A collection of inducible transcription factor-glucocorticoid receptor fusion lines for functional analyses in Arabidopsis thaliana.

Arabidopsis possesses approximately 2000 transcription factors (TFs) in its genome. They play pivotal roles in various biological processes but analysis of their function has been hampered by the overlapping nature of their activities. To uncover clues to their function, we generated inducible TF lines using glucocorticoid receptor (GR) fusion techniques in Arabidopsis. These TF-GR lines each express one of 1255 TFs as a fusion with the GR gene. An average 14 lines of T2 transgenic TF-GR lines were generated for each TF to monitor their function. To evaluate these transcription lines, we induced the TF-GR lines of phytochrome-interacting factor 4, which controls photomorphogenesis, with synthetic glucocorticoid dexamethasone. These phytochrome-interacting factor 4-GR lines showed the phenotype described in a previous report. We performed screening of the other TF-GR lines for TFs involved in light signaling under blue and far-red light conditions and identified 13 novel TF candidates. Among these, we found two lines showing higher anthocyanin accumulation under light conditions and we examined the regulating genes. These results indicate that the TF-GR lines can be used to dissect functionally redundant genes in plants and demonstrate that the TF-GR line collection can be used as an effective tool for functional analysis of TFs.

Physiological function of photoreceptor UVR8 in UV-B tolerance in the liverwort Marchantia polymorpha.

MAIN CONCLUSION: The physiological importance of MpUVR8 in UV-B resistance and translocation in a UV-B-dependent manner from the cytosol into the nucleus is characterized in Marchantia polymorpha. UV RESISTANCE LOCUS 8 (UVR8) is an ultraviolet-B (UV-B) light receptor functioning for UV-B sensing and tolerance in Arabidopsis thaliana and other species. It is unclear whether UVR8 physiologically functions in UV-B-induced defense responses in Marchantia polymorpha, which belongs to the earliest diverging group of embryophyte lineages. Here, we demonstrate that UVR8 has a physiological function in UV-B tolerance and that there is a UVR8-dependent pathway involved. In addition, a UVR8-independent pathway is revealed. We examine the tissue-specific expression pattern of M. polymorpha UVR8 (MpUVR8), showing that it is highly expressed in the apical notch in thalli and gametangiophores, as well as in antheridial and archegonial heads. Furthermore, Mpuvr8KO plant transformants, in which the MpUVR8 locus was disrupted, were produced and analyzed to understand the physiological and molecular function of MpUVR8. Analysis using these plants indicates the important roles of MpUVR8 and MpUVR8-regulated genes, and of MpUVR8-independent pathways in UV-B tolerance. Subcellular localization of Citrine-fused MpUVR8 in M. polymorpha cells was also investigated. It was found to translocate from the cytosol into the nucleus in response to UV-B irradiation. Our findings indicate strong conservation of the physiological function of UVR8 and the molecular mechanisms for UVR8-dependent signal transduction through regulation of gene expression in embryophytes.

The rice CYP78A gene BSR2 confers resistance to Rhizoctonia solani and affects seed size and growth in Arabidopsis and rice.

The fungal pathogen Rhizoctonia solani causes devastating diseases in hundreds of plant species. Among these, R. solani causes sheath blight, one of the three major diseases in rice. To date, few genes have been reported that confer resistance to R. solani. Here, rice-FOX Arabidopsis lines identified as having resistance to a bacterial pathogen, Pseudomonas syringae pv. tomato DC3000, and a fungal pathogen, Colletotrichum higginsianum were screened for disease resistance to R. solani. BROAD-SPECTRUM RESISTANCE2 (BSR2), a gene encoding an uncharacterized cytochrome P450 protein belonging to the CYP78A family, conferred resistance to R. solani in Arabidopsis. When overexpressed in rice, BSR2 also conferred resistance to two R. solani anastomosis groups. Both Arabidopsis and rice plants overexpressing BSR2 had slower growth and produced longer seeds than wild-type control plants. In contrast, BSR2-knockdown rice plants were more susceptible to R. solani and displayed faster growth and shorter seeds in comparison with the control. These results indicate that BSR2 is associated with disease resistance, growth rate and seed size in rice and suggest that its function is evolutionarily conserved in both monocot rice and dicot Arabidopsis.

Biosynthesis of riccionidins and marchantins is regulated by R2R3-MYB transcription factors in Marchantia polymorpha.

