Dormant Grains Research Articles

ACTIVITY AND COMPONENT COMPOSITION OF DEFENSE PROTEINS IN GRAIN AND SEEDLINGS OF WHEAT, BARLEY AND OATS

The aim of the work was a comparative study of the activity level and composition of β-1,3-glucanase, chitinase, peroxidase and protease inhibitors in grains and seedlings of wheat, barley and oats. These proteins play an important physiological role and are also involved in plant defense against pathogens. Experiments on germination, isolation, and analysis of proteins were carried out under the same conditions, which contributed to an objective assessment of the defense potential of the studied cereals. Barley grains and seedlings were characterized by the highest activity and degree of heterogeneity at IEF β-1,3-glucanase, while oats were characterized by a relatively low level of the enzyme. Oat grains and seedlings had high chitinase activity. The activity and isozymic composition of POX varied greatly among the three cereals, both in dormant grains and in the organs of the seedling. Wheat grains had the highest content of protease inhibitors, whereas oats had the lowest content. The studied properties of enzymes and inhibitors in normal conditions as a constitutive can be useful in studying their variability and induction of new isoforms under stressful conditions, including those caused by pathogens. These data can also be used to evaluate some quality characteristics of grain.

In-Depth Proteomic Analysis of the Secondary Dormancy Induction by Hypoxia or High Temperature in Barley Grains.

In barley, incubation of primary dormant (D1) grains on water under conditions that do not allow germination, i.e. 30°C in air and 15°C or 30°C in 5% O2, induces a secondary dormancy (D2) expressed as a loss of the ability to germinate at 15°C in air. The aim of this study was to compare the proteome of barley embryos isolated from D1 grains and D2 ones after induction of D2 at 30°C or in hypoxia at 15°C or 30°C. Total soluble proteins were analyzed by 2DE gel-based proteomics, allowing the selection of 130 differentially accumulated proteins (DAPs) among 1,575 detected spots. According to the protein abundance profiles, the DAPs were grouped into six abundance-based similarity clusters. Induction of D2 is mainly characterized by a down-accumulation of proteins belonging to cluster 3 (storage proteins, proteases, alpha-amylase inhibitors and histone deacetylase HD2) and an up-accumulation of proteins belonging to cluster 4 (1-Cys peroxiredoxin, lipoxygenase2 and caleosin). The correlation-based network analysis for each cluster highlighted central protein hub. In addition, most of genes encoding DAPs display high co-expression degree with 19 transcription factors. Finally, this work points out that similar molecular events accompany the modulation of dormancy cycling by both temperature and oxygen, including post-translational, transcriptional and epigenetic regulation.

Characterization of the enzyme activity in barley anthocyanin pathway and reaction of these pigments to electromagnetic irradiation

The enzymatic activities were indirectly investigated in the grain anthocyanin synthesis of blue barley by the HPLC data. These results were then evaluated together with absorbed light energy levels of the pigments. In addition, in two photochemical experiments, the change in aleurone pigmentation and the water activation was studied at the dormant grains, using focused sunlight and heat radiation. In the middle stage of the synthesis (at day after flowering, DAF 26), there was a significant cooling period in the weather. Differences of synthesis dynamics were found before and after the cooling period. The anthocyanin production after the cooling period (DAF 26–33) was more intense compared to the beginning of synthesis (17–22). In addition, a more intense degradation was detectable during the cooling period (22–26) than what was observed at the end of seed maturation (33–39). The most efficient light energy binders glucosides (delphinidin- and cyanidin-3-glu.) produced and degraded more dynamically than their more complex forms (malonylglucosides, rutinoside). Furthermore, among the pigments, the cyanidins are able to provide greater energy absorption. Differences in the synthesis dynamism of compounds indicate that individual enzymes and not a multienzyme complex operate in the last phase of the anthocyanin pathway, and by their operation, they can change the energy absorption level of aleurone. In irradiation of blue grains with focused sunlight (~ 400 to ~ 2500 nm, 2–3 min), the aleurone anthocyanins facilitated the vitrified water activation. During intense heat irradiation (~ 8000 nm, 1 min), the laser light scattering associated with water content decreased more intensively within the blue grains compared to the white, indicating the IR absorption surplus for the pigments. Observation suggests that the blue pigments in aleurone can contribute to energy transfer in the direction of water, so they may have role in enhancing energy dissipation.

