A. Michele Stanca

Hv-WRKY38: a new transcription factor involved in cold- and drought-response in barley

WRKY proteins constitute a large family of plant specific transcription factors implicated in many different processes. Here we describe Hv-WRKY38,a barley gene coding for a WRKY protein, whose expression is involved in cold and drought stress response. Hv-WRKY38 was early and transiently expressed during exposure to low non-freezing temperature, in ABA-independent manner. Furthermore, it showed a continuous induction during dehydration and freezing treatments. A WRKY38:YFP fusion protein was found to localise into the nucleus upon introduction into epidermal onion cells. Bacterially expressed Hv-WRKY38 was able to bind in vitro to the W-box element (T)TGAC(C/T) also recognisable by other WRKY proteins. Hv-WRKY38genomic DNA was sequenced and mapped onto the centromeric region of the barley chromosome 6H. Arabidopsis and rice sequences homologous to Hv-WRKY38 were also identified. Our results indicate that Hv-WRKY38 transcription factor may play a regulatory role in abiotic stress response.

Chromosome regions and stress-related sequences involved in resistance to abiotic stress in Triticeae

Drought, low temperature and salinity are the most important abiotic stress factors limiting crop productivity. A genomic map of major loci and QTLs affecting stress tolerance in Triticeae identified the crucial role of the group 5 chromosomes, where the highest concentration of QTLs and major loci controlling plants adaptation to the environment (heading date, frost and salt tolerance) has been found. In addition, a conserved region with a major role in drought tolerance has been localized to the group 7 chromosomes. Extensive molecular biological studies have led to the cloning of many stress-related genes and responsive elements. The expression of some stress-related genes was shown to be linked to stress-tolerant QTLs, suggesting that these genes may represent the molecular basis of stress tolerance. The development of suitable genetic tools will allow the role of stress-related sequences and their relationship with stress-tolerant loci to be established in the near future.

cor Gene Expression in Barley Mutants Affected in Chloroplast Development and Photosynthetic Electron Transport

The expression of several barley (Hordeum vulgare) cold-regulated (cor) genes during cold acclimation was blocked in the albino mutanta n, implying a chloroplast control on mRNAs accumulation. By using albino and xanthamutants ordered according to the step in chloroplast biogenesis affected, we show that the cold-dependent accumulation ofcor14b, tmc-ap3, andblt14 mRNAs depends on plastid developmental stage. Plants acquire the ability to fully express cor genes only after the development of primary thylakoid membranes in their chloroplasts. To investigate the chloroplast-dependent mechanism involved in cor gene expression, the activity of a 643-bp cor14b promoter fragment was assayed in wild-type and albino mutant a n leaf explants using transient β-glucuronidase reporter expression assay. Deletion analysis identified a 27-bp region between nucleotides −274 and −247 with respect to the transcription start point, encompassing a boundary of some element that contributes to the cold-induced expression of cor14b. However, cor14bpromoter was equally active in green and in albinoa n leaves, suggesting that chloroplast controls cor14b expression by posttranscriptional mechanisms. Barley mutants lacking either photosystem I or II reaction center complexes were then used to evaluate the effects of redox state of electron transport chain components on COR14b accumulation. In the mutants analyzed, the amount of COR14b protein, but not the steady-state level of the corresponding mRNA, was dependent on the redox state of the electron transport chain. Treatments of thevir-zb63 mutant with electron transport chain inhibitors showed that oxidized plastoquinone promotes COR14b accumulation, thus suggesting a molecular relationship between plastoquinone/plastoquinol pool and COR14b.

The accumulation of a cold-regulated chloroplastic protein is light-dependent

The protein encoded by cDNA clone pt59 and induced in barley (Hordeum vulgare L.) by cold was overexpressed in coli to produce the matching antibody, which in vivo recognized a cold-induced protein of 14 kDa (COR 14) that was found in the chloroplast stroma. The accumulation of COR14 occurred only at low temperatures after even a brief exposure of the plants to light. Plants grown and fully hardened in the dark accumulated a reduced amount of pt59-corresponding mRNA and only traces of COR14. Light exposure for as short as 5 min was enough to normalize the expression of pt59-corresponding mRNA and increase the accumulation of COR14. These findings indicate that one or more light-dependent factors are involved in transcription of the gene and accumulation of the protein. The COR14 protein was stored in amounts only slightly greater in the resistant barley cultivar Onice than in the susceptible cultivar Gitane, although the former had a higher induction-temperature threshold for COR14 than the latter. This fact is an evolutionary advantage, enabling the resistant varieties in the field to prepare for the cold well ahead of the susceptible ones.

First Survey of the Wheat Chromosome 5A Composition through a Next Generation Sequencing Approach

Wheat is one of the worlds most important crops and is characterized by a large polyploid genome. One way to reduce genome complexity is to isolate single chromosomes using flow cytometry. Low coverage DNA sequencing can provide a snapshot of individual chromosomes, allowing a fast characterization of their main features and comparison with other genomes. We used massively parallel 454 pyrosequencing to obtain a 2x coverage of wheat chromosome 5A. The resulting sequence assembly was used to identify TEs, genes and miRNAs, as well as to infer a virtual gene order based on the synteny with other grass genomes. Repetitive elements account for more than 75% of the genome. Gene content was estimated considering non-redundant reads showing at least one match to ESTs or proteins. The results indicate that the coding fraction represents 1.08% and 1.3% of the short and long arm respectively, projecting the number of genes of the whole chromosome to approximately 5,000. 195 candidate miRNA precursors belonging to 16 miRNA families were identified. The 5A genes were used to search for syntenic relationships between grass genomes. The short arm is closely related to Brachypodium chromosome 4, sorghum chromosome 8 and rice chromosome 12; the long arm to regions of Brachypodium chromosomes 4 and 1, sorghum chromosomes 1 and 2 and rice chromosomes 9 and 3. From these similarities it was possible to infer the virtual gene order of 392 (5AS) and 1,480 (5AL) genes of chromosome 5A, which was compared to, and found to be largely congruent with the available physical map of this chromosome.

