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Abscisic acid (ABA) induces genes that are highly expressed during late embryogenesis, but suppresses gibberellin (GA)-responsive genes essential for seed germination and seedling growth. Promoter elements necessary and sufficient for ABA up- and down-regulation of gene expression have been previously defined in barley aleurone layers. We have studied the effect of a protein phosphatase 2C, ABI1, an ABA-inducible protein kinase, PKABA1, and a transcription factor, VP1, on ABA action in a barley aleurone transient expression system. The observations have allowed us to dissect ABA signal transduction pathways leading to either induction or suppression of gene expression. The ABA induction of embryogenesis genes is highly inhibited in the presence of a mutated protein phosphatase 2C, encoded by the abi1-1 dominant mutant gene that is known to block ABA responses in Arabidopsis. However, the abi1-1 gene product has no effect on the ABA suppression of a GA-responsive alpha-amylase gene. On the other hand, PKABA1 suppresses the expression of alpha-amylase genes, but has little effect on ABA up-regulated genes. Therefore, it appears that ABA induction and suppression follow two separate signal transduction pathways with the former inhibited by ABI1 and the latter modulated by PKABA1. The presence of VP1 enhances the ABA induction of late embryogenesis genes, but also suppresses germination specific genes. A schematic model based on these observations is presented to explain the effect of these regulatory proteins on ABA-mediated gene expression.

We have identified a new wheat PKABA1-like protein kinase gene, TaPK3, that is expressed in greening wheat seedlings. TaPK3 has high sequence homology (97% similarity with some sequence diversity at the 3' end) to the wheat PKABA1 protein kinase mRNA, which is upregulated by cold-temperature treatment, dehydration and abscisic acid (ABA). Use of a TaPK3 gene-specific probe has revealed that TaPK3 is differentially expressed with respect to PKABA1. TaPK3 mRNA accumulates in greening shoot tissue of wheat, but is not affected by dehydration, cold-temperature treatment or ABA. Based on sequence and expression differences, we conclude that expression of the PKABA1-like protein kinases is not limited to stress responses.

A cDNA clone, pMA1949, detects two mRNA species in wheat seedling tissue that are late embryogenesis-abundant (LEA) and dehydration stress-inducible. Sequence analysis of the pMA1949 clone shows it to be a 991 bp partial cDNA encoding a polypeptide of 317 amino acids with homology to two group 3 LEA proteins, carrot (DC8) and a soybean protein encoded by pGmPM2 cDNA. Molecular analysis of the deduced protein reveals a 33 kDa acidic and extremely hydrophilic protein with potential amphiphilic alpha-helical regions. In addition, the protein contains eleven similar, contiguous repeats of 11 amino acids, which are separated by 118 amino acids from two additional and unique repeats of 36 residues each at the carboxyl end of the protein. Comparisons of sequences of reported group 3 LEA proteins revealed that there are two types, separable by sequence similarity of the 11 amino acid repeating motifs and by the presence or absence of a certain amino acid stretch at the carboxyl terminus. Based on results from these comparisons, we propose a second type of group 3 LEA proteins, called group 3 LEA (II).

Dormant seeds do not germinate when imbibed in water even when conditions are favorable for germination. These hydrated seeds remain viable, but growth-arrested for weeks due to unknown restrictions within the embryo. As a model system for the study of the molecular processes occurring in dormant seeds, we have chosen to examine gene expression in Bromus secalinas, a grass species that produces seeds with high levels of embryonic dormancy. Using differential screening for mRNAs present in hydrated dormant embryos, we have identified a cDNA clone, pBS128, that encodes a mRNA transcript found in the embryos of hydrated seeds of B. secalinus as well as in embryos from mature dry seeds. Striking differences in pBS128 transcript levels appear upon hydration of dormant and nondormant seeds. Upon imbibition pBS128 transcript levels increase over four-fold in dormant seeds, but rapidly decline and disappear in nondormant seeds, which subsequently germinate. The pBS128 transcript appears to be embryo-specific since the transcript is not detectable in either non-stressed or dehydrated seedling tissue. Application of 50 microM ABA to nondormant seeds arrests germination and enhances pBS128 transcript levels. The nucleotide sequence of the nearly full-length pBS128 cDNA shows no homology to other reported genes, and the putative protein sequence does not exhibit the hydrophilic characteristics of the ABA-responsive LEA (late embryogenesis abundant) proteins.

A cDNA clone (pMA2005) of a Group 3 LEA (late embryogenesis abundant) protein has been sequenced from wheat. The wheat cDNA clone codes for a protein with ten tandem repeats of an 11 amino acid sequence and has homology to other Group 3 LEAs reported in barley, carrot, cotton and rape (L. Dure et al., Plant Mol Biol 12: 475-486, 1989). The deduced amino acid sequence indicates that the wheat protein has a molecular weight of 23,000 and is a basic, hydrophilic protein. Northern analysis with the cDNA clone shows that dehydration of wheat shoot tissue results in increased transcript levels that correlate with increases in endogenous ABA.