Evaluation of malting properties and activities of three enzymes from sorghum (Sorghum bicolor) during malting

Sorghum (Sorghum bicolor (L) Moench), is used as human food such as bread, malt drinks and beers; as livestock feed, renewable energy source such as bioethanol. The experimental monitoring of enzyme activity during malting permits the detection of germination-specific genes. The products of α- amylase (simple sugars), β-amylase (maltose), protease (free amino nitrogen) and polyphenol oxidase (quinone), combined during kilning and mashing, with the development of characteristics flavor and coloured malt. In this study, we investigate malting properties of white and yellow varieties of sorghum while the variation in the activity of the three enzymes namely polyphenol oxidases, amylases and proteases, were monitored as the malting progresses. The result obtained were 95% and 86% germinative energy, 1.49 g and 1.09 g water absorption, 0.751 mg/ml and 0.639 mg/ml crude protein for yellow and white sorghum respectively. The activity of the three enzymes was higher in dormant yellow sorghum than dormant white sorghum. For both varieties, polyphenol oxidase activities were lowest during steeping than in dormant grain, while the highest activities were obtained during germination. Amylases activity did not changed significantly from dormant to germination. The proteases activities were highest during steeping than in dormant while gradually decreased to zero level during germination.

A role for jasmonates in the release of dormancy by cold stratification in wheat.

Hydration at low temperatures, commonly referred to as cold stratification, is widely used for releasing dormancy and triggering germination in a wide range of species including wheat. However, the molecular mechanism that underlies its effect on germination has largely remained unknown. Our previous studies showed that methyl-jasmonate, a derivative of jasmonic acid (JA), promotes dormancy release in wheat. In this study, we found that cold-stimulated germination of dormant grains correlated with a transient increase in JA content and expression of JA biosynthesis genes in the dormant embryos after transfer to 20 (o)C. The induction of JA production was dependent on the extent of cold imbibition and precedes germination. Blocking JA biosynthesis with acetylsalicylic acid (ASA) inhibited the cold-stimulated germination in a dose-dependent manner. In addition, we have explored the relationship between JA and abscisic acid (ABA), a well-known dormancy promoter, in cold regulation of dormancy. We found an inverse relationship between JA and ABA content in dormant wheat embryos following stratification. ABA content decreased rapidly in response to stratification, and the decrease was reversed by addition of ASA. Our results indicate that the action of JA on cold-stratified grains is mediated by suppression of two key ABA biosynthesis genes, TaNCED1 and TaNCED2.

Identification of Quantitative Trait Locus for Seed Dormancy and Expression Analysis of Four Dormancy-Related Genes in Sorghum

Seed dormancy is an important trait during the domestication of major crops. Dormant seeds are unable to germinate under conditions normally suitable for non-dormant seeds. Quantitative trait loci (QTLs) and genes affecting seed dormancy in sorghum remain largely unknown. To identify the genomic regions controlling seed dormancy in sorghum, we produced two F2 segregating populations from two crosses between a deep dormant weedy line B140 (as the female parent) and two weak dormant grain lines CK60B and MS138B (as the male parents). A genetic linkage map of the B140/CK60B population was constructed with 216 simple sequence repeat (SSR) markers spanning 1710.3 cM. One QTL on chromosome 4 and two QTLs on chromosome 7 were identified in the B140/CK60B population. They accounted for 17.9 to 24.9% of the phenotypic variance using a simple interval mapping method. The QTL on chromosome 4 was verified in another F2 mapping population (MS138B/B140). To understand the mechanism of dormancy, we conducted an expression analysis of four sorghum genes encoding homologs of dormancy genes of other plant species: Vp1 (maize), DOG1 (Arabidopsis), qSD12 and Sdr4 (rice). We demonstrated that there were clear differences in the expression level of all these four genes between the two parental lines with different seed dormancy level, and their expression were also tissue-specific with the expression levels of qSD12 low throughout all growth stages and tissues in B140. Our findings will help to define the genetic mechanism of seed dormancy in sorghum, and the identified QTLs may be useful biomarker for selection in sorghum improvement programs.

A role for barley CRYPTOCHROME1 in light regulation of grain dormancy and germination.