R2R3-MYB transcription factors constitute the largest gene family among plant transcription factor families. They became largely divergent during the evolution of land plants and regulate various biological processes. The functions of R2R3-MYBs are mostly characterized in seed plants but are poorly understood in non-seed plants. Here, we examined the function of two R2R3-MYB genes of Marchantia polymorpha (Mapoly0073s0038 and Mapoly0006s0226) that are closely related to subgroup 4 of the R2R3-MYB family. We performed LC/MS/MS metabolomics, RNA-seq analysis and expression analysis in overexpressors and knockout mutants of MpMYB14 and MpMYB02. Overexpression of MpMYB14 remarkably increased the amount of riccionidins, which are specific anthocyanins in liverworts and a few flowering plants. In contrast, overexpression of MpMYB02 increased the amount of several marchantins, which are characteristic cyclic bis (bibenzyl ether) compounds in M. polymorpha and related liverworts. Knockouts of MpMYB14 and MpMYB02 abolished the accumulation of riccionidins and marchantins, respectively. The expression of MpMYB14 was up-regulated by UV-B irradiation, N deficiency, and NaCl treatment, whereas the expression of MpMYB02 was down-regulated by NaCl treatment. Our results suggest that the regulatory framework of phenolic metabolism by R2R3-MYB was already established in early land plants.

Repression of Nitrogen Starvation Responses by Members of the Arabidopsis GARP-Type Transcription Factor NIGT1/HRS1 Subfamily.

Nitrogen (N) is often a limiting nutrient whose availability determines plant growth and productivity. Because its availability is often low and/or not uniform over time and space in nature, plants respond to variations in N availability by altering uptake and recycling mechanisms, but the molecular mechanisms underlying how these responses are regulated are poorly understood. Here, we show that a group of GARP G2-like transcription factors, Arabidopsis thaliana NITRATE-INDUCIBLE, GARP-TYPE TRANSCRIPTIONAL REPRESSOR1/HYPERSENSITIVE TO LOW Pi-ELICITED PRIMARY ROOT SHORTENING1 proteins (NIGT1/HRS1s), are factors that bind to the promoter of the N starvation marker NRT2.4 and repress an array of N starvation-responsive genes under conditions of high N availability. Transient assays and expression analysis demonstrated that NIGT1/HRS1s are transcriptional repressors whose expression is regulated by N availability. We identified target genes of the NIGT1/HRS1s by genome-wide transcriptome analyses and found that they are significantly enriched in N starvation response-related genes, including N acquisition, recycling, remobilization, and signaling genes. Loss of NIGT1/HRS1s resulted in deregulation of N acquisition and accumulation. We propose that NIGT1/HRS1s are major regulators of N starvation responses that play an important role in optimizing N acquisition and utilization under fluctuating N conditions.

Genetic Nature of Elemental Contents in Wheat Grains and Its Genomic Prediction: Toward the Effective Use of Wheat Landraces from Afghanistan.

Profiling elemental contents in wheat grains and clarifying the underlying genetic systems are important for the breeding of biofortified crops. Our objective was to evaluate the genetic potential of 269 Afghan wheat landraces for increasing elemental contents in wheat cultivars. The contents of three major (Mg, K, and P) and three minor (Mn, Fe, and Zn) elements in wheat grains were measured by energy dispersive X-ray fluorescence spectrometry. Large variations in elemental contents were observed among landraces. Marker-based heritability estimates were low to moderate, suggesting that the elemental contents are complex quantitative traits. Genetic correlations between two locations (Japan and Afghanistan) and among the six elements were estimated using a multi-response Bayesian linear mixed model. Low-to-moderate genetic correlations were observed among major elements and among minor elements respectively, but not between major and minor elements. A single-response genome-wide association study detected only one significant marker, which was associated with Zn, suggesting it will be difficult to increase the elemental contents of wheat by conventional marker-assisted selection. Genomic predictions for major elemental contents were moderately or highly accurate, whereas those for minor elements were mostly low or moderate. Our results indicate genomic selection may be useful for the genetic improvement of elemental contents in wheat.

Analysis of grain elements and identification of best genotypes for Fe and P in Afghan wheat landraces.

