Differential expression of the Arabidopsis genes coding for Em-like proteins

Late embryogenesis abundant (lea) genes are a large and diverse group of genes highly expressed during late stages of seed development. Five major groups of LEA proteins have been described. Two Em genes (group I lea genes) are present in the genome of Arabidopsis thaliana L., AtEm1 and AtEm6. Both genes encode for very similar proteins which differ basically in the number of repetitions of a highly hydrophilic amino acid motif. The spatial patterns of expression of the two Arabidopsis Em genes have been studied using in situ hybridization and transgenic plants transformed with the promoters of the genes fused to the beta-glucuronidase reporter gene (uidA). In the embryo, AtEm1 is preferentially expressed in the pro-vascular tissues and in meristems. In contrast, AtEm6 is expressed throughout the embryo. The activity of both promoters disappears rapidly after germination, but is ABA-inducible in roots of young seedlings, although in different cells: the AtEm1 promoter is active in the internal tissues (vasculature and pericycle) whereas the AtEm6 promoter is active in the external tissues (cortex, epidermis and root hairs). The AtEm1 promoter, but not AtEm6, is also active in mature pollen grains and collapsed nectaries of young siliques. These data indicate that the two Em proteins could carry out at least slightly different functions and that the expression of AtEm1 and AtEm6 is controlled at, at least, three different levels: temporal, spatial and hormonal (ABA).     

mRNAs for two ribosomal proteins are preferentially translated in the chloroplast of Chlamydomonas reinhardtii under conditions of reduced protein synthesis

Previous studies have identified a set of highly phosphorylated proteins of 23–25 kDa accumulated during normal embryogenesis of Zea mays L. and which disappear in early germination. They can be induced precociously in embryos by abscisic acid (ABA) treatment. Here the synthesis and accumulation of this group of proteins and their corresponding mRNAs were examined in ABA-deficient viviparous embryos at different developmental stages whether treated or not with ABA, and in water-stressed leaves of both wild-type and viviparous mutants.During embryogenesis and precocious germination of viviparous embryos the pattern of expression of the 23–25 kDa proteins and mRNAs closely resembles that found in non-mutant embryo development. They are also induced in young viviparous embryos by ABA treatment. In contrast, leaves of ABA-deficient mutants fail to accumulate mRNA in water stress, yet do respond to applied ABA. In water-stressed leaves of wild type plants the mRNAs are induced and translated into 4 proteins with a molecular weight and isoelectric point identical to those found in embryos.These results indicate that the 23–25 kDa protein set is a new member of the recently described class or proteins involved in generalized plant ABA responses.The different pattern of expression for the ABA-regulated 23–25 kDa proteins and mRNAs found in embryo and in vegetative tissues of viviparous mutants is discussed.     

Proteins in development and germination of a desiccation sensitive (recalcitrant) seed species

In the recalcitrant seeds of Avicennia marina, protein content and the rates of protein synthesis increase during histodifferentiation. This is similar to the situation in desiccation tolerant seeds. During the stage of reserve accumulation the protein content and rates of synthesis remain constant and there is no de novo synthesis of proteins which might qualify as storage proteins. There is also no change in the nature of proteins present in either axis or cotyledonary tissues during development or germination. Similarly, fluorographs of axis proteins show only very limited changes in the patterns of protein synthesis during development and germination, at least until the onset of root growth. Heat-stable proteins are present from an early developmental stage. However, no late embryogenic abundant (LEA) proteins are synthesised during the late stages of development, indicating that seedling establishment is independent of such maturation proteins. It is suggested that the lack of desiccation tolerance of A. marina seeds might be related to the absence of desiccation-related LEAs. Although the rate of protein synthesis increases during germination, protein metabolism appears to remain qualitatively the same as that occurring during development. The present results suggest that in these desiccation sensitive seeds, protein metabolism characterising development changes imperceptibly into that of germination.     

