RiceFOX: a database of Arabidopsis mutant lines overexpressing rice full-length cDNA that contains a wide range of trait information to facilitate analysis of gene function

Identification of gene function is important not only for basic research but also for applied science, especially with regard to improvements in crop production. For rapid and efficient elucidation of useful traits, we developed a system named FOX hunting (Full-length cDNA Over-eXpressor gene hunting) using full-length cDNAs (fl-cDNAs). A heterologous expression approach provides a solution for the high-throughput characterization of gene functions in agricultural plant species. Since fl-cDNAs contain all the information of functional mRNAs and proteins, we introduced rice fl-cDNAs into Arabidopsis plants for systematic gain-of-function mutation. We generated >30,000 independent Arabidopsis transgenic lines expressing rice fl-cDNAs (rice FOX Arabidopsis mutant lines). These rice FOX Arabidopsis lines were screened systematically for various criteria such as morphology, photosynthesis, UV resistance, element composition, plant hormone profile, metabolite profile/fingerprinting, bacterial resistance, and heat and salt tolerance. The information obtained from these screenings was compiled into a database named 'RiceFOX'. This database contains around 18,000 records of rice FOX Arabidopsis lines and allows users to search against all the observed results, ranging from morphological to invisible traits. The number of searchable items is approximately 100; moreover, the rice FOX Arabidopsis lines can be searched by rice and Arabidopsis gene/protein identifiers, sequence similarity to the introduced rice fl-cDNA and traits. The RiceFOX database is available at http://ricefox.psc.riken.jp/.     

A genome-wide gain-of-function analysis of rice genes using the FOX-hunting system

The latest report has estimated the number of rice genes to be approximately 32,000. To elucidate the functions of a large population of rice genes and to search efficiently for agriculturally useful genes, we have been taking advantage of the Full-length cDNA Over-eXpresser (FOX) gene-hunting system. This system is very useful for analyzing various gain-of-function phenotypes from large populations of transgenic plants overexpressing cDNAs of interest and others with unknown or important functions. We collected the plasmid DNAs of 13,980 independent full-length cDNA (FL-cDNA) clones to produce a FOX library by placing individual cDNAs under the control of the maize Ubiquitin-1 promoter. The FOX library was transformed into rice by Agrobacterium-mediated high-speed transformation. So far, we have generated approximately 12,000 FOX-rice lines. Genomic PCR analysis indicated that the average number of FL-cDNAs introduced into individual lines was 1.04. Sequencing analysis of the PCR fragments carrying FL-cDNAs from 8615 FOX-rice lines identified FL-cDNAs in 8225 lines, and a database search classified the cDNAs into 5462 independent ones. Approximately 16.6% of FOX-rice lines examined showed altered growth or morphological characteristics. Three super-dwarf mutants overexpressed a novel gibberellin 2-oxidase gene,confirming the importance of this system. We also show here the other morphological alterations caused by individual FL-cDNA expression. These dominant phenotypes should be valuable indicators for gene discovery and functional analysis.     

FOX-superroots of Lotus corniculatus, overexpressing Arabidopsis full-length cDNA, show stable variations in morphological traits

Using the full-length cDNA overexpressor (FOX) gene-hunting system, we have generated 130 Arabidopsis FOX-superroot lines in bird's-foot trefoil (Lotus corniculatus) for the systematic functional analysis of genes expressed in roots and for the selection of induced mutants with interesting root growth characteristics. We used the Arabidopsis-FOX Agrobacterium library (constructed by ligating pBIG2113SF) for the Agrobacterium-mediated transformation of superroots (SR) and the subsequent selection of gain-of-function mutants with ectopically expressed Arabidopsis genes. The original superroot culture of L. corniculatus is a unique host system displaying fast root growth in vitro, allowing continuous root cloning, direct somatic embryogenesis and mass regeneration of plants under entirely hormone-free culture conditions. Several of the Arabidopsis FOX-superroot lines show interesting deviations from normal growth and morphology of roots from SR-plants, such as differences in pigmentation, growth rate, length or diameter. Some of these mutations are of potential agricultural interest. Genomic PCR analysis revealed that 100 (76.9%) out of the 130 transgenic lines showed the amplification of single fragments. Sequence analysis of the PCR fragments from these 100 lines identified full-length cDNA in 74 of them. Forty-three out of 74 full-length cDNA carried known genes. The Arabidopsis FOX-superroot lines of L. corniculatus, produced in this study, expand the FOX hunting system and provide a new tool for the genetic analysis and control of root growth in a leguminous forage plant.     

