Calreticulin: conserved protein and diverse functions in plants.

Calreticulin (CRT) is a key Ca2+-binding protein mainly resident in the endoplasmic reticulum (ER), which is highly conserved and extensively expressed in all eukaryotic organisms investigated. The protein plays important roles in a variety of cellular processes including Ca2+ signaling and protein folding. Although calreticulin has been well characterized in mammalian systems, increased investigations have demonstrated that plant CRTs have a number of specific properties different from their animal counterparts. Recent developments on plant CRTs have highlighted the significance of CRTs in plants growth and development as well as biotic and abiotic stress responses. There are at least two distinct groups of calreticulin isoforms in higher plants. Glycosylation of CRT was uniquely observed in plants. In this article, we will describe our current understanding of plant calreticulin gene family, protein structure, cellular localization, and diverse functions in plants. We also discuss the prospects of using this information for genetic improvements of crop plants.

Identification of quantitative trait loci and environmental interactions for accumulation and remobilization of water-soluble carbohydrates in wheat (Triticum aestivum L.) stems.

Genetic analyses of nine traits associated with stem water-soluble carbohydrate (SWSC) accumulation and remobilization at grain-filling period under drought stress (DS) and well-watered (WW) conditions were undertaken using doubled haploid lines (DHLs) derived from two Chinese common wheat cultivars. Some significantly and very significantly positive correlation was observed among nine traits associated with SWSC. Higher phenotypic values for most traits were detected under DS. Broad sense heritabilities (h(B)(2)) of the traits showed wide fluctuations between two water treatments. A total of 48 additive and 62 pairs of epistatic QTL for nine traits were identified as distributing on all 21 chromosomes. A majority of QTL involved significant additive and epistatic effects with interactions of QTL and environments (QEIs). Two additive and two pairs of epistatic loci involved only QEIs without corresponding significant additive or epistatic effects. The contributions of the additive QEIs were two- to fourfolds higher than those of their corresponding additive QTL. Most of the additive QEIs for traits associated with SWSC interacted with DS. In addition, some QTL for the grain-filling efficiencies and thousand-grain weight were colocated in the same or adjacent chromosome intervals with QTL for accumulation and remobilization efficiency of SWSC before 14 days after flowering.

Molecular cloning and characterization of wheat calreticulin (CRT) gene involved in drought-stressed responses.

Calreticulin (CRT) is a highly conserved and ubiquitously expressed Ca(2+)-binding protein in multicellular eukaryotes. CRT plays a crucial role in many cellular processes including Ca(2+) storage and release, protein synthesis, and molecular chaperone activity. To elucidate the function of CRTs in plant responses against drought, a main abiotic stress limiting cereal crop production worldwide, a full-length cDNA encoding calreticulin protein namely TaCRT was isolated from wheat (Triticum aestivum L.). The deduced amino acid sequence of TaCRT shares high homology with other plant CRTs. Phylogenetic analysis indicates that TaCRT cDNA clone encodes a wheat CRT3 isoform. Southern analysis suggests that the wheat genome contains three copies of TaCRT. Subcellular locations of TaCRT were the cytoplasm and nucleus, evidenced by transient expression of GFP fused with TaCRT in onion epidermal cells. Enhanced accumulation of TaCRT transcript was observed in wheat seedlings in response to PEG-induced drought stress. To investigate further whether TaCRT is involved in the drought-stress response, transgenic plants were constructed. Compared to the wild-type and GFP-expressing plants, TaCRT-overexpressing tobacco (Nicotiana benthamiana) plants grew better and exhibited less wilt under the drought stress. Moreover, TaCRT-overexpressing plants exhibited enhanced drought resistance to water deficit, as shown by their capacity to maintain higher WUE (water use efficiency), WRA (water retention ability), RWC (relative water content), and lower MDR (membrane damaging ratio) (P < or = 0.01) under water-stress conditions. In conclusion, a cDNA clone encoding wheat CRT was successfully isolated and the results suggest that TaCRT is involved in the plant response to drought stress, indicating a potential in the transgenic improvements of plant water-stress.

[Comparison of methods to construct a full-length cDNA library].

The use of full-length cDNA libraries is an effective tool to obtain complete gene information in a high-efficiency, high-throughput manner, especially in organisms with huge genomes that are not amenable to whole genome sequencing. In this review, we outlined several methods of full-length cDNA library construction and compared their advantages and disadvantages based on their respective principles. Drawing on our own experience, we described the Cap-trapper method in detail, with an emphasis on its application in wheat full-length cDNA library construction as well as the determination of the ratio of full-length cDNA in a library.