It is well known that abscisic acid (ABA) plays a central role in the regulation of seed dormancy and that transcriptional regulation of genes encoding ABA biosynthetic and degradation enzymes is responsible for determining ABA content. However, little is known about the upstream signaling pathways impinging on transcription to ultimately regulate ABA content or how environmental signals (e.g., light and cold) might direct such expression in grains. Our previous studies indicated that light is a key environmental signal inhibiting germination in dormant grains of barley (Hordeum vulgare), wheat (Triticum aestivum), and Brachypodium distachyon and that this effect attenuates as after-ripening progresses further. We found that the blue component of the light spectrum inhibits completion of germination in barley by inducing the expression of the ABA biosynthetic gene 9-cis-epoxycarotenoid dioxygenase and dampening expression of ABA 8'-hydroxylase, thus increasing ABA content in the grain. We have now created barley transgenic lines downregulating the genes encoding the blue light receptors CRYTOCHROME (CRY1) and CRY2. Our results demonstrate that CRY1 is the key receptor perceiving and transducing the blue light signal in dormant grains.

In vitro binding of Sorghum bicolor transcription factors ABI4 and ABI5 to a conserved region of a GA 2-OXIDASE promoter: possible role of this interaction in the expression of seed dormancy.

The precise adjustment of the timing of dormancy release according to final grain usage is still a challenge for many cereal crops. Grain sorghum [Sorghum bicolor (L.) Moench] shows wide intraspecific variability in dormancy level and susceptibility to pre-harvest sprouting (PHS). Both embryo sensitivity to abscisic acid (ABA) and gibberellin (GA) metabolism play an important role in the expression of dormancy of the developing sorghum grain. In previous works, it was shown that, simultaneously with a greater embryo sensitivity to ABA and higher expression of SbABA-INSENSITIVE 4 (SbABI4) and SbABA-INSENSITIVE 5 (SbABI5), dormant grains accumulate less active GA4 due to a more active GA catabolism. In this work, it is demonstrated that the ABA signalling components SbABI4 and SbABI5 interact in vitro with a fragment of the SbGA 2-OXIDASE 3 (SbGA2ox3) promoter containing an ABA-responsive complex (ABRC). Both transcription factors were able to bind the promoter, although not simultaneously, suggesting that they might compete for the same cis-acting regulatory sequences. A biological role for these interactions in the expression of dormancy of sorghum grains is proposed: either SbABI4 and/or SbABI5 activate transcription of the SbGA2ox3 gene in vivo and promote SbGA2ox3 protein accumulation; this would result in active degradation of GA4, thus preventing germination of dormant grains. A comparative analysis of the 5′-regulatory region of GA2oxs from both monocots and dicots is also presented; conservation of the ABRC in closely related GA2oxs from Brachypodium distachyon and rice suggest that these species might share the same regulatory mechanism as proposed for grain sorghum.

Roles for blue light, jasmonate and nitric oxide in the regulation of dormancy and germination in wheat grain (Triticum aestivum L.)

Abscisic acid (ABA) plays a central role in seed dormancy and transcriptional regulation of genes coding for ABA biosynthetic and degradation enzymes is responsible for control of ABA content. However, little is known about signalling both before and after ABA regulation, in particular, how environmental signals are perceived and transduced. We are interested in these processes in cereal grains, particularly in relation to the development of strategies for controlling pre-harvest sprouting in barley and wheat. Our previous studies have indicated possible components of dormancy control and here we present evidence that blue light, nitric oxide (NO) and jasmonate are major controlling elements in wheat grain. Using microarray and pharmacological studies, we have found that blue light inhibits germination in dormant grain and that methyl jasmonate (MJ) and NO counteract this effect by reducing dormancy. We also present evidence that NO and jasmonate play roles in dormancy control in vivo. ABA was reduced by MJ and this was accompanied by reduced levels of expression of TaNCED1 and increased expression of TaABA8'OH-1 compared with dormant grain. Similar changes were caused by after-ripening. Analysis of global gene expression showed that although jasmonate and after-ripening caused important changes in gene expression, the changes were very different. While breaking dormancy, MJ had only a small number of target genes including gene(s) encoding beta-glucosidase. Our evidence indicates that NO and MJ act interdependently in controlling reduction of ABA and thus the demise of dormancy.