Identification of boron transporter genes likely to be responsible for tolerance to boron toxicity in wheat and barley

Tolerance to boron (B) toxicity in cereals is known to be associated with reduced tissue accumulation of B. Genes from roots of B-tolerant cultivars of wheat and barley with high similarities to previously reported B efflux transporters from Arabidopsis and rice were cloned. Expression of these genes was strongly correlated with the ability of tolerant genotypes to lower the concentration of B in roots. The gene from barley located to chromosome 4. Backcross lines containing a B tolerance locus on chromosome 4 showed tolerance in proportion to the level of expression of the transporter gene, whereas those lacking the locus were sensitive to B and had very low levels of gene expression. The results are consistent with a widespread mechanism of tolerance to high B based on efflux of B from root cells.     

Functional gene-mining for salt-tolerance genes with the power of Arabidopsis

Here we report on a functional gene-mining method developed to isolate stress tolerance genes without any prior knowledge of the genome or genetic mapping of the source germplasms. The feasibility of this approach was demonstrated by isolating novel salt stress tolerance genes from salt cress (Thellungiella halophila), an extremophile that is adapted to a harsh saline environment and a close relative of the model plant Arabidopsis thaliana. This gene-mining method is based on the expression of salt cress cDNA libraries in Arabidopsis. A cDNA expression library of the source germplasm, salt cress, was constructed and used to transform Arabidopsis via Agrobacterium-mediated gene transfer. A transgenic seed library consisting of >125,000 independent lines was generated and screened for salt-tolerant lines via a high-throughput genetic screen. A number of salt-tolerant lines were isolated, and the salt cress cDNAs were identified by PCR amplification and sequencing. Among the genes isolated, several novel small protein-encoding genes were discovered. The homologs of these genes in Arabidopsis have not been experimentally analyzed, and their functions remain unknown. The function of two genes isolated by this method, ST6-66 and ST225, and their Arabidopsis homologs, were investigated in Arabidopsis using gain- and loss-of-function analyses, and their importance in salt tolerance was demonstrated. Thus, our functional gene-mining method was validated by these results. Our method should be applicable for the functional mining of stress tolerance genes from various germplasms. Future improvements of the method are also discussed.     

Rice-<Emphasis Type="Italic">Arabidopsis</Emphasis> FOX line screening with FT-NIR-based fingerprinting for GC-TOF/MS-based metabolite profiling

The full-length cDNA over-expressing (FOX) gene hunting system is useful for genome-wide gain-of-function analysis. The screening of FOX lines requires a high-throughput metabolomic method that can detect a wide range of metabolites. Fourier transform-near-infrared (FT-NIR) spectroscopy in combination with the chemometric approach has been used to analyze metabolite fingerprints. Since FT-NIR spectroscopy can be used to analyze a solid sample without destructive extraction, this technique enables untargeted analysis and high-throughput screening focusing on the alteration of metabolite composition. We performed non-destructive FT-NIR-based fingerprinting to screen seed samples of 3000 rice-Arabidopsis FOX lines; the samples were obtained from transgenic Arabidopsis thaliana lines that overexpressed rice full-length cDNA. Subsequently, the candidate lines exhibiting alteration in their metabolite fingerprints were analyzed by gas chromatography-time-of-flight/mass spectrometry (GC-TOF/MS) in order to assess their metabolite profiles. Finally, multivariate regression using orthogonal projections to latent structures (O2PLS) was used to elucidate the predictive metabolites obtained in FT-NIR analysis by integration of the datasets obtained from FT-NIR and GC-TOF/MS analyses. FT-NIR-based fingerprinting is a technically efficient method in that it facilitates non-destructive analysis in a high-throughput manner. Furthermore, with the integrated analysis used here, we were able to discover unique metabotypes in rice-Arabidopsis FOX lines; thus, this approach is beneficial for investigating the function of rice genes related to metabolism.     

