Expression of a cold-responsive Lt-Cor gene and development of freezing tolerance during cold acclimation in wheat (Triticum aestivum L.).

Time-courses of the development of freezing tolerance and the expression of a cold-responsive gene wlt10 were monitored during cold acclimation in wheat (Triticum aestivum L.). Bioassay showed that cold acclimation conferred much higher freezing tolerance on a winter cultivar than a spring cultivar. Northern blot analysis showed that the expression of wlt10 encoding a novel wheat member of a cereal-specific LT-COR protein family was specifically induced by low temperature. A freezing-tolerant winter cultivar accumulated the mRNA more rapidly and for a longer period than a susceptible spring cultivar. The increase in the amount of mRNA was temporary but the peak occurred at the time when the maximum level of freezing tolerance was attained. The mRNA accumulated more in the leaves than in the roots, and different light/dark regimes modulated the level of mRNA accumulation. Genomic Southern blot analyses using the nulli-tetrasomic series showed that the wlt10 homologues were located on the homologous group 2 chromosomes.     

Nucleotide sequence and molecular analysis of the low temperature induced cereal gene, BLT4

Summary The nucleotide sequence and derived amino acid sequence of a cDNA clone (BLT4) for a low temperature induced barley gene were determined. This gene, together with a small family of related genes, was shown to reside on chromosome 3. The BLT4 clone has homology with genes in wheat and oats. Its expression was studied in oats and in barley doubled haploid lines segregating for spring/winter habit and for frost hardiness. These analyses show that elevated steady state levels of BLT4 mRNA are produced in shoot meristematic tissue after 3 days low positive temperature treatment. The low temperature response was found in all barley doubled haploid lines and was therefore not associated specifically with either the spring/winter habit or frost hardiness. Elevated levels of BLT4 mRNA were also seen in drought-stressed barley and it is likely that this is a gene encoding a low molecular weight protein that is responsive to dehydrative stresses, such as cold and drought.The EMBL accession number for BLT4 is X56547 H. vulgare cDNA     

Cold-induced accumulation of protein in the leaves of spring and winter barley cultivars

Electrophoretic pattern and quantitative changes in soluble proteins were determined in the leaves of spring and winter cultivars of barley (Hordeum vulgare L., cv. Makouei and cv. Reyhan, respectively) exposed to 4 degrees C for 14 d. Seedlings were grown in a controlled growth chamber for 2 weeks at a constant air temperature of 20 degrees C and then transferred to constant 4 degrees C for 14 d followed by returning to 20 degrees C (cold treatment), or they were maintained throughout at 20 degrees C during the experimental period of 40 d (control treatment). Plants were sampled every 48 h for leaf fresh weight measurements. Total leaf soluble proteins were extracted and their concentration was either determined by a colorimetric method, or size-fractionated on SDS-PAGE. Low temperature-induced increases in protein amount occurred over the second week of exposure to cold treatment irrespective of cultivar: the winter cultivar was 2 d prior in this response. The protein patterns and their density showed differences between-cultivars and between-temperature treatments. A new cold-induced polypeptide was recognized in the leaves of winter barley cultivar on day 22 (8 d at 4 degrees C) compared to the control. This polypeptide was produced earlier over the first 48 h of low temperature in the winter cultivar compared with the spring one, recognizing in the leaves of cold-treated seedling until day 26. This more rapid response to a low temperature by the winter barley cultivar indicates a more sensitive response compared with the spring barley, probably cold-shock protein is a component of this cold-induced response.     