Osmotic stress and abscisic acid induce expression of the wheat Em genes

The early-methionine-labelled (Em) polypeptide is the single most abundant cytosolic protein of dry wheat embryos. It is encoded by messenger RNA which accumulates during the later (maturation) stages of embryogenesis. The accumulation of Em mRNA can be induced in isolated developing embryos, in culture, by the application of the plant growth regulator, abscisic acid, which prevents precocious germination. Precocious germination is also inhibited by the culture of embryos under conditions of osmotic stress when accumulation of Em mRNA is induced. This induction occurs in the absence of any significant increase in the endogenous levels of embryonic abscisic acid although there is a requirement for the continued presence of the growth regulator. Additionally, expression of Em genes can be repeated during early germination, if imbibing embryos are subjected to osmotic stress. Induction of Em-gene expression by osmotic stress is consistent with the proposed role of the Em polypeptide in mediating the remarkable tolerance of cereal embryos to the programmed desiccation undergone during their maturation.     

Fungus- and wound-induced accumulation of mRNA containing a class II chitinase of the pathogenesis-related protein 4 (PR-4) family of maize

Pathogenesis-related (PR) proteins are plant proteins that are induced in response to pathogen attack. PR proteins are grouped into independent families based on their sequences and properties. The PR-4 family comprises class I and class II chitinases. We have isolated a full-length cDNA encoding a chitinase from maize which shares a high degree of nucleotide and amino acid sequence homology with the class II chitinases of the PR-4 family of PR proteins. Our results indicate that fungal infection, and treatment either with fungal elicitors or with moniliformin, a mycotoxin produced by the fungus Fusarium moniliforme, increase the level of ZmPR4 mRNA. In situ mRNA hybridization analysis in sections obtained from fungus-infected germinating embryos revealed that ZmPR4 mRNA accumulation occurs in those cell types that first establish contact with the pathogen. ZmPR4 mRNA accumulation is also stimulated by treatment with silver nitrate whereas the application of the hormones gibberellic acid or acetylsalicylic acid has no effect. Wounding, or treatment with abscisic acid or methyl jasmonate, results in accumulation of ZmPR4 mRNA in maize leaves. Furthermore, the ZmPR4 protein was expressed in Escherichia coli, purified and used to obtain polyclonal antibodies that specifically recognized ZmPR4 in protein extracts from fungus-infected embryos. Accumulation of ZmPR4 mRNA in fungus-infected maize tissues was accompanied by a significant accumulation of the corresponding protein. The possible implications of these findings as part of the general defence response of maize plants against pathogens are discussed.     

Cellular desiccation and hydration: developmentally regulated proteins, and the maturation and germination of seed embryos.

Little more than a decade ago, 2-dimensional mapping of proteins and biochemical study of their allied coding elements (mRNA and DNA) were first used to probe possible changes in the embryo during seed germination. Because specification was of primary importance, our attention was initially directed toward the characterization of individual proteins and coding elements which, in preliminary surveys of the germinating wheat embryo, were found to be conspicuously subject to developmental regulation. Three of the proteins have become subjects of comprehensive investigations in this and other laboratories: the Em protein, the Ec protein, and germin. The Em and Ec proteins are encoded by the conserved mRNA 'stored' in the mature embryos of dry, field-ripened seeds but germin is encoded by the nascent mRNA formed after mature embryos are germinated in water. The Ec protein is the only bona fide Zn metallothionein yet found in higher plants. Studies of their biology and molecular biology suggest that the Em protein has a role in hormone-mediated (abscisic acid) cellular desiccation and that germin has a role in hormone-mediated (auxin) cellular hydration. It is projected that further studies of the Em protein may help elucidate the molecular basis for a loss of dessication tolerance during germination, and that further studies of germin may help elucidate the molecular basis of plant cell enlargement.     

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.     

Molecular expression of <Emphasis Type="Italic">PsPIN1</Emphasis>, a putative auxin efflux carrier gene from pea (<Emphasis Type="Italic">Pisum sativum</Emphasis> L.)