This study was carried out with the aim of developing the methodology to determine elemental composition in wheat and identify the best germplasm for further research. Orphan and genetically diverse Afghan wheat landraces were chosen and EDXRF was used to measure the content of some of the elements to establish elemental composition in grains of 266 landraces using 10 reference lines. Four elements, K, Mg, P, and Fe, were measured by standardizing sample preparation. The results of hierarchical cluster analysis using elemental composition data sets indicated that the Fe content has an opposite pattern to the other elements, especially that of K. By systematic analysis the best wheat germplasms for P content and Fe content were identified. In order to compare the sensitivity of EDXRF, the ICP method was also used and the similar results obtained confirmed the EDXRF methodology. The sampling method for measurement using EDXRF was optimized resulting in high-throughput profiling of elemental composition in wheat grains at low cost. Using this method, we have characterized the Afghan wheat landraces and isolated the best genotypes that have high-elemental content and have the potential to be used in crop improvement.

Overexpression of rice OsREX1-S, encoding a putative component of the core general transcription and DNA repair factor IIH, renders plant cells tolerant to cadmium- and UV-induced damage by enhancing DNA excision repair.

Screening of 40,000 Arabidopsis FOX (Full-length cDNA Over-eXpressor gene hunting system) lines expressing rice full-length cDNAs brings us to identify four cadmium (Cd)-tolerant lines, one of which carried OsREX1-S as a transgene. OsREX1-S shows the highest levels of identity to Chlamydomonas reinhardtii REX1-S (referred to as CrREX1-S, in which REX denotes Required for Excision) and to yeast and human TFB5s (RNA polymerase II transcription factor B5), both of which are components of the general transcription and DNA repair factor, TFIIH. Transient expression of OsREX1-S consistently localized the protein to the nucleus of onion cells. The newly generated transgenic Arabidopsis plants expressing OsREX1-S reproducibly displayed enhanced Cd tolerance, confirming that the Cd-tolerance of the initial identified line was conferred solely by OsREX1-S expression. Furthermore, transgenic Arabidopsis plants expressing OsREX1-S exhibited ultraviolet-B (UVB) tolerance by reducing the amounts of cyclobutane pyrimidine dimers produced by UVB radiation. Moreover, those transgenic OsREX1-S Arabidopsis plants became resistant to bleomycin (an inducer of DNA strand break) and mitomycin C (DNA intercalating activity), compared to wild type. Our results indicate that OsREX1-S renders host plants tolerant to Cd, UVB radiation, bleomycin and mitomycin C through the enhanced DNA excision repair.

Identification and characterization of transcription factors regulating Arabidopsis HAK5.

Potassium (K) is an essential macronutrient for plant growth and reproduction. HAK5, an Arabidopsis high-affinity K transporter gene, plays an important role in K uptake. Its expression is up-regulated in response to K deprivation and is rapidly down-regulated when sufficient K levels have been re-established. To identify transcription factors regulating HAK5, an Arabidopsis TF FOX (Transcription Factor Full-length cDNA Over-eXpressor) library containing approximately 800 transcription factors was used to transform lines previously transformed with a luciferase reporter gene whose expression was driven by the HAK5 promoter. When grown under sufficient K levels, 87 lines with high luciferase activity were identified, and endogenous HAK5 expression was confirmed in 27 lines. Four lines overexpressing DDF2 (Dwarf and Delayed Flowering 2), JLO (Jagged Lateral Organs), TFII_A (Transcription initiation Factor II_A gamma chain) and bHLH121 (basic Helix-Loop-Helix 121) were chosen for further characterization by luciferase activity, endogenous HAK5 level and root growth in K-deficient conditions. Further analysis showed that the expression of these transcription factors increased in response to low K and salt stress. In comparison with controls, root growth under low K conditions was better in each of these four TF FOX lines. Activation of HAK5 expression by these four transcription factors required at least 310 bp of upstream sequence of the HAK5 promoter. These results indicate that at least these four transcription factors can bind to the HAK5 promoter in response to K limitation and activate HAK5 expression, thus allowing plants to adapt to nutrient stress.

The Arabidopsis COP9 SIGNALOSOME INTERACTING F-BOX KELCH 1 protein forms an SCF ubiquitin ligase and regulates hypocotyl elongation.

