M. K. Walker-Simmons

Gibberellin/Abscisic Acid Antagonism in Barley Aleurone Cells: Site of Action of the Protein Kinase PKABA1 in Relation to Gibberellin Signaling Molecules

The antagonism between gibberellins (GA) and abscisic acid (ABA) is an important factor regulating the developmental transition from embryogenesis to seed germination. In barley aleurone layers, the expression of genes encoding α-amylases and proteases is induced by GA but suppressed by ABA. It has been shown that an ABA-induced protein kinase, PKABA1, mediates the ABA suppression of α-amylase expression. Using a barley aleurone transient expression system, we have now localized the site of action of PKABA1 relative to other signal transduction components governing the expression of α-amylase. The expression of α-amylase can be transactivated by the transcription factor GAMyb, which is itself induced by GA. A truncated GAMyb containing the DNA binding domain but lacking the transactivation domain prevents the GA induction of α-amylase, further supporting the notion that GAMyb mediates the GA induction of α-amylase expression. Although ABA and PKABA1 strongly inhibit the GA induction of α-amylase, they have no effect on GAMyb-transactivated α-amylase expression. Using a GAMyb promoter–β-glucuronidase construct, we also show that both ABA and PKABA1 repress the GA induction of GAMyb. In the slender mutant, GAMyb and α-amylase are highly expressed, even in the absence of GA. However, this constitutive expression can still be inhibited by ABA, PKABA1, or an inhibitor of cGMP synthesis. On the basis of these observations, we suggest that PKABA1 acts upstream from the formation of functional GAMyb but downstream from the site of action of the Slender gene product. Because PKABA1 inhibits the GA induction of the GAMyb promoter–β-glucuronidase construct, it appears that at least part of the action of PKABA1 is to downregulate GAMyb at the transcriptional level.

The abscisic acid-responsive kinase PKABA1 interacts with a seed-specific abscisic acid response element-binding factor, TaABF, and phosphorylates TaABF peptide sequences.

The abscisic acid (ABA)-induced protein kinase PKABA1 is present in dormant seeds and is a component of the signal transduction pathway leading to ABA-suppressed gene expression in cereal grains. We have identified a member of the ABA response element-binding factor (ABF) family of basic leucine zipper transcription factors from wheat (Triticum aestivum) that is specifically bound by PKABA1. This protein (TaABF) has highest sequence similarity to the Arabidopsis ABA response protein ABI5. In two-hybrid assays TaABF bound only to PKABA1, but not to a mutant version of PKABA1 lacking the nucleotide binding domain, suggesting that binding of TaABF requires prior binding of ATP as would be expected for binding of a protein substrate by a protein kinase. TaABF mRNA accumulated together with PKABA1 mRNA during wheat grain maturation and dormancy acquisition and TaABFtranscripts increased transiently during imbibition of dormant grains. In contrast to PKABA1 mRNA, TaABF mRNA is seed specific and did not accumulate in vegetative tissues in response to stress or ABA application. PKABA1 produced in transformed cell lines was able to phosphorylate synthetic peptides representing three specific regions of TaABF. These data suggest that TaABF may serve as a physiological substrate for PKABA1 in the ABA signal transduction pathway during grain maturation, dormancy expression, and ABA-suppressed gene expression.

Chitosans and pectic polysaccharides both induce the accumulation of the antifungal phytoalexin pisatin in pea pods and antinutrient proteinase inhibitors in tomato leaves

The Proteinase Inhibitor Inducing Factor, PIIF, a pectic polysaccharide that induces synthesis and accumulation of proteinase inhibitor proteins in tomato and potato leaves, is an effective elicitor of the phytoalexin pisatin in pea pod tissues. The levels of pisatin induced by PIIF, and the time course of elicitation, are similar to those induced by chitosans, beta-1,4 glucosamine polymers, which are potent elicitors of pisatin in pea pods. Similarly, the chitosans, found in both insect and fungal cell walls, are the most potent inducers yet found of proteinase inhibitor accumulation in excised tomato cotyledons. The similarity in the induction of synthesis of proteinase inhibitors in tomato cotyledons and of pisatin in pea pods by pectic polysaccharides and chitosans suggests that the two polysaccharide types may be triggering a similar fundamental system present in pea and tomato plants that regulates the expression of genes for natural protection systems.

Group 3 Late Embryogenesis Abundant Proteins in Desiccation-Tolerant Seedlings of Wheat (Triticum aestivum L.)

Dormant seeds and young seedlings of wheat (Triticum aestivum L.) tolerate desiccation. A transcript expressed in this desiccation-tolerant tissue has been cloned and sequenced (J. Curry, C.F. Morris, M.K. Walker-Simmons [1991] Plant Mol Biol 16: 1073–1076). This wheat cDNA clone encodes a protein that is homologous to other group 3 late embryogenesis abundant (LEA) proteins. In this report, we describe the production of polyclonal antibodies to the protein product of the cDNA clone and assess group 3 LEA protein levels in desiccation-tolerant tissue. The group 3 LEA antibodies detected four major proteins in wheat with molecular masses from 27 to 30.5 kD. The genes for these proteins mapped to wheat chromosomes 1A, 1B, and 1D. The group 3 LEA proteins were present in mature seed embryos and were maintained when growth-arrested, dormant seeds were hydrated for 111 h. However, in germinating seeds the group 3 LEA proteins declined and were no longer detectable by 111 h. We severely dehydrated seedlings (more than 90% water loss) to assess group 3 LEA transcript and protein accumulation in tissues of these desiccation-tolerant plants. In response to dehydration, abscisic acid (ABA) levels increased dramatically and group 3 LEA mRNAs were induced in root, shoot, and scutellar tissue. However, group 3 LEA proteins were detected only in shoot and scutellar tissue and not in root tissue. Treatment of nonstressed seedlings with 20 [mu]M ABA resulted in low levels of group 3 LEA proteins in the roots, whereas higher levels were found in the shoot and scutellar tissue. When dehydrated seedlings were rehydrated, shoot and scutellar tissue resumed growth. The roots did not resume growth and subsequently died. New roots developed later from the scutellar tissue. Thus, in severely dehydrated wheat seedlings, the accumulation of high levels of group 3 LEA proteins is correlated with tissue dehydration tolerance.

