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.

The maize abscisic acid (ABA)-responsive rabl7 mRNA and Rabl7 protein distribution in maize embryo tissues was investigated by in situ hybridization and immunocytochemistry. rabl7 mRNA and Rabl7 protein were found in all cells of embryo tissues. Synthesis of rabl7 mRNA occurred initially in the embryo axis. As maturation progressed, rabl7 mRNA was detectable in the scutellum and accumulated in axis cells and provascular tissues. However, the response to exogenous ABA differed in various embryo cell types. The Rabl7 protein was located in the nucleus and in the cytoplasm, and qualitative differences in the phosphorylation states of the protein were found between the two subcellular compartments. Based on the similar domain arrangements of Rabl7 and a nuclear localization signal (NLS) binding phosphoprotein, Noppl40, interaction of Rabl7 with NLS peptides was studied. We found specific binding of Rabl7 to the wild-type NLS of the SV40 T antigen but not to an import incompetent mutant peptide. Moreover, binding of the NLS peptide to Rabl7 was found to be dependent upon phosphorylation. These results suggest that Rabl7 may play a role in nuclear protein transport.

The maize abscisic acid responsive protein Rab17 is a highly phosphorylated late embryogenesis abundant protein involved in plant responses to stress. In this study, we provide evidence of the importance of Rab17 phosphorylation by protein kinase CK2 in growth-related processes under stress conditions. We show the specific interaction of Rab17 with the CK2 regulatory subunits CK2␤-1 and CK2␤-3, and that these interactions do not depend on the phosphorylation state of Rab17. Live-cell fluorescence imaging of both CK2 and Rab17 indicates that the intracellular dynamics of Rab17 are regulated by CK2 phosphorylation. We found both CK2␤ subunits and Rab17 distributed over the cytoplasm and nucleus. By contrast, catalytic CK2␣ subunits and a Rab17 mutant protein (mRab17) that is not a substrate for CK2 phosphorylation remain accumulated in the nucleoli. A dual-color image shows that the CK2 holoenzyme accumulates mainly in the nucleus. The importance of Rab17 phosphorylation in vivo was assessed in transgenic plants. The overexpression of Rab17, but not mRab17, arrests the process of seed germination under osmotic stress conditions. Thus, the role of Rab17 in growth processes is mediated through its phosphorylation by protein kinase CK2. T he plant hormone abscisic acid (ABA) plays a major role in adaptation to osmotic stress and induces a number of genes that encode proteins generally assumed to be involved in protecting the cell and promoting recovery from stress. Proteins responsive to ABA accumulate during seed maturation; they naturally disappear during seed germination and can be induced to reappear by ABA treatment or osmotic stress in vegetative tissues (1, 2).Late embryogenesis abundant proteins (Lea) from group 2, responsive to ABA (Rab), or dehydrins are among the most common plant proteins involved in adaptation to water or osmotic stress. Several hypothetical roles have been proposed for Rab͞dehydrin proteins based on different experimental evidence, including binding to phosphate or sulfate ions (3), nuclear localization signal (NLS) peptides (4), calcium (5), and lipid vesicles containing acidic phospholipids (6), among others. All are aimed toward a protective role as chaperones to stabilize molecules or structures under stress conditions (4,7,8).Many proteins included in this Lea family contain the S domain (8), consisting of a tract of serines with several phosphorylation sites (9). Maize Rab17 protein is one of the most heavily phosphorylated proteins in mature embryos and is found both in nucleus and cytoplasm (4). The S domain of Rab17 is followed by a protein kinase CK2 phosphorylation consensus site (9). We previously established that Rab17 is phosphorylated by CK2 in serine residues of the S domain (10). Phosphorylation͞ dephosphorylation is an important mechanism that may regulate the function of Rab17 in the cell; however, the actual physiological function for Rab͞dehydrin proteins is still unknown, and a precise understanding of the function of Rab17 and the importance of its phosphorylation...