High expression level of a gene coding for a chloroplastic amino acid selective channel protein is correlated to cold acclimation in cereals

A cold-regulated gene (cor tmc-ap3) coding for a putative chloroplastic amino acid selective channel protein was isolated from cold-treated barley leaves combining the differential display and the 5-RACE techniques. Cor tmc-ap3 is expressed at low level under normal growing temperature, and its expression is strongly enhanced after cold treatment. A positive correlation between the expression of cor tmc-ap3 and frost tolerance was found both among barley cultivars and among cereal species. The COR TMC-AP3 protein was expressed in vitro, purified and used to raise a polyclonal antibody. Western analysis showed that the cor tmc-ap3 gene product is localized to the chloroplastic outer envelope fraction, supporting its putative function. The frost-resistant winter cultivar Onice accumulated COR TMC-AP3 more rapidly and at a higher level than the frost-susceptible spring cultivar Gitane. After 28 days of cold acclimation the winter cultivar had about 2-fold more protein than the spring genotype. All these results suggest that an increased amount of a chloroplastic amino acid selective channel protein could be required for cold acclimation in cereals. Hypotheses about the role of COR TMC-AP3 during the hardening process are discussed.     

A minimum requirements method to isolate large quantities of highly purified DNA from one drop of poultry blood

Requirement of vernalization is an important factor which plays a crucial role in cereals to transit from vegetative to reproductive phase. There are three types of growth habit in barley: winter, spring and facultative types; in which spring type does not require vernalization but winter and facultative genotypes require full and partial vernalization, respectively. Combination of two loci, Vrn-h1 and Vrn-h2, regulates vernalization in barley genotypes. Specific DNA markers have been identified for growth habit regulator genes in barley. In this study, we examined 24 barley genotypes using specific primers for detecting Vrn-h1 and Vrn-h2 loci. Results showed that among all differently suggested primer combinations, a few markers were precisely correlated with seasonal growth habit in barley. The specific markers of 600, 600 and 200 bps were verified for ZCCT-Ha, ZCCT-Hb and ZCCT-Hc loci, respectively. Our field growth habit test showed that cultivar Bahman as a winter growth habit, where all the others genotypes exhibited spring growth habit. By using specific primers for Vrn-h1, only Bahman cultivar produced 616 bp and 830 bp fragments and spring genotypes showed 574 bp or 616 bp alleles without any amplification for 830 bp fragments. Therefore, presence of 616 bp and 830 bp alleles together in each genotype can be considered as an informative marker for winter growth habit in barley. These informative markers can be used easily in barley breeding programmes for detection of growth habit types in the seedling stage.     

Molecular and Structural Characterization of Barley Vernalization Genes

Vernalization, the requirement of a period of low temperature to induce transition from the vegetative to reproductive state, is an evolutionarily and economically important trait in the Triticeae. The genetic basis of vernalization in cultivated barley (Hordeum vulgare subsp. vulgare) can be defined using the two-locus VRN-H1/VRN-H2 model. We analyzed the allelic characteristics of HvBM5A, the candidate gene for VRN-H1, from ten cultivated barley accessions and one wild progenitor accession (subsp. spontaneum), representing the three barley growth habits – winter, facultative, and spring. We present multiple lines of evidence, including sequence, linkage map location, and expression, that support HvBM5A being VRN-H1. While the predicted polypeptides from different growth habits are identical, spring accessions contain a deletion in the first intron of HvBM5A that may be important for regulation. While spring HvBM5A alleles are typified by the intron-localized deletion, in some cases, the promoter may also determine the allele type. The presence/absence of the tightly linked ZCCT-H gene family members on chromosome 4H perfectly correlates with growth habit and we conclude that one of the three ZCCT-H genes is VRN-H2. The VRN-H2 locus is present in winter genotypes and deleted from the facultative and spring genotypes analyzed in this study, suggesting the facultative growth habit (cold tolerant, vernalization unresponsive) is a result of deletion of the VRN-H2 locus and presence of a winter HvBM5A allele. All reported barley vernalization QTLs can be explained by the two-locus VRN-H1/VRN-H2 model based on the presence/absence of VRN-H2 and a winter vs. spring HvBM5A allele. Electronic Supplementary Material Electronic Supplementary material is available for this article at and accessible for authorised users.     