A cDNA coding for a putative auxin efflux carrier was amplified from Pisum sativum seedling shoot tips by RT-PCR and the corresponding full-length cDNA, PsPIN1, was subsequently obtained by RACE-PCR. The deduced amino acid sequence (599 residues) showed the three domain topology typical of the other PIN proteins. The PsPIN1 protein structure prediction possessed five transmembrane domains at both the N-(7-150) and C-(450-575) termini and a hydrophylic region in the middle. PsPIN1 showed highest similarity to Medicago, MtPIN4. Using the Genome Walking technique, a 1511 bp upstream region for PsPIN1 gene was sequenced. This PsPIN1 upstream region possessed multiple putative auxin, GA and light regulatory elements. The PsPIN1 mRNA was ubiquitously expressed throughout the pea plant, especially in growing tissues. Auxin induced PsPIN1 mRNA in dark grown pea seedling shoot tips. It was induced by 4-chloro-IAA, which is also an active auxin in pea, and by gibberellin (GA₃). Interestingly, the PsPIN1 mRNA was down-regulated by light treatment, possibly because light negatively regulates auxin and, especially GA levels in pea. Thus PIN1-mediated auxin efflux is a highly regulated process, not only at the level of protein localization, but also at the level of mRNA accumulation.     

Purification and properties of an endospermic protein of maize associated with the Opaque-2 and Opaque-6 genes

Maize endosperms accumulate during development a large amount of storage proteins (zeins). The rate of zein accumulation is under the control of several regulatory genes. Two of these, the opaque-2 and opaque-6 mutants, lower the zein level, thus improving the nutritional quality of maize meals. An endosperm protein of Mr 32 000 (b-32) appears to be correlated with the zein level. The b-32 protein is encoded by the opaque-6 gene which, in turn, is activated by opaque-2. We report the purification, amino-acid composition and peptide map of b-32 protein. Furthermore we demonstrate that the protein exists as a monomer likely located in the soluble cytoplasm. As a step towards the isolation of a complementary-DNA clone for b-32 protein, the purification of its corresponding mRNA is described.Abbreviations b-32 endosperm protein of M_r 32000 - cDNA complementary DNA - EDTA ethylenediaminetetraacetic acid - O2, O6 opaque 2, opaque-6 genes - PMSF phenylmethylsulfonylfluoride - RSP reduced soluble proteins - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis     

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.     

Aspetti Enzimatici Della Maturazione e Germinazione dei Semi di Ricino

Abstract

Enzyme levels during ripening and germination of castor bean seeds. — During the development of the endosperm of castor bean seeds two distinct phases can be recognized: pre-maturation and germination. The former is characterized, metabolically, by the rapid conversion of carbohydrates into lipids, and storage proteins. The latter is characterized by the reconversion of these storage materials into sugars. Both these processes are dependent upon the activity of the glycolytic pathway; for this reason the behaviour of some enzymes of this pathway and, in general, of the carbohydrate metabolism have been studied during the two phases. The changes (during the evolution of the seeds) of the following enzymes have been studied:

Gl-6-P-dehydrogenase, 6-P-gluconate dehydrogenase, P-glucomutase, Hexokinase Hexoseisomerase, Aldolase, alcaline and acid Phosphatase, Pyrophosphatase and ATP-ase.

All these activities have been measured in the 20.000 × g supernatant fraction of cell homogenates.

The results show that all the enzymes activities measured increase rapidly during the period of accumulation of storage materials. In the following period all of these activities decrease until the stage of ripeness of the seed. During the first few days of germination the activities increase again rapidly. A particular behaviour is the one of Fr-1-6-P-phosphatase (the enzyme cleaving the phosphate bond in C ¹ position). This enzyme reaches during germination a level much higher than the maximal observed during the ripening process. This might be an important fact correlated with the inversion of the glycolytic reactions during germination.     

Expression of genes for cell-wall proteins in dividing and wounded tissues ofZea mays L.

Hydroxyproline-rich glycoproteins (HRGPs) fromZea mays have been immunolocalized in the cell wall of root tip cells using ultrathin sections and antibodies ellicited against the purified protein. The accumulation of mRNA corresponding to this protein was studied using the cDNA probe. Maximum accumulation of the mRNA was found in tissues with a high proportion of dividing cells such as those in the root tip of young maize seedlings and a close relationship with cellular division was also observed in in-vitro cultures. However, the level of the mRNA in elongating tissues was minimal, as shown by studies carried out on the elongation zones of root tips and coleoptiles. The mRNA was induced by stress conditions, particularly by wounding young leaves and coleoptiles. It is concluded that in maize this group of proline-rich cell-wall proteins accumulates during cell division and not during cell elongation or differentiation, and participates in the stress-response mechanisms of the plant.