Induction of secondary dormancy by hypoxia in barley grains and its hormonal regulation

In barley, primary dormant grains did not germinate at 30 °C in air and at 15 °C in an atmosphere containing less than 10% O2, while they germinated easily at 15 °C in air. O2 tension in embryos measured with microsensors was 15.8% at 15 °C but only 0.3% at 30 °C. Incubation of grains at 30 °C is known to induce secondary dormancy in barley, and it was shown here that secondary dormancy was also induced by a 3 d treatment in O2 tensions lower than 10% at 15 °C. After such treatments, the grains lost their ability to germinate subsequently at 15 °C in air. During seed treatment in 5% O2, embryo abscisic acid (ABA) content decreased more slowly than in air and was not altered after transfer into air. Hypoxia did not alter the expression of ABA metabolism genes after 1 d, and induction of HvNCED2 occurred only after 3 d in hypoxia. Embryo sensitivity to ABA was similar in both primary and hypoxia-induced secondary dormant grains. Gibberellic acid (GA) metabolism genes were highly regulated and regulated earlier by the hypoxia treatment, with major changes in HvGA2ox3, HvGA3ox2 and HvGA20ox1 expression after 1 d, resulting in reduced GA signalling. Although a high temperature has an indirect effect on O2 availability, the data showed that it did not affect expression of prolyl-4-hydroxylases and that induction of secondary dormancy by hypoxia at 15 °C or by high temperature in air involved separate signalling pathways. Induction by hypoxia at 15 °C appears to be more regulated by GA and less by ABA than the induction by high temperature.

S phase of the cell cycle: a key phase for the regulation of thermodormancy in barley grain

The aim of the present work was to investigate the occurrence of the cell cycle during germination as related to thermodormancy in barley (Hordeum vulgare L., cv. Pewter) grains in relation with abscisic acid (ABA) by: (i) flow cytometry to determine the progression of the cell cycle; and (ii) reverse transcription-PCR to characterize the expression of some important genes involved in cell-cycle regulation. In dry embryos, cells are mostly (82%) arrested in G1 phase of the cell cycle, the remaining cells being in the G2 (17%) or S phase (0.9%). Germination at 20 °C was associated with an increase in the nuclei population in G2 and S (up to 32.5–44.5 and 9.2–11.3%, respectively, after 18–24h). At 30 °C, partial reactivation of the cell cycle occurred in embryos of dormant grains that did not germinate. Incubation with 50mM hydroxyurea suggests that thermodormancy resulted in a blocking of the nuclei in the S phase. In dry dormant grains, transcripts of CDKA1, CYCA3, KRP4, and WEE1 were present, while those of CDKB1, CDKD1, CYCB1, and CYCD4 were not detected. Incubation at 30 °C resulted in a strong reduction of CDKB1, CYCB1, and CYCD4 expression and overexpression of CDK1 and KRP4. ABA had a similar effect as incubation at 30 °C on the expression of CDKB1, CYCB1, and CYCD4, but did not increase that of CDK1 and KRP4. Patterns of gene expression are discussed with regard to thermodormancy expression and ABA.

Differential expression of abscisic acid metabolism and signalling genes induced by seed-covering structures or hypoxia in barley (Hordeum vulgare L.) grains

AbstractDormant barley grains cannot germinate at 30°C and this inability to germinate is imposed mostly by the glumellae which have been suggested to limit oxygen supply to the embryo. Hypoxia imposed either artificially or by the glumellae to embryos from dormant grains, increases embryo sensitivity to abscisic acid (ABA) and promotes the accumulation of ABA during the first hours after imbibition. Expression of candidate genes involved in ABA synthesis (HvNCED), catabolism (HvABA8OH1) and signalling (HvABI5, HvVP1 and HvPKABA) was analysed in embryos isolated from dormant whole or de-hulled grains incubated in air or in hypoxia (5% oxygen). The presence of the glumellae enhanced the expression of genes involved in ABA metabolism and signalling with respect to that observed in de-hulled grains incubated in air. These results suggest that at least part of the observed physiological responses to the presence of the glumellae are regulated at the level of gene expression. However, hypoxia imposed on dormant de-hulled grains did not mimic the presence of the glumellae in terms of expression of candidate genes. Hypoxia mimics the presence of the glumellae in terms of dormancy maintenance and ABA accumulation and sensitivity, but its effects appear to operate through different mechanisms.

Screening for grain dormancy in segregating generations of dormant × non-dormant crosses in white-grained wheat (Triticum aestivum L.)