Effect of the 5R(5A) Alien Chromosome Substitution on the Growth Habit and Winter Hardiness of Wheat

The growth habit, ear emergence time, and frost tolerance of wheat/rye substitution lines have been studied in cultivars Rang and Mironovskaya Krupnozernaya whose chromosome 5A is substituted with chromosome 5R of Onkhoyskaya rye. Hybrid analysis has demonstrated that the spring habit of the recipient cultivars Rang and Mironovskaya Krupnozernaya is controlled by dominant gene Vrn-A1 located in chromosome 5A. Onokhoyskaya rye has a dominant gene for the spring habit (Sp1) located in chromosome 5R. It has been found that the resultant 5R(5A) alien-substitution lines have a winter type of development and ears do not emerge during summer in plants sown in spring. The change in growth habit has been shown to be related to the absence of the rye Sp1 gene expression in the substitution lines. The winter hardiness of winter 5R(5A) alien-substitution lines has been studied under the environmental conditions of Novosibirsk. Testing the lines in the first winter demonstrated that their winter survival is 20–27%. The possible presence of the frost resistance gene homeoallelic to the known genes Fr1 and Fr2 of the common wheat located on chromosomes 5A and 5D, respectively, is discussed.     

Low-temperature tolerance and genetic potential in wheat (Triticum aestivum L.): response to photoperiod, vernalization, and plant development

It is frequently observed that winter habit types are more low-temperature (LT) tolerant than spring habit types. This raises the question of whether this is due to pleiotropic effects of the vernalization loci or to the linkage of LT-tolerance genes to these vernalization loci. Reciprocal near-isogenic lines (NILs) for alleles at the Vrn-A1 locus, Vrn-A1 and vrn-A1, determining spring and winter habit respectively, in two diverse genetic backgrounds of wheat (Triticum aestivum L.) were used to separate the effects of vernalization, photoperiod, and development on identical, or near identical, genetic backgrounds. The vrn-A1 allele in the winter lines allowed full expression of genotype dependent LT tolerance potential. The winter allele (vrn-A1) in a very cold tolerant genetic background resulted in 11°C, or a 2.4-fold, greater LT tolerance compared to the spring allele. Similarly, the delay in development caused by short-day (SD) versus long-day (LD) photoperiod in the identical spring habit NIL resulted in an 8.5°C or 2.1-fold, increase in LT tolerance. The duration of time in early developmental stages was shown to underlie full expression of genetic LT-tolerance potential. Therefore, pleiotropic effects of the vernalization loci can explain the association of LT tolerance and winter habit irrespective of either the proposed closely linked Fr-A1 or the more distant Fr-A2 LT-tolerance QTLs. Plant development progressively reduced LT-acclimation ability, particularly after the main shoot meristem had advanced to the double ridge reproductive growth stage. The Vrn-1 genes, or other members of the flowering induction pathway, are discussed as possible candidates for involvement in LT-tolerance repression.     

The change of chlorophyll fluorescence parameters in winter barley during recovery after freezing shock and as affected by cold acclimation and irradiance

The change of chlorophyll fluorescence parameters in froze leaves of 3 leaf-age seedlings were examined using two winter barley cultivars (Chumai 1 and Mo 103) differing in cold tolerance to investigate physiological response to low temperature as affected by cold acclimation (under 3/1 degrees C, day/night for 5 days before freezing treatment) and irradiation size (high irradiance: 380+/-25 micromol m(-2)s(-1) and low irradiance: 60+/-25 micromol m(-2)s(-1)) during recovery. The results showed that non-lethal freezing shock (exposed to -8 degrees C for 18 h) did not obviously affect maximum quantum efficiency in photosystem II (PSII), but dramatically increased non-photochemical quenching and reduced effective quantum yield in PSII. Cold acclimation significantly improved stability of photosynthetic function of leaves after freezing stress through buffering excessive energy and alleviating photoinhibition during recovery, indicating it increased recovery ability of barley plants from freezing injury. High irradiance was quite harmful to the stability of PSII in barley plants during recovery from freezing injury. The electron transport rate of PSII varied with cold-acclimation, irradiance and genotype. Cold acclimation caused significant increase in electron transport rate of PSII for relatively tolerant cultivar Mo 103, but not for relatively sensitive cultivar Chumai 1. It can be concluded that some chlorophyll fluorescence parameters during recovery from freezing shock may be used as the indicators in identification and evaluation of cold tolerance in barley.