Construction and evaluation of cDNA libraries for large-scale expressed sequence tag sequencing in wheat (Triticum aestivum L.).

A total of 37 original cDNA libraries and 9 derivative libraries enriched for rare sequences were produced from Chinese Spring wheat (Triticum aestivum L.), five other hexaploid wheat genotypes (Cheyenne, Brevor, TAM W101, BH1146, Butte 86), tetraploid durum wheat (T. turgidum L.), diploid wheat (T. monococcum L.), and two other diploid members of the grass tribe Triticeae (Aegilops speltoides Tausch and Secale cereale L.). The emphasis in the choice of plant materials for library construction was reproductive development subjected to environmental factors that ultimately affect grain quality and yield, but roots and other tissues were also included. Partial cDNA expressed sequence tags (ESTs) were examined by various measures to assess the quality of these libraries. All ESTs were processed to remove cloning system sequences and contaminants and then assembled using CAP3. Following these processing steps, this assembly yielded 101,107 sequences derived from 89,043 clones, which defined 16,740 contigs and 33,213 singletons, a total of 49,953 "unigenes." Analysis of the distribution of these unigenes among the libraries led to the conclusion that the enrichment methods were effective in reducing the most abundant unigenes and to the observation that the most diverse libraries were from tissues exposed to environmental stresses including heat, drought, salinity, or low temperature.

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 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.

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 alpha-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 alpha-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 alpha-amylase. The expression of alpha-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 alpha-amylase, further supporting the notion that GAMyb mediates the GA induction of alpha-amylase expression. Although ABA and PKABA1 strongly inhibit the GA induction of alpha-amylase, they have no effect on GAMyb-transactivated alpha-amylase expression. Using a GAMyb promoter--beta-glucuronidase construct, we also show that both ABA and PKABA1 repress the GA induction of GAMyb. In the slender mutant, GAMyb and alpha-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--beta-glucuronidase construct, it appears that at least part of the action of PKABA1 is to downregulate GAMyb at the transcriptional level.

The influence of wheat dehydration-induced proteins on the function of turkey spermatozoa after twenty-four-hour in vitro storage.

Dehydration-induced proteins and other osmo-protectants enable plants to survive dehydration stress. The object of this study was to determine whether dehydration-induced proteins from wheat seeds could protect turkey sperm and improve survival and function after liquid storage. A partially purified heat-soluble protein extract was isolated from dry, mature wheat seed embryos and added to semen diluent. Hens were inseminated with either fresh or semen stored 24 h at 5 C and fertility and hatchability data collected. The addition of 10% wheat protein extract to semen stored 24 h at 5 C improved fertility and hatchability of eggs over semen stored in diluent alone (P < 0.05). The heat-soluble proteins isolated from wheat seed embryos are capable of protecting turkey sperm during in vitro storage and could potentially improve long-term storage of sperm from other species.

An abscisic acid-induced protein kinase, PKABA1, mediates abscisic acid-suppressed gene expression in barley aleurone layers.

The phytohormone abscisic acid (ABA) induces genes-encoding proteins involved in desiccation tolerance and dormancy in seeds, but ABA also suppresses gibberellin (GA)-responsive genes encoding hydrolytic enzymes essential for postgermination growth. A unique serine/threonine protein kinase, PKABA1 mRNA, up-regulated by ABA in seeds, has been identified. In this report, the effect of PKABA1 on the signal transduction pathway mediating ABA induction and suppression of genes has been determined in aleurone layers of barley seeds. Two groups of gene constructs were introduced to barley aleurone layers by using particle bombardment: the reporter constructs containing the coding sequence of beta-glucuronidase gene linked to hormone-responsive promoters and the effector constructs containing the coding region of protein kinases linked to a constitutive promoter. Constitutive expression of PKABA1 drastically suppressed expression of low- and high-pI alpha-amylase and protease genes induced by GA. However, the presence of PKABA1 had only a small effect on the ABA induction of a gene encoding a late embryogenesis abundant protein, HVA1. Our results indicate that PKABA1 acts as a key intermediate in the signal transduction pathway leading to the suppression of GA-inducible gene expression in cereal aleurone layers.

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.

The wheat protein kinase gene, TaPK3, of the PKABA1 subfamily is differentially regulated in greening wheat seedlings.

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.

The Wheat Abscisic Acid-Responsive Protein Kinase mRNA, PKABA1, Is Up-Regulated by Dehydration, Cold Temperature, and Osmotic Stress.

The effects of dehydration, cold-temperature treatment, and osmotic and salt stress on the expression of an abscisic acid-responsive protein kinase mRNA (PKABA1) were determined in wheat (Triticum aestivum L.) seedlings. The PKABA1 transcript was detectable at basal levels in tissues of nonstressed plants and accumulated to higher levels in shoot, scutellar, and root tissues of stressed plants. PKABA1 transcript accumulated rapidly within 2 h following dehydration and within 24 h following other treatments (cold, osmotic stress, and high salt). The accumulation of PKABA1 mRNA could not be separated temporally from that of a wheat group 3 late embryogenesis abundant mRNA during dehydration and cold treatment. High PKABA1 mRNA levels were observed in field-grown plants growing under cold winter conditions but not under warmer summer conditions. A recent GenBank data base search indicated that other plant protein kinases with similar acidic amino acid stretches as in PKABA1 have been identified, and some of these kinases are responsive to environmental signals. These results suggest that PKABA1 may be part of general environmental stress responses in wheat.

The 7[prime]-Methyl Group of Abscisic Acid Is Critical for Biological Activity in Wheat Embryo Germination.

