Over-expression of OsPT2 under a rice root specific promoter Os03g01700.

Identification of root-specific promoters is a good method to drive root-specific gene expression for nutrient uptake. Constitutive over-expression of OsPT2 may have negative effects on the growth of rice seedlings under high Pi condition. Thus, characterization and utilization of root-specific promoters are critical for genetic breeding. Here, a root-specific promoter (Os03g01700) with a number of specific regulatory elements has been confirmed. Interestingly, cis-regulatory element S449 is significantly enriched in the -1475∼-2013 bp and -1077∼-1475 bp regions of Os03g01700 promoter. The activities of several deletion derivatives of Os03g01700 promoter were analyzed using both transient expression and genetic transformation system. The results showed that the root-specific cis-acting elements might be present in the -2013 bp～-1475 bp and -1077 bp～-561 bp regions of Os03g01700 promoter. To determine the actual effect of root-specific expression of OsPT2, a construction consisting of Os03g01700 promoter and OsPT2 CDS was used to transform rice. Under Pi-sufficient condition, there were a series of symptoms of phosphorus toxicity in the shoots of OsPT2 over-expressing (Ov-OsPT2) seedlings. Under Pi-deficient condition, more soluble Pi was accumulated in the shoots of Ov-OsPT2 seedlings than that in the wild type. Our data provide a candidate root-specific promoter in the breeding of rice with high phosphorus uptake variety.

Mutational Evidence for the Critical Role of CBF Transcription Factors in Cold Acclimation in Arabidopsis.

The three tandemly arranged CBF genes, CBF1, CBF2, and CBF3, are involved in cold acclimation. Due to the lack of stable loss-of-function Arabidopsis (Arabidopsis thaliana) mutants deficient in all three CBF genes, it is still unclear whether the CBF genes are essential for freezing tolerance and whether they may have other functions besides cold acclimation. In this study, we used the CRISPR/Cas9 system to generate cbf single, double, and triple mutants. Compared to the wild type, the cbf triple mutants are extremely sensitive to freezing after cold acclimation, demonstrating that the three CBF genes are essential for cold acclimation. Our results show that the three CBF genes also contribute to basal freezing tolerance. Unexpectedly, we found that the cbf triple mutants are defective in seedling development and salt stress tolerance. Transcript profiling revealed that the CBF genes regulate 414 cold-responsive (COR) genes, of which 346 are CBF-activated genes and 68 are CBF-repressed genes. The analysis suggested that CBF proteins are extensively involved in the regulation of carbohydrate and lipid metabolism, cell wall modification, and gene transcription. Interestingly, like the triple mutants, cbf2 cbf3 double mutants are more sensitive to freezing after cold acclimation compared to the wild type, but cbf1 cbf3 double mutants are more resistant, suggesting that CBF2 is more important than CBF1 and CBF3 in cold acclimation-dependent freezing tolerance. Our results not only demonstrate that the three CBF genes together are required for cold acclimation and freezing tolerance, but also reveal that they are important for salt tolerance and seedling development.

Isolation and characterization of two novel root-specific promoters in rice (Oryza sativa L.).

Novel root-specific promoters are important for developing methods to drive root-specific gene expression for nutrient and water absorption. RT-PCR (reverse transcription polymerase chain reaction) analysis identified high expression levels of Os03g01700 and Os02g37190 in root tissues across developmental stages in comparison with the constitutive genes OsAct1 (rice Actin1 gene), OsUbi1 (rice polyubiquitin rubi1 gene), and OsCc1 (rice cytochrome c gene). The copy numbers of Os03g01700 and Os02g37190 were evaluated by qRT-PCR. The results showed that Os03g01700 and Os02g37190 transcripts were highly accumulated in the examined root tissues but were not detected in young embryos or leaves at the indicated days after germination or in the panicle, in contrast to the ubiquitous expression of OsAct1, OsUbi1, and OsCc1. Additionally, the promoter regions of these two genes were linked to the GUSplus reporter gene and transformed into rice. GUS staining of the transgenic plants showed that the Os03g01700 and Os02g37190 promoters were active in primary and secondary roots throughout the developmental stages, except in root hairs. The GUSPlus transcript levels were also highly root-specific in the transgenic rice. Overall, the two promoters are highly active in the root tissues of rice and can be useful for the root-specific enhancement of target gene(s).

OsDGL1, a homolog of an oligosaccharyltransferase complex subunit, is involved in N-glycosylation and root development in rice.

A leaky rice mutant was isolated from an ethylmethane sulfonate (EMS)-mutagenized rice library based on its short root phenotype. The map-based cloning results showed that the mutant was due to a point mutation in the intron of OsDGL1 (LOC_Os07g10830), which encodes the dolichyl-diphosphooligosaccharide-protein glycosyltransferase 48 kDa subunit precursor. The mutation results in premature termination of protein synthesis. OsDGL1 is an ortholog of Arabidopsis DGL1, human OST48 and yeast WBP1, an essential protein subunit of the oligosaccharyltransferase (OST) complex, which is involved in N-glycosylation in eukaryotes. The leaky rice mutant, Osdgl1, displayed a change of matrix polysaccharides in its root cell wall, shorter root cell length, smaller root meristem and cell death in the root. Consistent with the known function of the OST complex in eukaryotes, the Osdgl1 mutation leads to a defect in N-glycosylation in the root. It was also found that reactive oxygen species (ROS) may be involved in this process.

[Gene expression in rice inflorescence development was monitored by using oligonucleotide microarray].

In this paper we chose 50 rice inflorescence genes from Internet,references. Rice oligonucleotide microarray was prepared by printing the target rice inflorescence genes oligonucleotide. Expression patterns of 50 genes from rice inflorescence in three different development phase were obtained by scanning using ScanArray3000 after array hybridization. The scatter plots and scale maps of the images were acquired after the acquired gene expression patterns were analyzed by ImaGene4.0 software. The scatter plots and scale maps show that there existed a significant difference in the expression of these candidate genes in rice inflorescences with different development phase. Further analysis of those candidate gene expression patterns will be helpful to understand the developmental mechanism of rice inflorescence.