Adela Goday

Gene sequence, developmental expression, and protein phosphorylation of RAB-17 in maize

The ABA-induced MA12 cDNA from maize, which encodes a set of highly phosphorylated embryo proteins, was used to isolate the corresponding genomic clone. This gene, called RAB-17 (responsive to ABA), encodes a basic, glycine-rich protein (mol. wt. 17 164) containing a cluster of 8 serine residues, seven of them contiguous. It is a homologue of the rice RAB-21 gene (Mundy J, Chua NH, EMBO J 7; 2279–2286, 1988). Phosphoamino acid analysis of the isolated protein indicates that only the serine residues are phosphorylated and a putative casein-type kinase phosphorylatable sequence was identified in the protein. The pattern of expression and in vivo phosphorylation of the RAB-17 protein was studied during maize embryo germination and in calli of both meristematic or embryonic origin. ABA treatment induced the synthesis of RAB-17 mRNA and protein in calli, however, the RAB-17 proteins were found to be highly phosphorylated only in embryos.

Regulation of the abscisic acid-responsive gene rab28 in maize viviparous mutants

SummaryWe have isolated a new maize gene, rab 28, that responds to abscisic acid (ABA) treatment. This gene has been characterized by determining the sequence of the cDNA and corresponding genomic copy, and by mapping the start site of its transcript. The rab 28 gene encodes a protein of predicted molecular weight 27713 Da which shows strong homology with the Lea D-34 protein identified in cotton. The proximal promoter region contains the conserved ABA-response element, CACGTGG, reported in other plant genes to be responsible for ABA induction. rab 28 mRNA has been identified as ABA-inducible in embryos and young leaves. It is also induced by water-stress in leaves of wild-type plants. Regulation of the rab 28 gene was studied in maize viviparous mutants. The results obtained with the ABA-insensitive vp1 mutant show that rab 28 transcripts do not accumulate to a significant level during embryogenesis. Surprisingly, induction of rab 28 mRNA can be achieved in young embryos by exogenous ABA treatment. Moreover, water-stressed or ABA-treated seedlings of vp1 contain significant levels of rab 28 mRNA which is not detectable in well-watered seedlings. Regulation of the rab 28 gene in excised young embryos of ABA-deficient vp2 mutants, in which influences of the maternal environment are absent, closely resembles that found in non-mutant excised young embryos. The significance of these results is discussed.

Plant responses to drought, from ABA signal transduction events to the action of the induced proteins

The plant hormone abscisic acid (ABA) is necessary for the regulation of several aspects during seed development and for the response to environmental stresses such as desiccation, salt and cold. An important part of the physiological response to ABA is achieved through gene expression. Although different signal molecules have been identified, the signal transduction pathway from ABA perception to the induction of specific genes is not very well established yet. In particular, phosphorylation and dephosphorylation events have been identified as important in ABA signalling. A great effort has been focused on analysing the promoters of ABA-responsive genes in order to identify cis- and trans-acting elements that mediate transcriptional regulation. Many different proteins accumulate in response to water deficit, some of them with a clear function in protecting the cells against dehydration. Other proteins, whose function is not completely understood, include LEA proteins that accumulate during late embryogenesis and in response to ABA.

Differential regulation of ABA-induced 23-25 kDa proteins in embryo and vegetative tissues of the viviparous mutants of maize.

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.

Proteomic analysis of wheat embryos with 2-DE and liquid-phase chromatography (ProteomeLab PF-2D) - a wider perspective of the proteome.

Cereal embryos are a model system to study desiccation tolerance due to their ability to survive extreme water loss during late embryogenesis. To identify proteins accumulating in mature embryos which can be used as potential markers for dehydration tolerance, we compared the embryo proteome from two durum wheat genotypes (Triticum durum Desf.), Mahmoudi (salt and drought sensitive) and Om Rabia3 (salt and drought tolerant). Total protein extracts from wheat embryos were analyzed by using conventional 2-DE and ProteomeLab PF-2D. Analysis using different pH ranges showed that a larger number of fractions were solved by LC, than by conventional 2-DE at extreme technical pHs (pH 4.0-5.0 and pH 6.5-8.0). In contrast, at intermediate pHs (pH 5.0-6.5), resolution was better in 2-DE gels. The two techniques were used in parallel to analyze total protein extracts from embryos of the two wheat varieties. Several proteins belonging to the seed storage family, LEA-type/heat shock proteins, enzyme metabolism and radical scavengers were identified by analysis of trypsin digested peptides via mass spectrometry. These proteins accumulate in different amounts in embryos of tolerant and sensitive wheat varieties. The differences in expression pattern were further validated by enzyme activity, western blotting analysis and correlated with their corresponding mRNA expression by RT-PCR analyses for the corresponding protein. We suggest that the differential expression pattern could be used as a basis for a biochemical screen of tolerance/sensitivity to drought and salt stress in wheat embryos and germplasm.