The regulation of protein turnover by the ubiquitin proteasome system (UPS) is a major posttranslational mechanism in eukaryotes. One of the key components of the UPS, the COP9 signalosome (CSN), regulates 'cullin-ring' E3 ubiquitin ligases. In plants, CSN participates in diverse cellular and developmental processes, ranging from light signaling to cell cycle control. In this work, we isolated a new plant-specific CSN-interacting F-box protein, which we denominated CFK1 (COP9 INTERACTING F-BOX KELCH 1). We show that, in Arabidopsis thaliana, CFK1 is a component of a functional ubiquitin ligase complex. We also show that CFK1 stability is regulated by CSN and by proteasome-dependent proteolysis, and that light induces accumulation of the CFK1 transcript in the hypocotyl. Analysis of CFK1 knockdown, mutant, and overexpressing seedlings indicates that CFK1 promotes hypocotyl elongation by increasing cell size. Reduction of CSN levels enhances the short hypocotyl phenotype of CFK1-depleted seedlings, while complete loss of CSN activity suppresses the long-hypocotyl phenotype of CFK1-overexpressing seedlings. We propose that CFK1 (and its regulation by CSN) is a novel component of the cellular mechanisms controlling hypocotyl elongation.

Role of the rice transcription factor JAmyb in abiotic stress response.

Plants have developed certain adaptive responses to environmental stresses that cause adverse effects on growth. To identify genes involved in the adaptive mechanisms, we constructed a large population of transgenic Arabidopsis expressing rice full-length cDNAs, and performed gain-of-function screening under high-salinity stress. In this study, we identified a rice R2R3-type MYB transcription factor gene, JAmyb, as a gene whose overexpression causes tolerance to high salinity. JAmyb overexpression in transgenic Arabidopsis improved tolerance to high-salinity stress during seed germination, seedling growth, and root elongation. In rice seedlings, JAmyb expression was induced by high-salinity and high-osmotic stresses and reactive oxygen species (ROS), suggesting that JAmyb is responsible for abiotic stress response. Microarray analysis showed that the overexpression of JAmyb stimulates the expression of several defense-associated genes, some of which have been predicted to be involved in osmotic adjustment, ROS removal, and ion homeostasis. Several transcription factors involved in the jasmonate (JA)-mediated stress response are also regulated by JAmyb. JAmyb has been reported to be associated with disease response. Our observations suggest that JAmyb plays a role in JA-mediated abiotic stress response in addition to biotic stress response in rice.

Arabidopsis PIZZA has the capacity to acylate brassinosteroids.

Brassinosteroids (BRs) affect a wide range of developmental processes in plants and compromised production or signalling of BRs causes severe growth defects. To identify new regulators of plant organ growth, we searched the Arabidopsis FOX (Full-length cDNA Over-eXpressor gene) collection for mutants with altered organ size and isolated two overexpression lines that display typical BR deficient dwarf phenotypes. The phenotype of these lines, caused by an overexpression of a putative acyltransferase gene PIZZA (PIZ), was partly rescued by supplying exogenous brassinolide (BL) and castasterone (CS), indicating that endogenous BR levels are rate-limiting for the growth of PIZ overexpression lines. Our transcript analysis further showed that PIZ overexpression leads to an elevated expression of genes involved in BR biosynthesis and a reduced expression of BR inactivating hydroxylases, a transcriptional response typical to low BR levels. Taking the advantage of relatively high endogenous BR accumulation in a mild bri1-301 background, we found that overexpression of PIZ results in moderately reduced levels of BL and CS and a strong reduction of typhasterol (TY) and 6-deoxocastasterone (6-deoxoCS), suggesting a role of PIZ in BR metabolism. We tested a set of potential substrates in vitro for heterologously expressed PIZ and confirmed its acyltransferase activity with BL, CS and TY. The PIZ gene is expressed in various tissues but as reported for other genes involved in BR metabolism, the loss-of-function mutants did not display obvious growth phenotypes under standard growth conditions. Together, our data suggest that PIZ can modify BRs by acylation and that these properties might help modulating endogenous BR levels in Arabidopsis.

Arabidopsis growth-regulating factor7 functions as a transcriptional repressor of abscisic acid- and osmotic stress-responsive genes, including DREB2A.