Development of analytical systems based on real-time PCR for Triticum species-specific detection and quantitation of bread wheat contamination in semolina and pasta

The cereal composition of specific foods is always a key factor in the quality and safety of the final product. It is important to introduce new control methods for certain special foods, such as those for coeliac consumers, where the contamination of different cereal species must be accurately determined. Moreover, pasta made from durum wheat is considered superior in several qualitative aspects to that manufactured with bread wheat or a mixture of the two species. This work has been directed toward the development of analytical systems for the qualitative and quantitative detection of specific cereals in food. More specifically, the primary aim of this work has been to develop analytical tools based on end-point and real-time PCR to detect the presence of Triticum species in flour and food. Furthermore, qualitative and quantitative PCR-based methods are proposed to detect hexaploid wheat adulteration in pasta.

Characterization of two barley genes that respond rapidly to dehydration stress

Abstract The present study investigates the barley cDNA clones paf93 and cdr29 which correspond to mRNAs regulated by low temperature and drought stress but not by exogenous ABA treatment. During the early stage of dehydration, these mRNAs are expressed before the induction of known ABA regulated genes such as dehydrins and when only a small increase occurs in ABA content. The cDNA clone cdr29 cross-hybridizes with two classes of mRNAs differing both in size and expression patterns. The sequence analysis revealed that paf93 encodes a protein homologous with the cold-regulated protein COR47 of Arabidopsis whereas cdr29 represents a plant gene homologous to yeast and mammalian sequences coding for acyl-coenzyme A oxidase.

Cold-regulated gene expression during winter in frost tolerant and frost susceptible barley cultivars grown under field conditions

Winterhardiness is a basic trait for successful winter survival barley (Hordeum vulgare L.) crop. Freezing tolerance, a fundamental component of winterhardiness, is based on an inducible process known as hardening or cold acclimation that occurs when plants are exposed to low non-freezing temperatures. In the recent years, many temperature-dependent genes specifically expressed during hardening have been isolated. Current data on relationship between gene expression and cold tolerance are mostly based on plants grown and hardened under environmentally controlled conditions and, usually, over a short period of time. In order to verify whether variations in the molecular response to cold are likely to be of significant adaptive value under natural environments, we have followed the accumulation of several COR genes ( pt59, pao86 and paf93) and proteins (COR14a and COR14b) during the 1996/97 and 97/98 winter seasons in barley cultivars with contrasting winterhardiness capacity grown under field conditions. In the 1996/97 experiment, a winter cultivar Onice and a spring cultivar Gitane were tested for the accumulation of the cold-regulated genes and proteins. The ability of the plants to promote a strong molecular response to cold was found to be associated with the winterhardiness capacity of the two cultivars. This result was further tested in the winter season 1997/98 using 10 barley varieties. All winter cultivars showed high accumulation of the cold-regulated proteins COR14a and COR14b, while some variations for this character were detected in the spring cultivars suggesting that the selection for winter survival has been effective to fix the high COR14 accumulation capacity. We conclude that a high level of COR14 may be a component the winter survival capacity of barley.

The transcripts of several components of the protein synthesis machinery are cold-regulated in a chloroplast-dependent manner in barley and wheat

Summary Nine clones coding different components of the protein synthesis machinery were selected from an EST barley library prepared from cold exposed plants and tested for their expression at low temperature. Northern analysis revealed that expression of elongation factor (EF) 1Bβ and two ribosomal protein (RP) genes S7 and L7A was enhanced following exposure to 3 °C, but not during dehydration or exogenous ABA treatment. The mRNA levels of EF1Bβ, RPS7, and RPL7A did not vary between cultivars with different frost tolerance, but differences in expression were found between different species. Experiments with an albino mutant and etiolated plants revealed that the cold-dependent regulation of EF1Bβ, RPS7, and RPL7A transcripts is controlled by a chloroplast-related pathway impaired in the mutant.

Preliminary study of the inheritance of temperature stress proteins in barley (hordeum vulgare L.)

Abstract Comparisons of three barley genotypes, two parentals and their F 1 offspring, were made for protein synthesis modifications upon exposure to a wide range of temperatures from 5° to 40°C. The two parental genotypes were selected for their differences in growth habit, geographical origin, spike type and cold hardiness characteristics. Moreover the two genotypes differed in specific proteins which appeared at low or at high temperatures. The F 1 progeny obtained showed the following characteristics: (a) some of the heat stress induced proteins (HSP) had similarity with those of the parentals; the highest number of similarities was found between F 1 and the Georgie parental; (b) the pattern of HSP induced was different at 34° and 40°C, a temperature dependency which is typical of Onice at the level of coleoptiles and specific of Georgie at the level of roots; (c) a relevant number of HSP in F 1 had no parental counterparts, i.e. they were F 1 or hybrid specific. Analysis of the cold induced proteins (CSP) showed the F 1 progeny inherited most of the CSP characteristics of both parentals, though with a preference of the Onice parental at least at the level of coleoptiles. These results, that few stress genes had an uniparental heredity, whereas other stress genes showed an F 1 specific pattern of expression may probably be a consequence of new regulatory interactions which took place in the hybrid between the stress genes of the two parentals.