Differential expression of three BOR1 genes corresponding to different genomes in response to boron conditions in hexaploid wheat (Triticum aestivum L.).

Boron (B) is an essential micronutrient for plants. Efflux-type B transporters, BORs, have been identified in Arabidopsis thaliana and rice. Here we identified BOR1 genes encoding B efflux transporters, from the hexaploid genome of wheat (Triticum aestivum L.). We cloned three genes closely related to OsBOR1 and named them TaBOR1.1, TaBOR1.2 and TaBOR1.3. All three TaBOR1s showed B efflux activities when expressed in tobacco BY-2 cells. TaBOR1-green fluorescent protein (GFP) fusion proteins were expressed in Arabidopsis leaf cells localized in the plasma membrane. The transcript accumulation patterns of the three genes differ in terms of tissue specificity and B nutrition responses. In roots, transcripts for all three genes accumulated abundantly while in shoots, the TaBOR1.2 transcript is the most abundant, followed by those of TaBOR1.1 and TaBOR1.3. Accumulation of TaBOR1.1 transcript is up-regulated under B deficiency conditions in both roots and shoots. In contrast, TaBOR1.2 transcript accumulation significantly increased in roots under excess B conditions. TaBOR1.3 transcript accumulation was reduced under excess B. Taken together, these results demonstrated that TaBOR1s are the B efflux transporters in wheat and, interestingly, the genes on the A, B and D genomes have different expression patterns.

AtIQM1, a novel calmodulin-binding protein, is involved in stomatal movement in Arabidopsis.

We recently identified a novel IQ motif-containing protein family, IQM, which shares sequence homology with a pea heavy metal-induced protein 6 and a ribosome inactivating protein, trichosanthin. Distinct expression patterns for each gene suggest that each IQM family member may play a different role in plant development and response to environmental cues. However functions of the IQM family members remain to be analyzed. IQM1 bound with calmodulin 5 (CaM5) in yeast two-hybrid assay via its IQ-motif. The CaM binding was Ca(2+)-independent in vitro, and was also observed in bimolecular fluorescence complementation analyses in onion epidermal cells. IQM1 was found to express strongly in guard cells and the cortex of roots. The T-DNA insertion mutants of IQM1 displayed a smaller stomatal aperture, a decreased water loss rate and a shorter primary root. Moreover, iqm1 did not change its stomatal aperture when treated with light, dark, ABA and chitin obviously. Microarray analyses showed that 243 and 28 genes were up- and down-regulated by more than twofold in iqm1-1, respectively. Interesting, 34 of 117 and 7 of 30 chitin-responsive transcriptional factor and ubiquitin ligase genes were up-regulated, respectively. Stomatal guard cells of iqm1-1 also showed enhanced expression of genes involved in production and signaling of reactive oxygen species (ROS). Consistently, increased ROS level was observed in the iqm1 guard cells.

OsGA20ox1, a candidate gene for a major QTL controlling seedling vigor in rice.

Seedling vigor is among the major determinants of stable stand establishment in direct-seeded rice (Oryza sativa L.) in temperate regions. Quantitative trait loci (QTL) for seedling vigor were identified using 250 recombinant inbred lines (RILs) derived from a cross between two japonica rice cultivars Kakehashi and Dunghan Shali. Seedling heights measured at 14 days after sowing were 20.3 and 29.4 cm for Kakehashi and Dunghan Shali, respectively. For the RILs, the height ranged from 14.1 to 31.7 cm. Four putative QTLs associated with seedling height were detected. qPHS3-2, the major QTL that was located on the long arm of chromosome 3, accounted for 26.2 % of the phenotypic variance. Using progeny of the near isogenic lines (NILs) produced by the backcross introduction of a chromosome segment carrying this major QTL into an elite cultivar Iwatekko, we fine-mapped qPHS3-2 to a 81-kb interval between two markers, ID_CAPS_01 and RM16227. Within this mapped region, we identified the gene OsGA20ox1, which is related to gibberellin (GA) biosynthesis. The relative expression levels of GA20ox1 in seedlings of Dunghan Shali and NILs were higher than that of Iwatekko. Concomitantly, the amount of endogenous active GA was higher in Dunghan Shali and the NILs compared to the level detected in Iwatekko. These results indicate that OsGA20ox1 is a strong candidate gene for major QTL controlling seedling vigor in rice.

