When rice gets the chills: comparative transcriptome profiling at germination shows WRKY transcription factor responses.

Rice is vital for food security. Due to its tropical origin, rice suffers from cold temperatures that affect its entire life cycle. Key genes have been identified involved in cold tolerance. WRKYs are generally downstream of the MAPK cascade and can act together with VQ proteins to regulate stress-responsive genes. Chilling treatment was applied at germination to two rice genotypes (tolerant and sensitive). Shoots at S3 stage were collected for RNA-seq to identify OsWRKY, OsMAPKs and OsVQs expression. Relationships among MAPKs, WRKYs and VQs were predicted through correlation analysis. OsWRKY transcriptional regulation was predicted by in silico analysis of cis-regulatory elements. A total of 39 OsWRKYs were differentially expressed. OsWRKY21, OsWRK24 and OsWRKY69 are potential positive regulators, while OsWRKY10, OsWRK47, OsWRKY62, OsWRKY72 and OsWRKY77 are potential negative regulators, of chilling tolerance. 12 OsMAPKs were differentially expressed. OsMAPKs were downregulated and negatively correlated with the upregulated OsWRKYs in the tolerant genotype. 19 OsVQs were differentially expressed, three and six OsVQs were positively correlated with OsWRKYs in the tolerant and sensitive genotypes, respectively. Seven differentially expressed OsWRKYs have cold-responsive elements in their promoters and five upregulated OsWRKYs in the tolerant genotype contained the W-box motif. Chilling causes changes in OsWRKY, OsMAPK and OsVQ gene expression at germination. OsWRKYs may not act downstream of the MAPK cascade to coordinate chilling tolerance, but OsWRKYs may act with VQs to regulate chilling tolerance. Candidate OsWRKYs are correlated and have a W-box in the promoter, suggesting an auto-regulation mechanism.

Iron excess in rice: from phenotypic changes to functional genomics of WRKY transcription factors.

Iron (Fe) is an essential microelement for all living organisms playing important roles in several metabolic reactions. Rice (Oryza sativa L.) is commonly cultivated in paddy fields, where Fe goes through a reduction reaction from Fe3+ to Fe2+. Since Fe2+ is more soluble, it can reach toxic levels inside plant cells, constituting an important target for studies. Here we aimed to verify morphological changes of different rice genotypes focusing on deciphering the underlying molecular network induced upon Fe excess treatments with special emphasis on the role of four WRKY transcription factors. The transcriptional response peak of these WRKY transcription factors in rice seedlings occurs at 4 days of exposition to iron excess. OsWRKY55-like, OsWRKY46, OsWRKY64, and OsWRKY113 are up-regulated in BR IRGA 409, an iron-sensitive genotype, while in cultivars Nipponbare (moderately resistant) and EPAGRI 108 (resistant) the expression profiles of these transcription factors show similar behaviors. Here is also shown that some cis-regulatory elements known to be involved in other different stress responses can be linked to conditions of iron excess. Overall, here we support the role of WRKY transcription factors in iron stress tolerance with other important steps toward finding why some rice genotypes are more tolerant than others.

Identification and characterization of the bZIP transcription factor involved in zinc homeostasis in cereals.

Members of the basic leucine zipper family, as bZIP19, are considered to be essential regulators of the adaptation to zinc deficiency. Knowing that this gene as well as its targets are conserved in the plant kingdom, we followed an in silico approach to identify and characterize the bzip19 gene in cereals. Through BLASTp in Phytozome database, 33 bzip19 genes were identified on the genomes of Oryza sativa, Sorghum bicolor, Zea mays, Glycine max, Triticum aestivum, and Brachypodium distachyon. The analysis of conserved motifs and bZIP domains was performed using MEME and PFAM databases. In 25 of these genes, CysHis-motifs at the basic N-terminal region were found. This motif is conserved in group bZIP and suggested to play a role as a Zn-sensor. Regarding their phylogeny, it was possible to infer orthologous groups and explore the evolutionary relationship between these BZIP19 proteins. Data mining allowed us to select eight putative orthologous whose expression profile was analyzed under stress conditions in the Genevestigator platform. The comparison between the expression profiles of these eight putative orthologous and the original Arabidopsis bzip19 also seems to indicate conserved transcriptional regulation. Thus, considering that modified expression of bZIP19 genes has already been confirmed as an efficient tool to improve Arabidopsis tolerance to Zn deficiency, and that these new bZIP orthologous have a high level of conservation when compared to the original bZIP19 sequence, they can be useful for the development of tolerant crops enabling plants to grow in areas of low zinc bioavailability.

Expression profile of rice Hsp genes under anoxic stress.