Simple RNAi vectors for stable and transient suppression of gene function in rice

Since the recent sequencing of the rice genome, the functional identification of rice genes has become increasingly important. Various tagged lines have been generated; however, the number of tagged genes available is not sufficient for extensive study of gene function. To help identify the functions of genes in rice, we developed a Gateway vector, pANDA, for RNA interference of rice genes. This vector can be used for Agrobacterium transformation of rice and allows easy and fast construction of efficient RNAi vectors. In the construct, hairpin RNA derived from a given gene is transcribed from a strong maize ubiquitin promoter, and an intron is placed 5' upstream of inverted repeats to enhance RNA expression. Analysis of rice genes using this vector showed that suppression of mRNA expression was observed in more than 90% of transgenic plants examined, and short interfering RNA indicative of RNA silencing was detected in each silenced plant. A similar vector, pANDA-mini, was also developed for direct transfer into leaf cells or protoplasts. This vector can be used for transient suppression of gene function in rice. These vectors should help identify the functions of rice genes whose tagged mutants are not available at present and complement existing methods for functional genomics of rice.     

Arabidopsis CBF3/DREB1A and ABF3 in transgenic rice increased tolerance to abiotic stress without stunting growth

Rice (Oryza sativa), a monocotyledonous plant that does not cold acclimate, has evolved differently from Arabidopsis (Arabidopsis thaliana), which cold acclimates. To understand the stress response of rice in comparison with that of Arabidopsis, we developed transgenic rice plants that constitutively expressed CBF3/DREB1A (CBF3) and ABF3, Arabidopsis genes that function in abscisic acid-independent and abscisic acid-dependent stress-response pathways, respectively. CBF3 in transgenic rice elevated tolerance to drought and high salinity, and produced relatively low levels of tolerance to low-temperature exposure. These data were in direct contrast to CBF3 in Arabidopsis, which is known to function primarily to enhance freezing tolerance. ABF3 in transgenic rice increased tolerance to drought stress alone. By using the 60 K Rice Whole Genome Microarray and RNA gel-blot analyses, we identified 12 and 7 target genes that were activated in transgenic rice plants by CBF3 and ABF3, respectively, which appear to render the corresponding plants acclimated for stress conditions. The target genes together with 13 and 27 additional genes are induced further upon exposure to drought stress, consequently making the transgenic plants more tolerant to stress conditions. Interestingly, our transgenic plants exhibited neither growth inhibition nor visible phenotypic alterations despite constitutive expression of the CBF3 or ABF3, unlike the results previously obtained from Arabidopsis where transgenic plants were stunted.     

Screening for resistance against Pseudomonas syringae in rice-FOX Arabidopsis lines identified a putative receptor-like cytoplasmic kinase gene that confers resistance to major bacterial and fungal pathogens in Arabidopsis and rice

Approximately 20,000 of the rice-FOX Arabidopsis transgenic lines, which overexpress 13,000 rice full-length cDNAs at random in Arabidopsis, were screened for bacterial disease resistance by dip inoculation with Pseudomonas syringae pv. tomato DC3000 (Pst DC3000). The identities of the overexpressed genes were determined in 72 lines that showed consistent resistance after three independent screens. Pst DC3000 resistance was verified for 19 genes by characterizing other independent Arabidopsis lines for the same genes in the original rice-FOX hunting population or obtained by reintroducing the genes into ecotype Columbia by floral dip transformation. Thirteen lines of these 72 selections were also resistant to the fungal pathogen Colletotrichum higginsianum. Eight genes that conferred resistance to Pst DC3000 in Arabidopsis have been introduced into rice for overexpression, and transformants were evaluated for resistance to the rice bacterial pathogen, Xanthomonas oryzae pv. oryzae. One of the transgenic rice lines was highly resistant to Xanthomonas oryzae pv. oryzae. Interestingly, this line also showed remarkably high resistance to Magnaporthe grisea, the fungal pathogen causing rice blast, which is the most devastating rice disease in many countries. The causal rice gene, encoding a putative receptor-like cytoplasmic kinase, was therefore designated as BROAD-SPECTRUM RESISTANCE 1. Our results demonstrate the utility of the rice-FOX Arabidopsis lines as a tool for the identification of genes involved in plant defence and suggest the presence of a defence mechanism common between monocots and dicots.     