Arabidopsis thaliana DEHYDRATION-RESPONSIVE ELEMENT BINDING PROTEIN2A (DREB2A) functions as a transcriptional activator that increases tolerance to osmotic and heat stresses; however, its expression also leads to growth retardation and reduced reproduction. To avoid these adverse effects, the expression of DREB2A is predicted to be tightly regulated. We identified a short promoter region of DREB2A that represses its expression under nonstress conditions. Yeast one-hybrid screening for interacting factors identified GROWTH-REGULATING FACTOR7 (GRF7). GRF7 bound to the DREB2A promoter and repressed its expression. In both artificial miRNA-silenced lines and a T-DNA insertion line of GRF7, DREB2A transcription was increased compared with the wild type under nonstress conditions. A previously undiscovered cis-element, GRF7-targeting cis-element (TGTCAGG), was identified as a target sequence of GRF7 in the short promoter region of DREB2A via electrophoretic mobility shift assays. Microarray analysis of GRF7 knockout plants showed that a large number of the upregulated genes in the mutant plants were also responsive to osmotic stress and/or abscisic acid. These results suggest that GRF7 functions as a repressor of a broad range of osmotic stress-responsive genes to prevent growth inhibition under normal conditions.

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.

Growth arrest by trehalose-6-phosphate: an astonishing case of primary metabolite control over growth by way of the SnRK1 signaling pathway.

The strong regulation of plant carbon allocation and growth by trehalose metabolism is important for our understanding of the mechanisms that determine growth and yield, with obvious applications in crop improvement. To gain further insight on the growth arrest by trehalose feeding, we first established that starch-deficient seedlings of the plastidic phosphoglucomutase1 mutant were similarly affected as the wild type on trehalose. Starch accumulation in the source cotyledons, therefore, did not cause starvation and consequent growth arrest in the growing zones. We then screened the FOX collection of Arabidopsis (Arabidopsis thaliana) expressing full-length cDNAs for seedling resistance to 100 mm trehalose. Three independent transgenic lines were identified with dominant segregation of the trehalose resistance trait that overexpress the bZIP11 (for basic region/leucine zipper motif) transcription factor. The resistance of these lines to trehalose could not be explained simply through enhanced trehalase activity or through inhibition of bZIP11 translation. Instead, trehalose-6-phosphate (T6P) accumulation was much increased in bZIP11-overexpressing lines, suggesting that these lines may be insensitive to the effects of T6P. T6P is known to inhibit the central stress-integrating kinase SnRK1 (KIN10) activity. We confirmed that this holds true in extracts from seedlings grown on trehalose, then showed that two independent transgenic lines overexpressing KIN10 were insensitive to trehalose. Moreover, the expression of marker genes known to be jointly controlled by SnRK1 activity and bZIP11 was consistent with low SnRK1 or bZIP11 activity in seedlings on trehalose. These results reveal an astonishing case of primary metabolite control over growth by way of the SnRK1 signaling pathway involving T6P, SnRK1, and bZIP11.

Functional analysis of two isoforms of leaf-type ferredoxin-NADP(+)-oxidoreductase in rice using the heterologous expression system of Arabidopsis.

Ferredoxin-NADP(+)-oxidoreductase (FNR) mediates electron transfer between ferredoxin (Fd) and NADP(+); therefore, it is a key enzyme that provides the reducing power used in the Calvin cycle. Other than FNR, nitrite reductase, sulfite reductase, glutamate synthase, and Fd-thioredoxin reductase also accept electrons from Fd, an electron carrier protein in the stroma. Therefore, the regulation of electron partitioning in the chloroplast is important for photosynthesis and other metabolic pathways. The regulatory mechanism of electron partitioning, however, remains to be elucidated. We found, by taking advantage of a gain-of-function approach, that expression of two rice (Oryza sativa) full-length cDNAs of leaf-type FNRs (OsLFNR1 and OsLFNR2) led to altered chlorophyll fluorescence and growth in Arabidopsis (Arabidopsis thaliana) and rice. We revealed that overexpression of the OsLFNR1 and OsLFNR2 full-length cDNAs resulted in distinct phenotypes despite the high sequence similarity between them. Expression of OsLFNR1 affected the nitrogen assimilation pathway without inhibition of photosynthesis under normal conditions. On the other hand, OsLFNR2 expression led to the impairment of photosynthetic linear electron transport as well as Fd-dependent cyclic electron flow around photosystem I. The endogenous protein level of OsLFNR was found to be suppressed in both OsLFNR1- and OsLFNR2-overexpressing rice plants, leading to changes in the stoichiometry of the two LFNR isoforms within the thylakoid and soluble fractions. Thus, we propose that the stoichiometry of two LFNR isoforms plays an important role in electron partitioning between carbon fixation and nitrogen assimilation.