Although flooding is one of the most important environmental stresses worldwide, not all plant species are intolerant to its effects. Species from semi-aquatic environments, such as rice, have the capacity to cope with flooding stress. Heat-shock proteins (Hsps) are thought to contribute to cellular homeostasis under both optimal and adverse growth conditions. Studies of gene expression in plants exposed to low levels of oxygen revealed the up-regulation of Hsp genes. However, it is not clear whether Hsp genes are transcribed as a function of tolerance or whether they represent a response to anoxic stress. Therefore, the accumulation of Hsp gene transcripts was investigated in two different cultivars, "Nipponbare" (flooding tolerant) and "IPSL 2070" (flooding sensitive), subjected to anoxic stress. Fifteen-day-old rice root seedlings from both cultivars were used. Four different treatments were performed: no anoxia (control); 24-h anoxia; 48-h anoxia; and 72-h anoxia. Anoxic stress was confirmed by the increased gene expression of alcohol dehydrogenase. The data obtained showed that both rice cultivars ("Nipponbare" and "IPSL 2070") accumulated Hsp gene transcripts under anoxic stress; however, the majority of the Hsp genes evaluated were responsive to anoxic stress in "IPSL 2070" (flooding sensitive), whereas in "Nipponbare" (flooding tolerant), only six genes were highly up-regulated. This suggests that although Hsps have an important role in the response to anoxia, they are not the major cause of tolerance.

Relationships between four measures of genetic distance and breeding behavior in spring wheat.

We estimated the genetic distances among 10 spring wheat genotypes based on pedigree data, morphological traits and AFLP markers, used individually and combined with morphological traits, to find the best predictors of general- and specific-combining abilities among parental genotypes. Ten wheat parents were crossed in a diallel form, disregarding reciprocal hybrids, totaling 45 combinations. The F1 hybrids, F2 populations and parents were evaluated in the field in 2007. The experimental plots consisted of 20 plants for F1 hybrids and 40 plants for parental and F2 populations. All methods (pedigree data, AFLP markers and morphological traits, used individually and combined) were found to be useful for the assessment of genetic diversity. The significant coefficient correlations ranged from low (0.45) to moderate (0.67) between the distance measures and hybrid performance. There was significant agreement between the distance measures based on AFLP markers vs morphological traits + AFLP markers (r = 0.47) and between pedigree data vs morphological traits + AFLP markers (r = 0.43). The pedigree distance was positively associated with traits 100-kernel weight and grain yield per plant in F1 (correlations of 0.67 and 0.62, respectively) and F2 (correlations of 0.62 and 0.59, respectively) generations. These correlation values indicate that the genetic distance, based on pedigree data, could replace diallel crosses for the selection of parents with higher combining ability and with moderate reliability.

The Hybaid Lecture. Microcollinearity and segmental duplication in the evolution of grass nuclear genomes.

Recent studies have shown that grass genomes have very similar gene compositions and regions of conserved gene order, as exemplified by collinear genetic maps of DNA markers. We have begun the detailed study of sequence organization in large (100-500 kb) segments of the nuclear genomes of maize, sorghum and rice. Our results indicate collinearity of genes in the regions homoeologous to the maize adh1 and sh2-a1 genes. Comparable genes were found to be physically closer to each other in grasses with small genomes (rice and sorghum) than they are in maize. In several instances, we have found evidence of tandem and 'distantly tandem' duplications of segments containing maize and sorghum genes. These duplications complicate characterizations of microcollinearity and could also interfere with some map-based approaches to gene isolation.

Effect of diets containing dry beans (Phaseolus vulgaris, L.) on the rat excretion of endogenous nitrogen.

Wistar rats of 60.5 +/- 5.0 g fasted for 24 h were injected intraperitoneally with 10 microCi of [14C]glycine. One to two hours after injection the rats were fed a diet containing 10.53 +/- 0.75% protein provided by dry beans or casein, or a protein-free diet and submitted to a 4-d nitrogen balance. Radioactivity in the feces of rats fed casein, cooked beans and raw beans was roughly 2, 5 and 10 times greater, respectively, than in the feces of those fed the protein-free diet. Apparent protein digestibility showed a strong negative linear correlation (r = -0.9805, P less than or equal to 0.01) with radioactivity in the feces. Positive correlation (P less than or equal to 0.01) was demonstrated between radioactivity and either total carbon or total nitrogen in the feces of rats injected with [14C]glycine. Mean value for the radioactivity in the urine of rats fed the different diets did not differ significantly (P less than or equal to 0.05). Endogenous nitrogen excretion of rats on bean diets was estimated by the ratio of total endogenous N to marker N, based on the protein-free diet. The results indicated that rats fed bean-containing diets excreted significantly more endogenous nitrogen than those fed the casein diet, even though the casein diet had stimulated twice as much endogenous excretion than the protein-free diet. As a consequence, apparent digestibility and biological value of bean protein are generally underestimated, although the "real" biological value was not affected by the endogenous nitrogen excretion of the rat.