Characterization of rice genes using a heterologous full-length cDNA expression system.

As a result of the progress in sequencing technology, many plant genomes have now been determined. Functional genomics is required to clarify gene function in many of these species. To identify useful genes easily and quickly, we have developed a FOX (full-length cDNA overexpressor) hunting system in which full-length cDNAs are overexpressed in Arabidopsis plants. This system was applied to high-throughput analysis of rice genes through heterologous expression in Arabidopsis (rice FOX Arabidopsis lines). We demonstrated that it is possible to carry out high-throughput analysis of gene function by utilizing rice FOX Arabidopsis lines. In this protocol, we describe how to isolate candidate rice FOX Arabidopsis lines and to determine the rice fl-cDNA that is responsible for the observed phenotype.

SD3, an Arabidopsis thaliana homolog of TIM21, affects intracellular ATP levels and seedling development.

It is poorly understood how plants control their growth by cell division, elongation, and differentiation. We have characterized a seedling-lethal mutant segregation distortion 3 (sd3) that showed a very dwarf phenotype when grown in the light and, in the dark, had short hypocotyls with reduced ploidy levels. The corresponding gene of SD3 encodes a protein with high similarity to yeast translocase on the inner mitochondrial membrane 21 (TIM21), which is a component of the TIM23 complex. Indeed, SD3 protein fused to GFP localized in the mitochondria. SD3 overexpression increased cotyledon size in the light and hypocotyl thickness in the dark. The expression of genes for several subunits of the respiratory-chain complexes III and IV was up-regulated in SD3-overexpressing plants. Furthermore, these plants showed high levels of ATP whereas those of sd3 were low. These results suggested that SD3 induced an increase in cell size by raising the expression of the respiratory-chain subunit genes and hence increased the intracellular ATP levels. We propose that intracellular ATP levels regulated by mitochondria control plant organ size.

Application of full-length cDNA resources to gain-of-function technology for characterization of plant gene function.

Generation and characterization of mutants are important for the investigation of gene function. Gain-of-function technology is one of the most useful approaches for the systematic production of mutant resources. Full-length cDNAs have been collected from various plant species and have become important resources for functional genomics. We have developed a novel gain-of-function technology for the identification of gene function using a full-length cDNA library, and this system has been named as FOX hunting system (Full-length cDNA Over-eXpressing gene hunting system). In this system, full-length cDNAs are randomly expressed in Arabidopsis. We also generated rice FOX Arabidopsis lines in which full-length cDNAs from rice were expressed in Arabidopsis, and we demonstrated that gene function derived from heterologous organisms can be analyzed systematically using the FOX hunting approach. In this protocol, we describe the process of generating Arabidopsis mutants expressing rice full-length cDNA libraries and the methods of identifying genes from the isolated mutants.

Full-length cDNA overexpressor gene hunting system (FOX hunting system).

Full-length cDNAs (fl-cDNAs) are important resources for the characterization of gene function, since they contain all the information required for the production of functional RNAs and proteins. Large sets of fl-cDNA clones have been collected from several plant species and have become available for functional genomic analysis. We have developed a system for the identification of gene function by screening for transgenic plants ectopically expressing fl-cDNAs and named it the FOX (fl-cDNA overexpressor gene) hunting system. This system can be applied to almost all plant species without prior knowledge of their genome sequences because only fl-cDNAs are required. For utilization of the FOX hunting system, Agrobacterium libraries and Arabidopsis seeds carrying rice and Arabidopsis fl-cDNAs are available. Here, we will describe the procedure followed in the FOX hunting system from the generation of expression vectors carrying fl-cDNAs to the confirmation of phenotype in retransformed plants.

A novel chloroplast protein, CEST induces tolerance to multiple environmental stresses and reduces photooxidative damage in transgenic Arabidopsis.

Environmental stresses are major factors in limiting plant growth and crop production. To find genes improving salt tolerance, the screening of a large population of transgenic Arabidopsis thaliana that expressed rice full-length cDNAs under salinity stress is reported here. In this study one of the isolated salt-tolerant lines, R07303 was analysed in detail. An uncharacterized rice gene CHLOROPLAST PROTEIN-ENHANCING STRESS TOLERANCE (OsCEST) was integrated in R07303. Newly constructed transgenic Arabidopsis that overexpressed OsCEST or its Arabidopsis homologue AtCEST showed improved tolerance to salinity stress. OsCEST and AtCEST were mainly transcribed in photosynthetic tissues. Green fluorescent protein-fused OsCEST and AtCEST proteins were localized to the chloroplast in the Arabidopsis leaf protoplasts. CEST-overexpressing Arabidopsis showed enhanced tolerance not only to salt stress but also to drought stress, high-temperature stress, and paraquat, which causes photooxidative stress. Under saline conditions, overexpression of CESTs modulated the stress-induced impairment of photosynthetic activity and the peroxidation of lipids. Reduced expression of AtCEST because of double-stranded RNA interference resulted in the impairment of photosynthetic activity, the reduction of green pigment, defects in chloroplast development, and growth retardation under light. This paper discusses the relationship between the chloroplast protein CEST and photooxidative damage.

