Youichi Kondou

Efficient Yeast One-/Two-Hybrid Screening Using a Library Composed Only of Transcription Factors in Arabidopsis thaliana

Yeast one-hybrid screening is widely used for the identification of transcription factors (TFs) that interact with specific DNA sequences. However, screening a whole cDNA library is not efficient for the identification of TFs because TF genes represent only a small percentage of clones in a cDNA library. Here, we present the development of an efficient yeast one-hybrid screening system using a prey library composed only of approximately 1,500 TF cDNAs of Arabidopsis thaliana. This library enabled us to isolate a TF that binds to a specific promoter sequence with high efficiency, even when the promoter region of the gene of interest was directly employed as bait. Furthermore, this library was also successfully applied as a yeast two-hybrid library to find TFs that interact with specific proteins. This efficient system will contribute to the elucidation of gene regulatory networks in plants.

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/.

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.

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.

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.

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

NIGT1/HRS1s are GARP-type transcription factors that repress nitrogen starvation responses in order to optimize nitrogen acquisition and utilization under fluctuating nitrogen availability and demand. 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.

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.

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.

Identification of Photosynthesis-Related Genes in Rice Using FOX Hunting System

Photosynthesis is one of the most important determinants of crop productivity. Although many studies have been conducted to improve yield, very little progress has been made. In this study, we used rice FOX (Full length cDNA over-expressing) lines that rice full-length cDNAs were over-expressed in Arabidopsis to identify novel photosynthesis-related genes of rice. We have used imaging of chlorophyll fluorescence to screen candidates. To confirm over-expression of transformed cDNAs cause the observed phenotype, we generated transformants overexpressing each cDNA, resulting that eight lines showed the same phenotype as original lines. Isolated candidates could be classified into four categories depending on chlorophyll fluorescence parameters. We analyzed gene expression profile of three candidate lines showing similar photosynthetic characteristics using DNA microarray. We found commonly regulated genes among three candidates, indicating that candidates have both shared and independent influences on gene expression.