Diethyldithiocarbamic Acid Inhibits the Octadecanoid Signaling Pathway for the Wound Induction of Proteinase Inhibitors in Tomato Leaves

The induction of proteinase inhibitor I synthesis in tomato (Lycopersicon esculentum) leaves in response to wounding is strongly inhibited by diethyldithiocarbamic acid (DIECA). DIECA also inhibits the induction of inhibitor I synthesis by the 18-amino acid polypeptide systemin, polygalac turonic acid (PGA), and linolenic acid, but not by jasmonic acid, suggesting that DIECA interferes with the octadecanoid signaling pathway. DIECA only weakly inhibited tomato lipoxygenase activity, indicating that DIECA action occurred at a step after the conversion of linolenic acid to 13(S)-hydroperoxylinolenic acid (HPOTrE). DIECA was shown to efficiently reduce HPOTrE to 13-hydroxylinolenic acid (HOTrE), which is not a signaling intermediate. Therefore, in vivo, DIECA is likely inhibiting the signaling pathway by shunting HPOTrE to HOTrE, thereby severely reducing the precursor pool leading to cyclization and eventual synthesis of jasmonic acid. Phenidone, an inhibitor of lipoxygenase, inhibited proteinase inhibitor I accumulation in response to wounding, further supporting a role for its substrate, linolenic acid, and its product, HPOTrE, as components of the signal-transduction pathway that induces proteinase inhibitor synthesis in response to wounding, systemin, and PGA.

CLoning and expression of an embryo-specific mRNA up-regulated in hydrated dormant seeds

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 50μM ABA to nondormant seeds arrests germination and enhances pBS 128 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.

Sequence analysis of a cDNA encoding a group 3 LEA mRNA inducible by ABA or dehydration stress in wheat.

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.

Dissection of abscisic acid signal transduction pathways in barley aleurone layers

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 α-amylase gene. On the other hand, PKABA1 suppresses the expression of α-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.

Heat-soluble proteins extracted from wheat embryos have tightly bound sugars and unusual hydration properties

Late embryogenesis abundant (LEA) proteins accumulate in developing seeds prior to maturation drying and are presumed to help protect embryos from desiccation stress. The unusual solubility properties of these proteins, such as resistance to heat coagulation, have led to suggestions that they alter the hydration properties of cellular constituents. Hydration characteristics and water potential range at which wheat heat-soluble LEA proteins were expressed have been determined. Levels of heatsoluble proteins decline in germinating seeds but can be induced by dehydration (ψ ≤ −0.5 MPa) in crown meristematic tissue that is desiccation tolerant. The heatsoluble extract from mature wheat embryos contained proteins and sugars at about a 1:1 (w/w) ratio. Only about half of the sugars could be removed by exhaustive dialysis; the rest appeared to be tightly associated with the proteins. The water sorption characteristics of undialysed and dialysed heat-soluble protein/sugar fractions were compared with other water-soluble proteins (bovine serum albumin, lysozyme or gluten) and with sucrose. At relative humidities greater than 50%, the amount of water absorbed by protein-sugar mixes was a function of the sugar content. For the same sugar content, the heat-soluble protein preparation absorbed 2–3 times more water than a lysozyme/sucrose preparation. The rate at which heat-soluble protein fractions dried was also different to desorption rates of lysozyme/sucrose mixes. While lysozyme/sucrose mixtures dried either very rapidly (within 20 min) or very slowly (about 2 months) depending on the sugar content, desorption rates of the heat soluble protein-sugar preparations were intermediate (2–10 days) and modulated by sugar concentration. Based on the presumption that the hydrophilic properties of LEA heat-soluble proteins are important to their function, it is suggested that these proteins function to control drying so that cells stay at critical water potentials for the proper time. In this respect, the heat-soluble LEA proteins do not prevent desiccation, but serve as hydration buffers.

8[prime]-Methylene Abscisic Acid (An Effective and Persistent Analog of Abscisic Acid)

We report here the synthesis and biological activity of a new persistent abscisic acid (ABA) analog, 8[prime]-methylene ABA. This ABA analog has one additional carbon atom attached through a double bond to the 8[prime]-carbon of the ABA molecule. (+)-8[prime]-Methylene ABA is more active than the natural hormone (+)-ABA in inhibiting germination of cress seed and excised wheat embryos, in reducing growth of suspension-cultured corn cells, and in reducing transpiration in wheat seedlings. The (+)-8[prime]-methylene analog is slightly weaker than (+)-ABA in increasing expression of ABA-inducible genes in transgenic tobacco, but is equally active in stimulating a transient elevation of the pH of the medium of corn cell cultures. In corn cells, both (+)-ABA and (+)-8[prime]-methylene ABA are oxidized at the 8[prime] position. ABA is oxidized to phaseic acid and (+)-8[prime]-methylene ABA is converted more slowly to two isomeric epoxides. The alteration in the ABA structure causes the analog to be metabolized more slowly than ABA, resulting in longer-lasting and more effective biological activity relative to ABA.