Pre-harvest sprouting (PHS) in wheat (Triticum aestivum L.) is a significant problem. Introgression of genes controlling grain dormancy into white-grained bread wheat is one means of improving resistance to PHS. In this study seven dormant (containing the SW95-50213 and AUS1408 sources) × non-dormant crosses were produced to investigate the effectiveness of selection for grain dormancy in early segregating generations. Each generation (F1–F4) was grown in a temperature controlled glasshouse with an extended photoperiod (i.e. continuous light). F2 and F3 generations were subject to selection. Five hundred harvest-ripe grains were tested for germination over a 14 day period, and the 100 most dormant grains were retained and grown-on to produce the next generation within each cross. The response to selection was assessed through analysis of the time to 50% germination (G50) in the F2, F3 and F4 generations. In addition, changes in marker class frequencies for two SSR markers (barc170 and gpw2279) flanking a known quantitative trait locus (QTL) for grain dormancy on chromosome 4A were assessed in DNA from F2 plants selected from early germinating (non-dormant) and late germinating (dormant) phenotypic extremes within each cross. Selection for grain dormancy in the F2 and F3 generations effectively recovered the dormant phenotype in all seven crosses, i.e. the F4 generation was not significantly different from the dormant parent. Further, selection based on individual F2 grains changed marker class frequencies for the 4A dormancy QTL; in most cases eliminating the marker class homozygous for the non-dormant alleles. Application of this screening method will enable breeders to better select for grain dormancy and may lead to development of new cultivars offering effective resistance to PHS in the near future.

Thermodormancy and ABA metabolism in barley grains

Incubation of barley primary dormant grains at 30°C, a temperature at which they cannot germinate results in a reinforcement of their sensitivity to temperature, and in particular in a loss of their ability to germinate at 15-20°C 1. Incubation of the grains at 30°C in the presence of GA3 (1 mM) or of isolated embryos prevents this induction of secondary dormancy. In such a condition, embryo ABA content was lower than that measured in embryos of seeds incubated at 30°C on water. Expression of genes involved in ABA metabolism (HvABA8’OH1, HvNCED1 and HvNCED2) was studied in isolated embryos incubated on water and in the presence of GA3 (1 mM). Expression of HvABA8’OH1, HvNCED1 and HvNCED2 was discussed in relation with ABA content and germination ability at 30°C.

Regulation of dormancy in barley by blue light and after-ripening: effects on abscisic acid and gibberellin metabolism.

White light strongly promotes dormancy in freshly harvested cereal grains, whereas dark and after-ripening have the opposite effect. We have analyzed the interaction of light and after-ripening on abscisic acid (ABA) and gibberellin (GA) metabolism genes and dormancy in barley (Hordeum vulgare 'Betzes'). Analysis of gene expression in imbibed barley grains shows that different ABA metabolism genes are targeted by white light and after-ripening. Of the genes examined, white light promotes the expression of an ABA biosynthetic gene, HvNCED1, in embryos. Consistent with this result, enzyme-linked immunosorbent assays show that dormant grains imbibed under white light have higher embryo ABA content than grains imbibed in the dark. After-ripening has no effect on expression of ABA biosynthesis genes, but promotes expression of an ABA catabolism gene (HvABA8'OH1), a GA biosynthetic gene (HvGA3ox2), and a GA catabolic gene (HvGA2ox3) following imbibition. Blue light mimics the effects of white light on germination, ABA levels, and expression of GA and ABA metabolism genes. Red and far-red light have no effect on germination, ABA levels, or HvNCED1. RNA interference experiments in transgenic barley plants support a role of HvABA8'OH1 in dormancy release. Reduced HvABA8'OH1 expression in transgenic HvABA8'OH1 RNAi grains results in higher levels of ABA and increased dormancy compared to nontransgenic grains.

Field studies on the regulation of abscisic acid content and germinability during grain development of barley: molecular and chemical analysis of pre-harvest sprouting

To investigate whether the regulation of abscisic acid (ABA) content was related to germinability during grain development, two cDNAs for 9-cis-epoxycarotenoid dioxygenase (HvNCED1 and HvNCED2) and one cDNA for ABA 8'-hydroxylase (HvCYP707A1), which are enzymes thought to catalyse key regulatory steps in ABA biosynthesis and catabolism, respectively, were cloned from barley (Hordeum vulgare L.). Expression and ABA-quantification analysis in embryo revealed that HvNCED2 is responsible for a significant increase in ABA levels during the early to middle stages of grain development, and HvCYP707A1 is responsible for a rapid decrease in ABA level thereafter. The change in the embryonic ABA content of imbibing grains following dormancy release is likely to reflect changes in the expression patterns of HvNCEDs and HvCYP707A1. A major change between dormant and after-ripened grains occurred in HvCYP707A1; the increased expression of HvCYP707A1 in response to imbibition, followed by a rapid ABA decrease and a high germination percentage, was observed in the after-ripened grains, but not in the dormant grains. Under field conditions, HvNCED2 showed the same expression level and pattern during grain development in 2002, 2003, and 2004, indicating that HvNCED2 expression is regulated in a growth-dependent manner in the grains. By contrast, HvNCED1 and HvCYP707A1 showed a different expression pattern in each year, indicating that the expression of these genes is affected by environmental conditions during grain development. The varied expression levels of these genes during grain development and imbibition, which would have effects on the activity of ABA biosynthesis and catabolism, might be reflected, in part, in the germinability in field-grown barley.