[A preliminary study on gene expression profile induced by water stress in wheat (Triticum aestivum L.) seedling].

In the present research, suppression subtractive hybridization (SSH) and high density membrane techniques were employed to analysis genes induced by water stress in wheat seedling at 2-leaf stage. The purpose was to comprehensively understand the genetic bases of drought resistance and to find the key genes related to drought resistance in wheat. A total of 181 positive clones were obtained by screening the SSH library including 1 530 individual recombinant clones. The result of the sequence homologous comparison and function querying showed that 83.2% differentially expressed genes were high homologous with those induced by abiotic or biotic stresses in plant and their functions were to protect the cell directly or indirectly from stress strain. Seventeen differentially expressed ESTs found first were registered in GenBank. After further identifying by reverse Northern, RT-PCR and Northern, the gene expression profile induced by water stress in wheat seedling was preliminarily revealed.

[Relationships of wheat leaf stomatal traits with wheat yield and drought-resistance].

Taking the DH population of wheat cultivar Hanxuan10/Lumai14 as test object, and by the methods of correlation analysis and path analysis, this paper studied the relationships of the flag leaf stomatal density (SD), stomatal length and width (SL and SW), stomatal conductance (g(s)), photosynthetic rate (P(n)), and transpiration rate (T(r)) on the 10th and 20th day after anthesis with the yield and the index of drought-resistance under the conditions of drought stress and normal irrigation. Under the two conditions, most of the test leaf traits on the 10th day after anthesis had less correlation with the yield and the index of drought-resistance, whereas the leaf traits on the 20th day after anthesis had significant positive correlations with thousand kernel weight but less correlation with grain number per ear, grain yield per plant, and index of drought-resistance. Path analysis showed that g(s), P(n), and T(r) were the main factors affecting the grain yield per plant (YPP) and the index of drought resistance (IDR), and the effects were stronger both in direct and in indirect ways. The direct and indirect effects of SD, SL, and SW on the YPP and IDR were lesser. Under both drought stress and normal irrigation, and on the 10th and 20th day after anthesis, there were significant correlations between SD and SL, and between SL and SW, g(s), P(n), and Tr, but the correlations of SD and SL with g(s), P(n), and T(r) changed with water condition or growth stage. Therefore, it would be not always a good means to select the leaf stomatal density and size as the targets for breeding to improve the leaf stomatal conductance, photosynthetic rate, and transpiration rate, and further, to promote the yield.

[Sequence polymorphism and mapping of wheat Ca2+-binding protein TaCRT-A gene].

Taking thirty-seven hexaploid wheat (AABBDD) accessions with different drought resistance at seedling stage, three wheat species with A genome (AA), and three tetraploid wheat species (AABB) as test materials, and by direct sequencing the single nucleotide polymorphism (SNP) in TaCRT-A, this paper analyzed the relationships of the SNP with the drought resistance of wheat ( Triticum aestivum) at its seedling stage, and mapped the TaCRT-A on the chromosome of wheat. The full-length sequence of the TaCRT-A genomic DNA was 3887 bp. A total of 202 nucleotide variant loci were observed in the full length sequence of 167141 bp, among which, 165 SNP and 37 InDel with the frequencies of 1 SNP/1013 bp and 1 InDel/4517 bp were detected, respectively. The nucleotide diversity (pi) in coding region of TaCRT-A was lower than that in non-coding region, suggesting that the selection pressure in coding region was stronger than that in non-coding region. The 43 accessions could be classified as 14 haplotypes (H1-H14) by haploid analysis, among which, H1, H2, and H13 all contained one accession which was the donor species of A genome in common wheat, H16 and H7 had one high drought-resistant accession, H8 comprised tetraploid wheat, drought-resistant accessions, and drought-sensitive accessions, whereas H11 included the wheat accessions with drought-resistance and medium-drought resistance. Though the expression of TaCRT was induced by water stress, no significant relationship was identified between TaCRT-A polymorphism and drought resistance. Using a population of recombinant inbred lines derived from a cross of Opata 85 x W7984, the TaCRT-A was mapped between SSR markers Xmwg30 and Xmwg570 on chromosome 3A, and the genetic distances were 10.5 cM and 49.6 cM from the flanking markers, respectively.

Isolation and characterization of a gene encoding a polyethylene glycol-induced cysteine protease in common wheat.