Wheat (Triticum aestivum L.) embryo germination is inhibited by natural (S)-(+)-abscisic acid (ABA). In this report we have determined critical structural features of the ABA molecule, particularly the methyl and ketone groups of the ABA ring, required for inhibitory activity. To examine the ring residues a series of new optically active ABA analogs have been synthesized in which the 4[prime]-keto, 7[prime]-, 8[prime]-, or 8[prime]- and 9[prime]-carbons have been replaced with hydrogen atoms. Each of the analogs was tested over a range of concentrations as a germination inhibitor. Enantiomers of the analogs altered at the 4[prime]-keto or 8[prime]- and 9[prime]-methyl groups were active, but less so than ABA. Both enantiomers of 7[prime]-demethylABA were inactive as germination inhibitors. The results show that the 7[prime]-methyl group is absolutely required for activity, but that the other residues are less critical for hormone recognition.

Mapping of abscisic acid responsive genes and vp1 to chromosomes in wheat and Lophopyrum elongatum.

The plant hormone abscisic acid (ABA) affects developmental and physiological processes that can impact crop production. These processes include germination, environmental stress responses in vegetative tissue, embryo maturation, and dormancy. Identification and molecular tagging of ABA-responsive genes, as well as the vp1 gene, required for ABA sensitivity, may be valuable in developing breeding strategies designed for manipulating ABA-regulated traits. Using aneuploid genetic stocks and alien substitution lines the chromosomal location of vp1 and seven abscisic acid (ABA) responsive genes was determined in wheat and Lophopyrum elongatum (Host) Löve. Clones (gene homology, if known, in parentheses) isolated from wheat hybridized to wheat and L. elongatum homoeologous chromosome groups as follows: pMA80 (dhn), chromosome 4; pMA1949 (group 3 LEA (1I)), chromosome 3S; pMA1951, chromosome 5S; pMA1959 (Em), chromosome 1L; pMA2005 (group 3 LEA), chromosome 1S; and PKABA1, chromosome 2L. Clone pBS128, corresponding to an ABA-responsive gene from Bromus secalinus, hybridized to wheat chromosome 2S. The cDNA clone pcvp23 that is homologous to vp1, a gene required for ABA responsiveness and the prevention of precocious germination in maize embryos, did not hybridize well with wheat but hybridized to chromosomes 3L and 7β in L. elongatum. Three of the clones mapped previously in barley by other researchers, pMA1949, pMA1951, pMA1959, were found here to be on the corresponding homoeologous chromosomes in wheat.

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.

Unusual sequence of group 3 LEA (II) mRNA inducible by dehydration stress in wheat.

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).

Isolation of a wheat cDNA clone for an abscisic acid-inducible transcript with homology to protein kinases.

Increases in the plant hormone abscisic acid (ABA) initiate water-stress responses in plants. We present evidence that a transcript with homology to protein kinases is induced by ABA and dehydration in wheat. A 1.2-kilobase cDNA clone (PKABA1) was isolated from an ABA-treated wheat embryo cDNA library by screening the library with a probe developed by polymerase chain reaction amplification of serine/threonine protein kinase subdomains VIb to VIII. The deduced amino acid sequence of the PKABA1 clone contains the features of serine/threonine protein kinases, including homology with all 12 conserved regions of the catalytic domain. PKABA1 transcript levels are barely detectable in growing seedlings but are induced dramatically when plants are subjected to dehydration stress. The PKABA1 transcript can also be induced by supplying low concentrations of ABA, and coordinate increases in ABA levels and PKABA1 mRNA occur when seedlings are water-stressed. Identification of this ABA-inducible transcript with homology to protein kinases provides a basis for examining the role of protein phosphorylation in plant responses to dehydration.

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 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.

Optically pure abscisic Acid analogs-tools for relating germination inhibition and gene expression in wheat embryos.

We report an examination of the structural requirements of the abscisic acid (ABA) recognition response in wheat dormant seed embryos using optically pure isomers of ABA analogs. These compounds include permutations to the ABA structure with either an acetylene or a trans bond at C-4 C-5, and either a single or double bond at the C-2' C-3' double bond. (R)-ABA and the three isomers with the same configuration at C-1' as natural ABA were found to be effective germination inhibitors. The biologically active ABA analogs exhibited differential effects on ABA-responsive gene expression. All the ABA analogs that inhibited germination induced two ABA-responsive genes, wheat group 3 lea and dhn (rab). However, (R)-ABA and (S)-dihydroABA were less effective in inducing the ABA-responsive gene Em within the time that embryonic germination was inhibited.

Production of polyclonal antibodies in rabbits is simplified using perforated plastic golf balls.

Production of polyclonal antibodies in the lumen of a perforated golf ball implanted surgically under the skin of a rabbit offers advantages over conventional techniques. Less stress is placed on the rabbit because bleeding is eliminated, complete adjuvants are not used and animal handling is minimized. The technique also offers the advantage that large amounts of antibody-containing fluid can be removed easily from the ball. In this report we describe the surgical protocol and demonstrate use of this technique to produce high-titered antibodies to plant and plant viral 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.

Molecular cloning and expression of abscisic Acid-responsive genes in embryos of dormant wheat seeds.

Hydrated dormant cereal seeds do not germinate even when environmental conditions are favorable for germination. By using cDNA cloning and differential screening, we have identified mRNAs from five gene families that are abundant in the embryos of imbibed, but developmentally arrested wheat (Triticum aestivum L.) seeds. Gene transcript levels of these mRNAs are maintained and even increase in embryos of imbibed dormant seeds for as long as the seeds remain dormant. In contrast, transcript levels decline in nondormant seeds after imbibition and disappear as germination occurs. All the identified genes are ABA responsive. Based on these data we conclude that wheat seeds in the hydrated dormant state exhibit prolonged expression of ABA-responsive genes.