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/.

High-throughput characterization of plant gene functions by using gain-of-function technology.

Gain-of-function approaches have been used as an alternative or complementary method to loss-of-function approaches as well as to confer new functions to plants. Gain-of-function is achieved by increasing gene expression levels through the random activation of endogenous genes by transcriptional enhancers or the expression of individual transgenes by transformation. The advantages of gain-of-function approaches compared to loss-of-function approaches for the characterization of gene functions include the abilities to (a) analyze individual gene family members, (b) characterize the function of genes from nonmodel plants using a heterologous expression system, and (c) identify genes that confer stress tolerance to plants that result from the introduction of transgenes. In this review, we describe the current status of gain-of-function mutagenesis and provide several examples of how gene functions have been characterized via high-throughput screening using gain-of-function technology.

Metabolomic screening applied to rice FOX Arabidopsis lines leads to the identification of a gene-changing nitrogen metabolism.

Plant metabolomics developed as a powerful tool to examine gene functions and to gain deeper insight into the physiology of the plant cell. In this study, we screened Arabidopsis lines overexpressing rice full-length (FL) cDNAs (rice FOX Arabidopsis lines) using a gas chromatography-time-of-flight mass spectrometry (GC-TOF/MS)-based technique to identify rice genes that caused metabolic changes. This screening system allows fast and reliable identification of candidate lines showing altered metabolite profiles. We performed metabolomic and transcriptomic analysis of a rice FOX Arabidopsis line that harbored the FL cDNA of the rice ortholog of the Lateral Organ Boundaries (LOB) Domain (LBD)/Asymmetric Leaves2-like (ASL) gene of Arabidopsis, At-LBD37/ASL39. The investigated rice FOX Arabidopsis line showed prominent changes in the levels of metabolites related to nitrogen metabolism. The transcriptomic data as well as the results from the metabolite analysis of the Arabidopsis At-LBD37/ASL39-overexpressor plants were consistent with these findings. Furthermore, the metabolomic and transcriptomic analysis of the Os-LBD37/ASL39-overexpressing rice plants indicated that Os-LBD37/ASL39 is associated with processes related to nitrogen metabolism in rice. Thus, the combination of a metabolomics-based screening method and a gain-of-function approach is useful for rapid characterization of novel genes in both Arabidopsis and rice.

Simple construction of plant RNAi vectors using long oligonucleotides.

RNA interference (RNAi) is one of the most important technologies currently available for the analysis of gene function. However, despite the development of various methods, it is still difficult to construct RNAi vectors for plants with the appropriate inverted repeat fragments to produce double-stranded RNA for knockdown experiments. To solve this problem we have developed an easy and simple method to make RNAi constructs using two long oligonucleotides consisting of partially complementary sequences without the need for PCR amplification and multiple cloning steps. CHS RNAi plants generated using this method showed yellow seed color and a decrease in antocyanin content--phenotypes typically observed in CHS loss-of-function mutants. Moreover, we demonstrated specific knockdown of both the PHYA and PHYB genes using a tandem RNAi construct. This method thus represents a powerful tool for gene knockdown in plants.

A T-DNA insertion mutant of AtHMA1 gene encoding a Cu transporting ATPase in Arabidopsis thaliana has a defect in the water-water cycle of photosynthesis.

The water-water cycle is the electron flow through scavenging enzymes for the reactive species of oxygen in chloroplasts, and is proposed to play a role in alternative electron sink in photosynthesis. Here we showed that the water-water cycle is impaired in the T-DNA insertion mutant of AtHMA1 gene encoding a Cu transporting ATPase in chloroplasts. Chlorophyll fluorescence under steady state was not affected in hma1, indicating that photosynthetic electron transport under normal condition was not impaired. Under electron acceptor limited conditions, however, hma1 showed distinguished phenotype in chlorophyll fluorescence characteristics. The most severe phenotype of hma1 could be observed in high (0.1%) CO(2) concentrations, indicating that hma1 has the defect other than photorespiration. The transient increase of chlorophyll fluorescence upon the cessation of the actinic light as well as the NPQ induction of chlorophyll fluorescence revealed that the two pathways of cyclic electron flow around PSI, NDH-pathway and FQR-pathway, are both intact in hma1. Based on the NPQ induction under 0% oxygen condition, we conclude that the water-water cycle is impaired in hma1, presumably due to the decreased level of Cu/Zn SOD in the mutant. Under high CO(2) condition, hma1 exhibited slightly higher NPQ induction than wild type plants, while this increase of NPQ in hma1 was suppressed when hma1 was crossed with crr2 having a defect in NDH-mediated PSI cyclic electron flow. We propose that the water-water cycle and NDH-mediated pathways might be regulated compensationally with each other especially when photorespiration is suppressed.

