of the gene encoding the PR-like protein PRms in germinating maize embryos

Summary The PRms protein is a pathogenesis-related (PR)-like protein whose mRNA accumulates during germination of maize seeds. Expression of the PRms gene is induced after infection of maize seeds with the fungus Fusarium moniliforme. To further our investigations on the expression of the PRms gene we examined the accumulation of PRms mRNA in different tissues of maize seedlings infected with E. moniliforme and studied the effect of fungal elicitors, the mycotoxin moniliformin, the hormone gibberellic acid, and specific chemical agents. Our results indicate that fungal infection, and treatment either with fungal elicitors or with moniliformin, a mycotoxin produced by F. monilforme, increase the steady-state level of PRms mRNA. PRms mRNA accumulation is also stimulated by the application of the hormone gibberellic acid or by treatment with silver nitrate, whereas acetylsalicylic acid has no effect. In situ RNA hybridization in isolated germinating embryo sections demonstrates that the PRms gene is expressed in the scutellum, particularly in a group of inner cells, and in the epithelium lying at the interface of the scutellum and the endosperm. The pattern of expression of the PRms gene closely resembles that found for hydrolytic enzymes, being confined to the scutellum and the aleurone layer of the germinating maize seed. Our results suggest that the PRms protein has a function during the normal process of seed germination that has become adapted to serve among the defence mechanisms induced in response to pathogens during maize seed germination.     

Acidification of the Medium Associated with Normal and Fusicoccin-Induced Seed Germination

Fusicoccin, a toxin stimulating cell enlargement and inducing proton extrusion in various plant tissues, has been shown to replace kinetin, gibberellic acid and red light in breaking seed dormancy. It also removes the inhibitory effect of abscisic acid. The present data also show that the stimulating effect of fucicoccin on embryo growth of decoated radish (Raphanus sativus L.) and maize (Zea mays) seeds and on the development of maize embryos is accompanied by an early, significant acidification of the medium. Acidification of the medium is also observed when fusicoccin reverses the abscisic acid-induced inhibition of germination. These results support the hypothesis that the mode of action of fusicoccin in promoting germination involves, as in stimulation of cell enlargement, the activation at the cell membrane level of proton extrusion processes. The physiological significance of fusicoccin-induced release of protons at the onset of germination is discussed in comparison with the results concerning the mechanism of action of fusicoccin on cell enlargement in other plant materials.     

Reserve mobilization in the Arabidopsis endosperm fuels hypocotyl elongation in the dark, is independent of abscisic acid, and requires PHOSPHOENOLPYRUVATE CARBOXYKINASE1

Arabidopsis thaliana is used as a model system to study triacylglycerol (TAG) accumulation and seed germination in oilseeds. Here, we consider the partitioning of these lipid reserves between embryo and endosperm tissues in the mature seed. The Arabidopsis endosperm accumulates significant quantities of storage lipid, and this is effectively catabolized upon germination. This lipid differs in composition from that in the embryo and has a specific function during germination. Removing the endosperm from the wild-type seeds resulted in a reduction in hypocotyl elongation in the dark, demonstrating a role for endospermic TAG reserves in fueling skotomorphogenesis. Seedlings of two allelic gluconeogenically compromised phosphoenolpyruvate carboxykinase1 (pck1) mutants show a reduction in hypocotyl length in the dark compared with the wild type, but this is not further reduced by removing the endosperm. The short hypocotyl phenotypes were completely reversed by the provision of an exogenous supply of sucrose. The PCK1 gene is expressed in both embryo and endosperm, and the induction of PCK1:beta-glucuronidase at radicle emergence occurs in a robust, wave-like manner around the embryo suggestive of the action of a diffusing signal. Strikingly, the induction of PCK1 promoter reporter constructs and measurements of lipid breakdown demonstrate that whereas lipid mobilization in the embryo is inhibited by abscisic acid (ABA), no effect is seen in the endosperm. This insensitivity of endosperm tissues is not specific to lipid breakdown because hydrolysis of the seed coat cell walls also proceeded in the presence of concentrations of ABA that effectively inhibit radicle emergence. Both processes still required gibberellins, however. These results suggest a model whereby the breakdown of seed carbon reserves is regulated in a tissue-specific manner and shed new light on phytohormonal regulation of the germination process.     

