Cristina Crosatti

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.

Photosynthetic Antenna Size in Higher Plants Is Controlled by the Plastoquinone Redox State at the Post-transcriptional Rather than Transcriptional Level

We analyze the effect of the plastoquinone redox state on the regulation of the light-harvesting antenna size at transcriptional and post-transcriptional levels. This was approached by studying transcription and accumulation of light-harvesting complexes in wild type versus the barley mutant viridis zb63, which is depleted in photosystem I and where plastoquinone is constitutively reduced. We show that the mRNA level of genes encoding antenna proteins is almost unaffected in the mutant; this stability of messenger level is not a peculiarity of antenna-encoding genes, but it extends to all photosynthesis-related genes. In contrast, analysis of protein accumulation by two-dimensional PAGE shows that the mutant undergoes strong reduction of its antenna size, with individual gene products having different levels of accumulation. We conclude that the plastoquinone redox state plays an important role in the long term regulation of chloroplast protein expression. However, its modulation is active at the post-transcriptional rather than transcriptional level.

Post-transcriptional and post-translational regulations of drought and heat response in plants: a spider’s web of mechanisms

Drought and heat tolerance are complex quantitative traits. Moreover, the adaptive significance of some stress-related traits is more related to plant survival than to agronomic performance. A web of regulatory mechanisms fine-tunes the expression of stress-related traits and integrates both environmental and developmental signals. Both post-transcriptional and post-translational modifications contribute substantially to this network with a pivotal regulatory function of the transcriptional changes related to cellular and plant stress response. Alternative splicing and RNA-mediated silencing control the amount of specific transcripts, while ubiquitin and SUMO modify activity, sub-cellular localization and half-life of proteins. Interactions across these modification mechanisms ensure temporally and spatially appropriate patterns of downstream-gene expression. For key molecular components of these regulatory mechanisms, natural genetic diversity exists among genotypes with different behavior in terms of stress tolerance, with effects upon the expression of adaptive morphological and/or physiological target traits.

Harden the chloroplast to protect the plant

The chloroplast is the central switch of the plants response to cold and light stress. The ability of many plant species to develop a cold tolerant phenotype is dependent on the presence of light and photosynthetic activity during low-temperature growth. Light exposure at low temperature stimulates an over-reduction of the plastoquinone pool as well as the accumulation of reactive oxygen species, and both metabolic conditions generate a retrograde signal controlling nuclear gene expression. At the same time the chloroplast is the target of many cold acclimation processes which are the results of the chloroplast-nucleus cross-talk. Often, the extent of cold acclimation of the chloroplast is tightly correlated with the overall plant tolerance to chilling and freezing temperatures, a finding suggesting that the chloroplast cold acclimation could be the rate limiting factor in the adaptation to low temperature.

Studies for assessing the influence of hardening on cold tolerance of barley genotypes

SummaryFrost tolerance of 30 barley (Hordeum vulgare L.) cultivars have been field evaluated in North Italy during the 1990/1991 winter season that was characterized by exceptionally low temperatures without snow cover. The results showed a significant correlation between cold injury and grain yield loss (r=0.61**). Five cultivars chosen for their varying degree of frost tolerance were further evaluated using laboratory tests. Measurements of survival rate and membrane damage were used to assess the influence of hardening on frost resistance. The reliability of the tests is shown by the high correlation to the field data. For both the laboratory temperature regimes and field conditions, the tested cultivars showed the same order of classification. The effect of a rise in temperature at the end of the hardening treatment on frost tolerance is also reported. The laboratory tests here proposed can be integrated in a breeding programme for improving frost tolerance in barley.

The rice Osmyb4 gene enhances tolerance to frost and improves germination under unfavourable conditions in transgenic barley plants

The Osmyb4 rice gene, coding for a transcription factor, proved to be efficient against different abiotic stresses as a trans(cis)gene in several plant species, although the effectiveness was dependent on the host genomic background. Eight barley transgenic lines carrying the rice Osmyb4 gene under the control of the Arabidopsis cold inducible promoter cor15a were produced to test the efficiency of this gene in barley. After a preliminary test, the best performing lines were subjected to freezing at −11°C and −12°C. Frost tolerance was assessed measured the Fv/Fm parameter widely used to indicate the maximum quantum yield of photosystem II photochemistry in the dark adapted state. Three transgenic lines showed significantly increased tolerance. These selected lines were further studied under a complex stress applying cold and hypoxia at germinating stage. In these conditions the three selected transgenic lines outperformed the wild type barley in terms of germination vigour. The transgenic plants also showed a significant modification of their metabolism under cold/hypoxia conditions as demonstrated through the assessment of the activity of key enzymes involved in anoxic stress response. None of the transgenic lines showed dwarfism, just a slight retarded growth. These results provide evidence that the cold dependent expression of Osmyb4 can efficiently improved frost tolerance and germination vigour at low temperature without deleterious effect on plant growth.

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.