Programmed induction of endoreduplication by DNA double-strand breaks in Arabidopsis.

Genome integrity is continuously threatened by external stresses and endogenous hazards such as DNA replication errors and reactive oxygen species. The DNA damage checkpoint in metazoans ensures genome integrity by delaying cell-cycle progression to repair damaged DNA or by inducing apoptosis. ATM and ATR (ataxia-telangiectasia-mutated and -Rad3-related) are sensor kinases that relay the damage signal to transducer kinases Chk1 and Chk2 and to downstream cell-cycle regulators. Plants also possess ATM and ATR orthologs but lack obvious counterparts of downstream regulators. Instead, the plant-specific transcription factor SOG1 (suppressor of gamma response 1) plays a central role in the transmission of signals from both ATM and ATR kinases. Here we show that in Arabidopsis, endoreduplication is induced by DNA double-strand breaks (DSBs), but not directly by DNA replication stress. When root or sepal cells, or undifferentiated suspension cells, were treated with DSB inducers, they displayed increased cell size and DNA ploidy. We found that the ATM-SOG1 and ATR-SOG1 pathways both transmit DSB-derived signals and that either one suffices for endocycle induction. These signaling pathways govern the expression of distinct sets of cell-cycle regulators, such as cyclin-dependent kinases and their suppressors. Our results demonstrate that Arabidopsis undergoes a programmed endoreduplicative response to DSBs, suggesting that plants have evolved a distinct strategy to sustain growth under genotoxic stress.

Metabolomics reveals comprehensive reprogramming involving two independent metabolic responses of Arabidopsis to UV-B light.

Because of ever-increasing environmental deterioration it is likely that the influx of UV-B radiation (280-320 nm) will increase as a result of the depletion of stratospheric ozone. Given this fact it is essential that we better understand both the rapid and the adaptive responses of plants to UV-B stress. Here, we compare the metabolic responses of wild-type Arabidopsis with that of mutants impaired in flavonoid (transparent testa 4, tt4; transparent testa 5, tt5) or sinapoyl-malate (sinapoylglucose accumulator 1, sng1) biosynthesis, exposed to a short 24-h or a longer 96-h exposure to this photo-oxidative stress. In control experiments we subjected the genotypes to long-day conditions as well as to 24- and 96-h treatments of continuous light. Following these treatments we evaluated the dynamic response of metabolites including flavonoids, sinapoyl-malate precursors and ascorbate, which are well known to play a role in cellular protection from UV-B stress, as well as a broader range of primary metabolites, in an attempt to more fully comprehend the metabolic shift following the cellular perception of this stress. Our data reveals that short-term responses occur only at the level of primary metabolites, suggesting that these effectively prime the cell to facilitate the later production of UV-B-absorbing secondary metabolites. The combined results of these studies together with transcript profiles using samples irradiated by 24-h UV-B light are discussed in the context of current models concerning the metabolic response of plants to the stress imposed by excessive UV-B irradiation.

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.

A high-throughput screening system for Arabidopsis transcription factors and its application to Med25-dependent transcriptional regulation.

The activities of transcription factors (TFs) require interactions with specific DNA sequences and other regulatory proteins. To detect such interactions in Arabidopsis, we developed a high-throughput screening system with a Gateway-compatible Gal4-AD-TF library of 1589 Arabidopsis TFs, which can be easily screened by mating-based yeast-one-hybrid (Y1H) and yeast-two-hybrid (Y2H) methods. The efficiency of the system was validated by examining two well-characterized TF-DNA and TF-protein interactions: the CHE-CCA1 promoter interaction by Y1H and NPR1-TGAs interactions by Y2H. We used this system to identify eight TFs that interact with a Mediator subunit, Med25, a key regulator in JA signaling. We identified five TFs that interacted with the GCC-box cis-element in the promoter of PDF1.2, a downstream gene of Med25. We found that three of these TFs, all from the AP2-EREBP family, interact directly both with Med25 and the GCC-box of PDF1.2, suggesting that Med25 regulates PDF1.2 expression through these three TFs. These results demonstrate that this high-throughput Y1H/Y2H screening system is an efficient tool for studying transcriptional regulation networks in Arabidopsis. This system will be available for other Arabidopsis researchers, and thus it provides a vital resource for the Arabidopsis community.