The molecular genetics of seed maturation in maize

The maturation phase of seed formation involves coordinated expression of multiple developmental pathways. These processes include abscisic acid regulated responses associated with the arrest of embryo development and induction of anthocyanin synthesis in embryo and aleurone tissues. Studies of the maturation defective vivaparous mutants of maize suggest that one gene, viviparous-1 ( vpl ), regulates both of these pathways in the developing seed. Mutations at vpl reduce the sensitivity of the developing embryo to abscisic acid. In addition, Vpl is required for expression of Cl , a regulatory gene for the anthocyanin pathway. This interaction is consistent with the idea that Vpl and Cl function as part of a regulatory hierarchy controlling seed development. Molecular studies of vpl mutations which separate control of embryo arrest and anthocyanin synthesis suggest that these functions map to discrete domains in the Vpl protein. Therefore, coordinate control of diverse maturation processes may be achieved through expression of a functionally complex regulatory molecule.     

Rapeseed embryo development in culture on high osmoticum is similar to that in seeds

The development of Brassica napus L. cv Tower embryos of different ages cultured in vitro with and without high osmoticum (0.48 and 0.69 molar sorbitol) was compared with normal development in situ to investigate the role of a drying environment in embryo maturation. Sensitivity to osmoticum was assayed in terms of its ability to mimic normal development, i.e. to both suppress germination and maintain 12 S storage protein (cruciferin) synthesis at levels comparable to those seen in the developing seed. The osmotic conditions used block germination of predesiccation stage embryos but were not sufficient to prevent desiccation stage embryos from taking up water and germinating. At all stages tested, the osmotically treated embryos had approximately normal levels of cruciferin mRNA. Measurements of endogenous abscisic acid (ABA) levels by radioimmunoassay indicated that the osmotic effects on germination and gene expression were not mediated by elevated embryonic ABA. Comparison of the kinetics of osmotic and ABA effects on gene expression showed that the osmotic effect is more rapid. These results are consistent with the hypothesis that ABA acts by inhibiting water uptake, which mechanically prevents germination and affects gene expression in some unknown manner.     

A mutant of Arabidopsis which is defective in seed development and storage protein accumulation is a new abi3 allele

In order to investigate the role of the plant hormones gibberellin (GA) and abscisic acid (ABA) in seed development and germination the GA biosynthetic inhibitor, Uniconazol, was used to isolate mutants with abnormal germination profiles. In one of these mutants, the ability to germinate on Uniconazol is due to a mutation in the ABI3 gene. However, unlike the previously reported abi3 mutant, this line displays an array of seed-specific developmental defects. The accumulation of seed reserve proteins is dramatically reduced due to reduced levels of the storage protein mRNA. The embryos remain green throughout development and are desiccation intolerant. However, immature seeds are completely non-dormant and grow normally. These results suggest the ABI3 gene is essential for the synthesis of seed storage proteins and for the protection of the embryo during desiccation.     

Interaction of nuclear factors with upstream sequences of a lipid body membrane protein gene from carrot.

To study the regulation of gene expression during embryo development, we isolated a gene, DC 59, expressed in embryos but not in mature carrot plants. Sequence and S1 analysis showed that the gene was composed of one exon encoding a polypeptide of 19 kilodaltons and was highly homologous to the lipid body membrane protein gene L3 from maize. The plant hormone abscisic acid regulated the accumulation of DC 59 mRNA. To understand the mechanism of embryo-specific and hormonal regulation of DC 59, 5' DNA fragments were incubated with nuclear proteins. Two adjacent regions (from -706 to -235) interacted with nuclear extracts from embryos, resulting in the formation of four complexes (C1, C2, C3, and C4). Factors involved in the formation of the C3 and C4 complexes could be competed with sequences upstream of DC 8, a gene that is coordinately expressed with DC 59 during embryo development. DNase I footprinting analysis revealed that nuclear extracts from embryos bound to four AT-rich sequences, and the protected motifs within fragment V were located in the highly homologous upstream regions of DC 59 and DC 8 genes.     

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.