Hypoxia interferes with ABA metabolism and increases ABA sensitivity in embryos of dormant barley grains

Two mechanisms have been suggested as being responsible for dormancy in barley grain: (i) ABA in the embryo, and (ii) limitation of oxygen supply to the embryo by oxygen fixation as a result of the oxidation of phenolic compounds in the glumellae. The aim of the present work was to investigate whether hypoxia imposed by the glumellae interferes with ABA metabolism in the embryo, thus resulting in dormancy. In dormant and non-dormant grains incubated at 20 degrees C and in non-dormant grains incubated at 30 degrees C (i.e. when dormancy is not expressed), ABA content in the embryo decreased dramatically during the first 5 h of incubation before germination was detected. By contrast, germination of dormant grains was less than 2% within 48 h at 30 degrees C and embryo ABA content increased during the first hours of incubation and then remained 2-4 times higher than in embryos from grains in which dormancy was not expressed. Removal of the glumellae allowed germination of dormant grains at 30 degrees C and the embryos did not display the initial increase in ABA content. Incubation of de-hulled grains under 5% oxygen to mimic the effect of glumellae, restored the initial increase ABA in content and completely inhibited germination. Incubation of embryos isolated from dormant grains, in the presence of a wide range of ABA concentrations and under various oxygen tensions, revealed that hypoxia increased embryo sensitivity to ABA by 2-fold. This effect was more pronounced at 30 degrees C than at 20 degrees C. Furthermore, when embryos from dormant grains were incubated at 30 degrees C in the presence of 10 microM ABA, their endogenous ABA content remained constant after 48 h of incubation under air, while it increased dramatically in embryos incubated under hypoxia, indicating that the apparent increase in embryo ABA responsiveness induced by hypoxia was, in part, mediated by an inability of the embryo to inactivate ABA. Taken together these results suggest that hypoxia, either imposed artificially or by the glumellae, increases embryo sensitivity to ABA and interferes with ABA metabolism.

Protein phosphorylation is suppressed when wheat embryos are hydrated and remain growth arrested

An early difference in net protein phosphorylation activity occurred in dormant and after-ripened wheat (Triticum aestivum L.) grains within the first hour of imbibition at 30°C. Embryos from dry, dormant and after-ripened caryopses exhibited a high degree of protein phosphorylation, particularly of four proteins (68, 54, 51 and 42 kDa). Upon hydration, protein phosphorylation activity in dormant embryos decreased within 1 h and reached minimal phosphorylation activity by 5 h. Protein phosphorylation activity in after-ripened (germinable) embryos was not suppressed and remained high during germination. If the hydrated dormant embryos were dried, protein phosphorylation activity was restored. Application of the protein phosphatase inhibitor, okadaic acid, partially overcame dormancy and slowed the decrease in phosphorylation activity. These results suggested that reduction of protein phosphorylation activity slowed the rate of germination.

Inhibition of α-Amylase Acting in Hexaploid Triticale Lines by Exogenous Abscisic Acid

Hexaploid triticale introgressive lines developed after recombination of A-genome with Am-genome of diploid wheat (Triticum monococcum) were analysed in respect of grains responsiveness to exogenous ABA treatment. This was assessed by in vivo bioassay as grain germination indices, and by α-amylase assay as quantity of synthesised α-amylase measured with the technique of radial diffusion in agarose gel. The results showed an important diminishing of seedling length caused by ABA (variable in different lines) as well as genotype dependant variability of α-amylase synthesis inhibition. The differences of ABA responsiveness were seen both in whole grains and in embryoless half-grains as a direct reaction of the aleurone layer. Variation of grain sensitivity to ABA treatment compared with two sprouting resistance indices showed a significant correlation with Falling Number values in grains, but not with a dormant grains germination in spikes. This is an evidence that in triticale precocious starch decompose in unripened and ungerminated grains is dependent on genotype ABA-responsiveness of the aleurone layer.

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 TaABF transcripts 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.