Drought signal transduction in plants

Water deficit is one of the most common environmental limitations of crop productivity by affecting growth through alterations in metabolism and gene expression. The mechanisms involved in drought perception and signal transduction pathways are poorly understood. The participation of the plant hormone abscisic acid (ABA) has been well established. ABA levels increase when there are changes in the environment that result in cellular dehydration. Different approaches have been taken to understanding the molecular responses to desiccation and how ABA regulates gene expression. Recent efforts have identified particular topics of importance in the dissection of the signal transduction pathway which are summarized as follows: physiological approaches: identification of signalling molecules. Genetic approaches: the use of mutants, and Molecular approaches: promoter analysis.

Arabidopsis thaliana atrab28: a nuclear targeted protein related to germination and toxic cation tolerance.

The Arabidopsis gene Atrab28 has been shown to be expressed during late embryogenesis. The pattern of expression of Atrab28 mRNA and protein during embryo development is largely restricted to provascular tissues of mature embryos, and in contrast to the maize Rab28 homologue it cannot be induced by ABA and dehydration in vegetative tissues.Here, we have studied the subcellular location of Atrab28 protein and the effect of its over-expression in transgenic Arabidopsis plants. The Atrab28 protein was mainly detected in the nucleus and nucleolus of cells from mature embryos. In frame fusion of Atrab28 to the reporter green fluorescent protein (GFP) directed the GFP to the nucleus in transgenic Arabidopsis and in transiently transformed onion cells. Analysis of chimeric constructs identified an N-terminal region of 60 amino acids containing a five amino acid motif QPKRP that was necessary for targeting GFP to the nucleus. These results indicate that Atrab28 protein is targeted to the nuclear compartments by a new nuclear localization signal (NLS). Transgenic Arabidopsis plants, with gain of Atrab28 function, showed faster germination rates under either standard or salt and osmotic stress conditions. Moreover, improved cation toxicity tolerance was also observed not only during germination but also in seedlings. These results suggest a role of Atrab28 in the ion cell balance during late embryogenesis and germination.

Regulation of the rab17 gene promoter in transgenic Arabidopsis wild-type, ABA-deficient and ABA-insensitive mutants

The abscisic acid-responsive gene rab17 is induced during maize embryo maturation and in vegetative tissues under water stress conditions. To investigate how ABA is involved in the induction of the rab17 gene, we present here a genetic approach to analyse the transcriptional regulation of the 1.3 kb rab17 promoter fragment in transgenic wild-type Arabidopsis and mutants which are deficient (aba) and insensitive (abi1, abi2 and abi3) to ABA. During seed development the rab17 promoter fragment confers similar temporal and spatial regulation on the reporter gene GUS, both in transgenic wild-type and ABA-deficient and ABA-insensitive mutants. The rab17 promoter was only active in embryo and endosperm during late seed development, although the ABA-deficient embryo mutant showed a reduction in the level of GUS activity. During germination rab17 promoter activity decreases, and GUS activity is not enhanced by water stress in transgenic wild-type and mutant plants. In contrast, transcription of the Arabidopsis endogenous rab gene is stimulated by water stress, both in wild-type and ABA-insensitive mutants. Our data suggest that different molecular mechanisms mediate seed-specific expression and ABA water stress induction of the rab17 gene and indicate strong conservation of the seed-specific regulatory mechanism for rab genes in monocot and dicot plants.

Molecular Biological Responses to Drought in Maize

Almost all plants can withstand extremes of osmotic stress at some stages of their life cycle. This is the case in cereal seed whose embryos can tolerate reductions in water content of about 80%. Such severe desiccation kills cells from other parts of the plant. During growth, plants also endure mild water deficits (10–15% reduction of their water content) in dry periods. However, the molecular mechanisms contributing to tolerance of dehydration are largely unresolved. The hormone, abscisic acid (ABA), appears to modulate the responses of plants under conditions of water deficit. Developmental studies of seeds and physiological studies of the effects of water stress indicate that ABA controls quantitative and qualitative aspects of the accumulation of specific mRNAs and proteins. Despite considerable research in this field, the function of the ABA- responsive proteins remains elusive and the mode of action of the hormone is unknown. The rab genes of maize are expressed in different plant tissues in response to ABA and osmotic stress. Current evidence indicates that more than one mechanism determines the level of expression of the rab genes. One level of control involves transcription of the rab genes. Specific sequence elements directing hormone responsiveness are localised in discrete regions of the rab gene promoters, rab mRNAs are induced in all tissues by ABA. In contrast, the level of rab proteins differs between embryo and vegetative tissues since they are strongly accumulated in dry embryos but poorly represented in the overall protein pattern of ABA- treated or water-stressed vegetative tissues. These data indicate that different levels of control are involved in the regulation of rab mRNA stability and protein accumulation during ABA-induced responses.

PLANT AP2/EREBP AND bZIP TRANSCRIPTION FACTORS: STRUCTURE AND FUNCTION

Regulation of gene expression by transcription factors is a central mechanism utilized among organisms and relies on interactions between sequence-specific DNA-binding proteins with cis-elements located in the promoter and enhancer regions of the corresponding genes. In plants, a large number of transcription factors has been identified the past years, with some of them exhibiting structural features unique to plant transcription factors. In this review we summarize the current knowledge on the AP2/EREBP and bZIP families of plant transcription factors stressing out putative ways of their mode of regulation and function.