Systematic approaches to using the FOX hunting system to identify useful rice genes.

Ectopic gene expression, or the gain-of-function approach, has the advantage that once the function of a gene is known the gene can be transferred to many different plants by transformation. We previously reported a method, called FOX hunting, that involves ectopic expression of Arabidopsis full-length cDNAs in Arabidopsis to systematically generate gain-of-function mutants. This technology is most beneficial for generating a heterologous gene resource for analysis of useful plant gene functions. As an initial model we generated more than 23,000 independent Arabidopsis transgenic lines that expressed rice fl-cDNAs (Rice FOX Arabidopsis lines). The short generation time and rapid and efficient transformation frequency of Arabidopsis enabled the functions of the rice genes to be analyzed rapidly. We screened rice FOX Arabidopsis lines for alterations in morphology, photosynthesis, element accumulation, pigment accumulation, hormone profiles, secondary metabolites, pathogen resistance, salt tolerance, UV signaling, high light tolerance, and heat stress tolerance. Some of the mutant phenotypes displayed by rice FOX Arabidopsis lines resulted from the expression of rice genes that had no homologs in Arabidopsis. This result demonstrated that rice fl-cDNAs could be used to introduce new gene functions in Arabidopsis. Furthermore, these findings showed that rice gene function could be analyzed by employing Arabidopsis as a heterologous host. This technology provides a framework for the analysis of plant gene function in a heterologous host and of plant improvement by using heterologous gene resources.

The mutant of sll1961, which encodes a putative transcriptional regulator, has a defect in regulation of photosystem stoichiometry in the cyanobacterium Synechocystis sp. PCC 6803.

In acclimation to changing light environments, photosynthetic organisms modulate the ratio of two photosynthetic reaction centers (photosystem I [PSI] and photosystem II). One mutant, which could not modulate photosystem stoichiometry upon the shift to high light, was isolated from mutants created by random transposon mutagenesis. Measurements of chlorophyll fluorescence and analysis of the reaction center subunits of PSI through western blotting in this mutant revealed that the content of PSI could not be suppressed under high-light condition. In the mutant, transposon was inserted to the sll1961 gene encoding a putative transcriptional regulator. DNA microarray analysis revealed that the expression of sll1773 was drastically induced in the sll1961 mutant upon exposure to high light for 3 h. Our results demonstrate that a transcriptional regulator, Sll1961, and its possible target proteins, including Sll1773, may be responsible for the regulation of photosystem stoichiometry in response to high light.

Over-reduced states of the Mn-cluster in cucumber leaves induced by dark-chilling treatment.

Oxygen evolution is inhibited when leaves of chilling-sensitive plants like cucumber are treated at 0 degrees C in the dark. The activity is restored by moderate illumination at room temperature. We examined the changes in the redox state of the Mn-cluster in cucumber leaves in the processes of dark-chilling inhibition and subsequent light-induced reactivation by means of thermoluminescence (TL). A TL B-band arising from S(2)Q(B)(-) charge recombination in PSII was observed upon single-flash illumination of untreated leaves, whereas four flashes were required to yield the B-band after dark-chilling treatment for 24 h. This three-step delay indicates that over-reduced states of the Mn-cluster such as the S(-2) state were formed during the treatment. Fitting analysis of the flash-number dependence of the TL intensities showed that the Mn-cluster was more reduced with a longer period of the treatment and that S(-3) was the lowest S-state detectable in the dark-chilled leaves. Measurements of the Mn content by atomic absorption spectroscopy showed that Mn atoms were gradually released from PSII during the dark-chilling treatment but re-bound to PSII by illumination at 30 degrees C. Thus, dark-chilling inhibition of oxygen evolution can be ascribed to the disintegration of the Mn-cluster due to its over-reduction. The observation of the S(-3) state in the present in vivo system strongly suggests that S(-3), which has been observed only by addition of exogenous reductants into in vitro preparations, is indeed a redox intermediate of the Mn-cluster in the processes of its disintegration and photoactivation.