Plant cysteine protease (CP) genes are induced by abiotic stresses such as drought, yet their functions remain largely unknown. We isolated the full-length cDNA encoding a Triticum aestivum CP gene, designated TaCP, from wheat by the rapid amplification of cDNA ends (RACE) method. Sequence analysis revealed that TaCP contains an open reading frame encoding a protein of 362 amino acids, which is 96% identical to barley cysteine protease HvSF42. The TaCP transcript level in wheat seedlings was upregulated during polyethylene glycol (PEG) stress, with a peak appearing around 12 h after treatment. TaCP expression level increased rapidly with NaCl treatment at 48 h. TaCP responded strongly to low temperature (4 degree C) treatment from 1 h post-treatment and reached a peak of about 40-fold at 72 h. However, it showed only a very slight response to abscisic acid (ABA). More than one copy of TaCP was present in each of the three genomes of hexaploid wheat and its diploid donors. TaCP fused with green fluorescent protein (GFP) was located in the plasma membrane of onion epidermis cells. Transgenic Arabidopsis plants overexpressing TaCP showed stronger drought tolerance and higher CP activity under water-stressed conditions than wild-type Arabidopsis plants. The results suggest that TaCP plays a role in tolerance to water deficit.

Cloning the PvP5CS gene from common bean (Phaseolus vulgaris) and its expression patterns under abiotic stresses.

A full-length cDNA denominated PvP5CS for Delta(1)-pyrroline-5-carboxylate synthetase (P5CS), an enzyme involved in the biosynthesis of proline, was cloned from common bean using a candidate gene approach. PvP5CS contains an open reading frame encoding a 716 amino acid polypeptide. Sequence analysis showed that PvP5CS shares 95.1% homology in nucleotide sequence and 93.2% identity in amino acid sequence with the mothbean (Vigna aconitifolia) P5CS. The expression patterns of PvP5CS in common bean treated with drought, cold (4 degrees C), and salt (200 mM NaCl) stresses were examined using real-time quantitative PCR. These abiotic stresses caused significant up-regulation of the expression of PvP5CS in leaves. The PvP5CS mRNA transcript increased to 2.5 times the control level after 4d drought stress. A rapid up-regulation of PvP5CS, to about 16.3 times the control at 2h post-treatment was observed under salt stress. A significant increase in PvP5CS expression (11.7-fold) was detected after 2h of cold stress. The peaks of proline accumulation appeared at 8d for drought, 24h for cold and 9h for salt stress, somewhat later than the peaks of PvP5CS expression. These results suggest that PvP5CS was a stress-inducible gene regulating the accumulation of proline in plants subjected to stress. Finally, subcellular localization assays showed that the PvP5CS protein was present in the nucleus and at the plasmalemma.

[Genetic characteristics of wheat functional leaves at filling stage under different water regimes].

Taking one hundred and fifty doubled haploid (DH) lines of winter wheat cultivars Hanxuan 10 x Lumai 14 as well as their parents as test materials, the genetic bases of top three leaves length, breadth, and angle at mid-filling stage, and their correlations with yield traits were studied under two water regimes rainfed (drought stress, DS) and well-irrigation (WI) in 2005-2007. Under DS, the leaf length and width of DH lines and their parents were significantly lower than those under WI, whereas the leaf angle had a miscellaneous variation. Under the two water regimes, all the test traits of DH lines had a transgressive segregation, with the variation coefficients ranged from 5.1% to 45.9%. The heritability and gene numbers for the given traits showed great differences. Under WI and DS, the heritability of flag leaf angle (FLA) was the highest (91% and 97%, respectively), and that of the third leaf angle (TLA) was the lowest (23% and 31%, respectively). However, the gene number for FLA (4 and 2 under WI and DS, respectively) was the least, and that of TLA (21 and 25, respectively) in 2007 was the most. Interactive effects were observed among the genes controlling FLA, second leaf angle (SLA), and TLA under WI and DS, and controlling third leaf length (TLL) under WI. The grain number per spike and the grain weight per spike were positively correlated with the length and width of top three leaves, and the thousand-grain weight and the yield per plant were significantly correlated with TLL, FLA, and SLA, but all correlation coefficients were smaller ( < 0.481). It was suggested that the selection of top three leaves length and width should be carried out in the early generations of breeding procedure, while that of leaf angles should be carried out in advanced generations. An appropriate soil moisture regime retained in the critical growth period of the leaves would promote their growth and contribute to the grain yield.