Expression of the Arabidopsis borate efflux transporter gene, AtBOR4, in rice affects the xylem loading of boron and tolerance to excess boron.

Boron is an essential nutrient for plants, but it is toxic in excess. Transgenic rice plants expressing an Arabidopsis thaliana borate efflux transporter gene, AtBOR4, at a low level exhibited increased tolerance to excess boron. Those lines with high levels of expression exhibited reduced growth. These findings suggest a potential of the borate transporter BOR4 for the generation of high-boron tolerant rice.

A multifaceted genomics approach allows the isolation of the rice Pia-blast resistance gene consisting of two adjacent NBS-LRR protein genes.

The Oryza sativa (rice) resistance gene Pia confers resistance to the blast fungus Magnaporthe oryzae carrying the AVR-Pia avirulence gene. To clone Pia, we employed a multifaceted genomics approach. First, we selected 12 R-gene analog (RGA) genes encoding nucleotide binding site-leucine rich repeats (NBS-LRRs) proteins from a region on chromosome 11 that shows linkage to Pia. By using seven rice accessions, we examined the association between Pia phenotypes and DNA polymorphisms in the 10 genes, which revealed three genes (Os11gRGA3-Os11gRGA5) exhibiting a perfect association with the Pia phenotypes. We also screened ethyl methane sulfonate (EMS)-treated mutant lines of the rice cultivar 'Sasanishiki' harboring Pia, and isolated two mutants that lost the Pia phenotype. DNA sequencing of Os11gRGA3-Os11gRGA5 from the two mutant lines identified independent mutations of major effects in Os11gRGA4. The wild-type 'Sasanishiki' allele of Os11gRGA4 (SasRGA4) complemented Pia function in both mutants, suggesting that SasRGA4 is necessary for Pia function. However, when the rice cultivar 'Himenomochi' lacking Pia was transfected with SasRGA4, the Pia phenotype was not recovered. An additional complementation study revealed that the two NBS-LRR-type R genes, SasRGA4 and SasRGA5, that are located next to each other and oriented in the opposite direction are necessary for Pia function. A population genetics analysis of SasRGA4 and SasRGA5 suggests that the two genes are under long-term balancing selection.

High-throughput SuperSAGE for digital gene expression analysis of multiple samples using next generation sequencing.

We established a protocol of the SuperSAGE technology combined with next-generation sequencing, coined "High-Throughput (HT-) SuperSAGE". SuperSAGE is a method of digital gene expression profiling that allows isolation of 26-bp tag fragments from expressed transcripts. In the present protocol, index (barcode) sequences are employed to discriminate tags from different samples. Such barcodes allow researchers to analyze digital tags from transcriptomes of many samples in a single sequencing run by simply pooling the libraries. Here, we demonstrated that HT-SuperSAGE provided highly sensitive, reproducible and accurate digital gene expression data. By increasing throughput for analysis in HT-SuperSAGE, various applications are foreseen and several examples are provided in the present study, including analyses of laser-microdissected cells, biological replicates and tag extraction using different anchoring enzymes.

Arabidopsis auxin response factor6 and 8 regulate jasmonic acid biosynthesis and floral organ development via repression of class 1 KNOX genes.