SummaryThe maize abscisic acid-responsive Rab17 protein localizes to the nucleus and cytoplasm in maize cells. In-frame fusion of Rab17 to the reporter protein β-glucuronidase (GUS) directed GUS to the nucleus and cytoplasm in transgenic Arabidopsis thaliana and in transiently transformed onion cells. Analysis of chimeric constructs identified one region between amino acid positions 66-96, which was necessary for targeting GUS to the nucleus. This region contains a serine cluster followed by a putative consensus site for protein kinase CK2 phosphorylation, and a stretch of basic amino acids resembling the simian virus 40 large T antigen-type nuclear localization signal (NLS). Mutation of two basic amino acids in the putative NLS had a weak effect on nuclear targeting in the onion cell system and did not modify the percentage of nuclear fusion protein in the Arabidopsis cells. The mutation of three amino acids in the consensus site for CK2 recognition resulted in the absence of in vitro phosphorylated forms of Rab17 and in a strong decrease of GUS enzymatic activity in isolated nuclei of transgenic Arabidopsis. These results suggest that phosphorylation of Rab17 by protein kinase CK2 is the relevant step for its nuclear location, either by facilitating binding to specific proteins or as a direct part of the nuclear targeting apparatus.

Late Embryogenesis Abundant (LEA) proteins, a group of hydrophilic proteins, have been linked to survival in plants and animals in periods of stress, putatively through safeguarding enzymatic function and prevention of aggregation in times of dehydration/heat. Yet despite decades of effort, the molecular-level mechanisms defining this protective function remain unknown. In this paper, we summarize and review research discoveries of the classification of the LEA protein groups based on their amino acid sequence similarity and on the presence of distinctive conserved motifs. Moreover, we focus on high correlation between their accumulation and water deficit, reinforcing their functional relevance under abiotic stresses. We also discuss the biochemical properties of LEA proteins arising from their hydrophilic nature and by amino acid composition. Although significant similarities have not been found between the members of the different groups, a unifying and outstanding feature of most of them is their high hydrophilicity and high content of glycine. Therefore, we have highlighted the biotechnological applications of LEA genes, and the effects of over-expressing LEA genes from all LEA groups from different species of origin into different plant hosts. Apart from agronomical purposes, LEA proteins could be useful for other biotechnological applications in relation to their capacity to prevent aggregation of proteins.

ABSTRACIWe have earlier identified a set of proteins of 23 to 25 kilodaltons (kD), covering an isoelectric point (pI) range of 6.2 to 8.2, which accumulate gradually during normal embryogenesis of Zea mays and disappear in early germination. These polypeptides can be induced prematurely in immature embryos by abscisic acid (ABA) treatment. We report here that the more acidic protein forms are due to post-translational phosphorylation of at least two polypeptides of 23 kD, pI 8.2 and 25 kD, pl 8.0. A polyclonal antiserum was obtained which recognizes all forms of both the 23-kD and 25-kD polypeptides. Recovery of cDNA clones coffesponding to these proteins was accomplished by hybridization with cDNA made from size-selected mRNA enriched for these sequences. Hybrid selection experiments demonstrate that clone MA12 specifically hybridizes with mRNAs encoding the 23-kD and 25-kD protein set which are recognized by the antiserum. By Northern hybridization analysis, the RNA encoded by clone MA12 is shown to accumulate in mature embryos and to be induced in young embryos upon ABA incubation.

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 of 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.

The maize abscisic acid (ABA)-responsive rab17 mRNA and Rab17 protein distribution in maize embryo tissues was investigated by in situ hybridization and immunocytochemistry. rab17 mRNA and Rab17 protein were found in all cells of embryo tissues. Synthesis of rab17 mRNA occurred initially in the embryo axis. As maturation progressed, rab17 mRNA was detectable in the scutellum and accumulated in axis cells and provascular tissues. However, the response to exogenous ABA differed in various embryo cell types. The Rab17 protein was located in the nucleus and in the cytoplasm, and qualitative differences in the phosphorylation states of the protein were found between the two subcellular compartments. Based on the similar domain arrangements of Rab17 and a nuclear localization signal (NLS) binding phosphoprotein, Nopp140, interaction of Rab17 with NLS peptides was studied. We found specific binding of Rab17 to the wild-type NLS of the SV40 T antigen but not to an import incompetent mutant peptide. Moreover, binding of the NLS peptide to Rab17 was found to be dependent upon phosphorylation. These results suggest that Rab17 may play a role in nuclear protein transport.