Overexpression of the mitogen-activated protein kinase gene OsMAPK33 enhances sensitivity to salt stress in rice (Oryza sativa L.)

Mitogen-activated protein kinases (MAPK) signalling cascades are activated by extracellular stimuli such as environmental stresses and pathogens in higher eukaryotic plants. To know more about MAPK signalling in plants, aMAPK cDNA clone, OsMAPK33, was isolated from rice. The gene is mainly induced by drought stress. In phylogenetic analysis, OsMAPK33 (Os02g0148100) showed approximately 47-93% identity at the amino acid level with other plant MAPKs. It was found to exhibit organ-specific expression with relatively higher expression in leaves as compared with roots or stems, and to exist as a single copy in the rice genome. To investigate the biological functions of OsMAPK33 in rice MAPK signalling, transgenic rice plants that either overexpressed or suppressed OsMAPK33 were made. Under dehydration conditions, the suppressed lines showed lower osmotic potential compared with that of wild-type plants, suggesting a role of OsMAPK33 in osmotic homeostasis. Nonetheless, the suppressed lines did not display any significant difference in drought tolerance compared with their wild-type plants. With increased salinity, there was still no difference in salt tolerance between OsMAPK33-suppressed lines and their wild-type plants. However, the overexpressing lines showed greater reduction in biomass accumulation and higher sodium uptake into cells, resulting in a lower K+/Na+ ratio inside the cell than that in the wild-type plants and OsMAPK33-suppressed lines. These results suggest that OsMAPK33 could play a negative role in salt tolerance through unfavourable ion homeostasis. Gene expression profiling of OsMAPK33 transgenic lines through rice DNA chip analysis showed that OsMAPK33 altered expression of genes involved in ion transport. Further characterization of downstream components will elucidate various biological functions of this novel rice MAPK.     

The FOX hunting system: an alternative gain-of-function gene hunting technique

We have developed a novel gain-of-function system that we have named the FOX hunting system (Full-length cDNA Over-eXpressing gene hunting system). We used normalized full-length cDNA and introduced each cDNA into Arabidopsis by in planta transformation. About 10 000 independent full-length Arabidopsis cDNAs were expressed independently under the CaMV 35S promoter in Arabidopsis. Each transgenic Arabidopsis contained on average 2.6 cDNA clones and was monitored under various categories such as morphological changes, fertility and leaf color. We found 1487 possible morphological mutants from 15 547 transformants. When 115 pale green T(1) mutants were analyzed, 59 lines represented the mutant phenotypes in more than 50% of the T(2) progeny. Characterization of two leaf color mutants revealed the significance of this approach. We also document mutants from several categories and their corresponding full-length cDNAs.     

LcFIN2, a novel chloroplast protein gene from sheepgrass,enhances tolerance to low temperature in Arabidopsis and rice

Adverse environmental stresses affect plant growth and crop yields. Sheepgrass (Leymus chinensis (Trin.) Tzvel), an important forage grass that is widely distributed in the east of Eurasia steppe, has high tolerance to extreme low temperature. Many genes that respond to cold stress were identified in sheepgrass by RNA‐sequencing, but more detailed studies are needed to dissect the function of those genes. Here, we found that LcFIN2, a sheepgrass freezing‐induced protein 2, encoded a chloroplast‐targeted protein. Expression of LcFIN2 was upregulated by freezing, chilling, NaCl and abscisic acid (ABA) treatments. Overexpression of LcFIN2 enhanced the survival rate of transgenic Arabidopsis after freezing stress. Importantly, heterologous expression of LcFIN2 in rice exhibited not only higher survival rate but also accumulated various soluble substances and reduced membrane damage in rice under chilling stress. Furthermore, the chlorophyll content, the quantum photochemistry efficiency of photosystem II (ΦPSII), the non‐photochemical quenching (NPQ), the net photosynthesis rate (Pn) and the expression of some chloroplast ribosomal‐related and photosynthesis‐related genes were higher in the transgenic rice under chilling stress. These findings suggested that the LcFIN2 gene could potentially be used to improve low‐temperature tolerance in crops.