Two mutations in Arabidopsis thaliana, auxin response factor6 (arf6) and arf8, concomitantly delayed the elongation of floral organs and subsequently delayed the opening of flower buds. This phenotype is shared with the jasmonic acid (JA)-deficient mutant dad1, and, indeed, the JA level of arf6 arf8 flower buds was decreased. Among JA biosynthetic genes, the expression level of DAD1 (DEFECTIVE IN ANTHER DEHISCENCE1) was markedly decreased in the double mutant, suggesting that ARF6 and ARF8 are required for activation of DAD1 expression. The double mutant arf6 arf8 also showed other developmental defects in flowers, such as aberrant vascular patterning and lack of epidermal cell differentiation in petals. We found that class 1 KNOX genes were expressed ectopically in the developing floral organs of arf6 arf8, and mutations in any of the class 1 KNOX genes (knat2, knat6, bp and hemizygous stm) partially suppressed the defects in the double mutant. Furthermore, ectopic expression of the STM gene caused a phenotype similar to that of arf6 arf8, including the down-regulation of DAD1 expression. These results suggested that most defects in arf6 arf8 are attributable to abnormal expression of class 1 KNOX genes. The expression of AS1 and AS2 was not affected in arf6 arf8 flowers, and as1 and arf6 arf8 additively increased the expression of class 1 KNOX genes. We concluded that ARF6 and ARF8, in parallel with AS1 and AS2, repress the class 1 KNOX genes in developing floral organs to allow progression of the development of these organs.

Overexpression of the RADICAL-INDUCED CELL DEATH1 (RCD1) gene of Arabidopsis causes weak rcd1 phenotype with compromised oxidative-stress responses.

rcd1 is a mutant of Arabidopsis thaliana that is more resistant to methyl viologen, but more sensitive to ozone than the wild type. rcd1-2 is caused by a single nucleotide substitution that results in a premature stop codon at Trp-332. The rcd1-2 mRNA level does not change significantly with the mutation. Since overexpression of rcd1-1 cDNA has been shown to bring about an rcd1-like phenotype, we created and examined the overexpression lines of RCD1 by the use of the cauliflower mosaic virus 35S promoter. The transgenic lines exhibited a weak rcd1-like phenotype, although no resistance to methyl viologen was observed. Further, they fully complemented the aberrant rcd1-2 phenotype. Subcellular localization of RCD1 was examined by transiently expressing green fluorescent protein (GFP) fused with RCD1 in onion epidermal cells. GFP signals are observed as aggregated foci in the inner nuclear matrix-like region.

A methyl viologen-resistant mutant of Arabidopsis, which is allelic to ozone-sensitive rcd1, is tolerant to supplemental ultraviolet-B irradiation.

To better understand the role of active oxygen species (AOS) in acquired resistance to increased levels of ultraviolet (UV)-B irradiation in plants, we isolated an Arabidopsis mutant that is resistant to methyl viologen, and its sensitivity to UV-B was investigated. A complementation test revealed that the obtained mutant was allelic to the ozone-sensitive radical-induced cell death1-1 (rcd1-1). Therefore, this mutant was named rcd1-2. rcd1-2 was recessive and nearly 4-fold more resistant to methyl viologen than wild type. It exhibited a higher tolerance to short-term UV-B supplementation treatments than the wild type: UV-B-induced formation of cyclobutane pyrimidine dimers was reduced by one-half after 24 h of exposure; the decrease in quantum yield of photosystem II was also diminished by 40% after 12 h of treatment. Furthermore, rcd1-2 was tolerant to freezing. Steady-state mRNA levels of plastidic Cu/Zn superoxide dismutase and stromal ascorbate peroxidase were higher in rcd1-2 than in wild type, and the mRNA level of the latter enzyme was enhanced by UV-B exposure more effectively in rcd1-2. UV-B-absorbing compounds were more accumulated in rcd1-2 than in wild type after UV-B exposure for 24 h. These findings suggest that rcd1-2 methyl viologen resistance is due to the enhanced activities of the AOS-scavenging enzymes in chloroplasts and that the acquired tolerance to the short-term UV-B exposure results from a higher accumulation of sunscreen pigments. rcd1 appears to be a mutant that constitutively shows stress responses, leading to accumulation of more pigments and AOS-scavenging enzymes without any stresses.