Full-length cDNA Over-eXpressor Research Articles

Construction of a Full-Length cDNA Over-Expressing Library to Identify Valuable Genes from Populus tomentosa.

Poplar wood is the main source of renewable biomass energy worldwide, and is also considered to be a model system for studying woody plants. The Full-length cDNA Over-eXpressing (FOX) gene hunting system is an effective method for generating gain-of-function mutants. Large numbers of novel genes have successfully been identified from many herbaceous plants according to the phenotype of gain-of-function mutants under normal or abiotic stress conditions using this system. However, the system has not been used for functional gene identification with high-throughput mutant screening in woody plants. In this study, we constructed a FOX library from the Chinese white poplar, Populus tomentosa. The poplar cDNA library was constructed into the plant expression vector pEarleyGate101 and further transformed into Arabidopsis thaliana (thale cress). We collected 1749 T1 transgenic plants identified by PCR. Of these, 593 single PCR bands from different transgenic lines were randomly selected for sequencing, and 402 diverse sequences of poplar genes were isolated. Most of these genes were involved in photosynthesis, environmental adaptation, and ribosome biogenesis based on Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway annotation. We characterized in detail two mutant lines carrying PtoCPCa or PtoWRKY13 cDNA insertions. Phenotypic characterization showed that overexpression of these genes in A. thaliana affected trichome development or secondary cell wall (SCW) deposition, respectively. Together, the Populus-FOX-Arabidopsis library generated in our experiments will be helpful for efficient discovery of novel genes in poplar.

Fox Hunting in Wild Apples: Searching for Novel Genes in Malus Sieversii.

Malus sieversii is considered the progenitor of modern apple (Malus pumila) cultivars and to represent a valuable source of genetic diversity. Despite the importance of M. sieversii as a source of disease resistance, stress tolerance, and novel fruit traits, little is known about gene function and diversity in M. sieversii. Notably, a publicly annotated genome sequence for this species is not available. In the current study, the FOX (Full-length cDNA OvereXpressing) gene hunting system was used to construct a library of transgenic lines of Arabidopsis in which each transgenic line overexpresses a full-length gene obtained from a cDNA library of the PI619283 accession of M. sieversii. The cDNA library was constructed from mRNA obtained from bark tissues collected in late fall–early winter, a time at which many abiotic stress-adaptative genes are expressed. Over 4000 apple FOX Arabidopsis lines have been established from the pool of transgenic seeds and cDNA inserts corresponding to various Gene Ontology (GO) categories have been identified. A total of 160 inserts appear to be novel, with no or limited homology to M. pumila, Arabidopsis, or poplar. Over 1300 lines have also been screened for freezing resistance. The constructed library of transgenic lines provides a valuable genetic resource for exploring gene function and diversity in Malus sieversii. Notably, no such library of t-DNA lines currently exists for any Malus species.

Asparaginyl-tRNA synthetase gene (<i>SYNC1</i>) characterized by <i>Lotus corniculatus</i> FOX-superroot lines has effects on plant morphology and amino acid contents of seed in soybean

The application of useful genes from model plants to crops is an important step to verify its agricultural usefulness. SYNC1, an asparaginyl-tRNA synthetase gene, was previously identified through the Full-length cDNA Over-eXpressor gene (FOX gene-hunting system) of Arabidopsis cDNA by using super-growing root (SR) culture of Lotus corniculatus, and was suggested to have a potential in increasing some amino acid contents and plant biomass. To identify the functionality of SYNC1 gene in a typical legume crop soybean, the effects of its overexpression in transgenic plants to agricultural traits and free amino acid contents were evaluated. The transgenic soybean plants were produced from infected half-seed explants of 1 day old seedlings with the suspension of Agrobacterium tumefaciens harboring expression vector pB7WG2D-SYNC1. The transgenic plants that overexpressed SYNC1 gene had increased asparagine and lysine contents in matured seeds, and increased aspartate, lysine, alanine and histidine contents in germinated seeds. The changes in those free amino acid contents affected plant morphology and led to significant increase in plant length, number of branches and number of branch nodes as yield components of soybean. The transgenic plants also showed a tendency of higher number of pods, seeds and total seed weight per plant. These results showed that the overexpression of SYNC1 gene contributes on the increase of plant free amino acid contents and biomass, and this approach is expected to be applicable in other legumes, grain and forage crops.

Screening of abiotic stress-responsive cotton genes using a cotton full-length cDNA overexpressing Arabidopsis library.

Cotton (Gossypium hirsutum L.) is a major crop and the main source of natural fiber worldwide. Because various abiotic and biotic stresses strongly influence cotton fiber yield and quality, improved stress resistance of this crop plant is urgently needed. In this study, we used Gateway technology to construct a normalized full-length cDNA overexpressing (FOX) library from upland cotton cultivar ZM12 under various stress conditions. The library was transformed into Arabidopsis to produce a cotton-FOX-Arabidopsis library. Screening of this library yielded 6,830 transgenic Arabidopsis lines, of which 757 were selected for sequencing to ultimately obtain 659 cotton ESTs. GO and KEGG analyses mapped most of the cotton ESTs to plant biological process, cellular component, and molecular function categories. Next, 156 potential stress-responsive cotton genes were identified from the cotton-FOX-Arabidopsis library under drought, salt, ABA, and other stress conditions. Four stress-related genes identified from the library, designated as GhCAS, GhAPX, GhSDH, and GhPOD, were cloned from cotton complementary DNA, and their expression patterns under stress were analyzed. Phenotypic experiments indicated that overexpression of these cotton genes in Arabidopsis affected the response to abiotic stress. The method developed in this study lays a foundation for high-throughput cloning and rapid identification of cotton functional genes.

Functional Identification of Salt-Stress-Related Genes Using the FOX Hunting System from Ipomoea pes-caprae.

Ipomoea pes-caprae is a seashore halophytic plant and is therefore a good model for studying the molecular mechanisms underlying salt and stress tolerance in plant research. Here, we performed Full-length cDNA Over-eXpressor (FOX) gene hunting with a functional screening of a cDNA library using a salt-sensitive yeast mutant strain to isolate the salt-stress-related genes of I. pes-caprae (IpSR genes). The library was screened for genes that complemented the salt defect of yeast mutant AXT3 and could grow in the presence of 75 mM NaCl. We obtained 38 candidate salt-stress-related full-length cDNA clones from the I. pes-caprae cDNA library. The genes are predicted to encode proteins involved in water deficit, reactive oxygen species (ROS) scavenging, cellular vesicle trafficking, metabolic enzymes, and signal transduction factors. When combined with the quantitative reverse transcription-polymerase chain reaction (qRT-PCR) analyses, several potential functional salt-tolerance-related genes were emphasized. This approach provides a rapid assay system for the large-scale screening of I. pes-caprae genes involved in the salt stress response and supports the identification of genes responsible for the molecular mechanisms of salt tolerance.

The chemical NJ15 affects hypocotyl elongation and shoot gravitropism via cutin polymerization.

Caf: cafenstrole; DEG: differentially expressed gene; FDR: false discovery rate; FOX: full length cDNA-overexpressor; VLCFA: very-long-chain fatty acid.

Overexpression of TIFY genes promotes plant growth in rice through jasmonate signaling.

Because environmental stress can reduce crop growth and yield, the identification of genes that enhance agronomic traits is increasingly important. Previous screening of full-length cDNA overexpressing (FOX) rice lines revealed that OsTIFY11b, one of 20 TIFY proteins in rice, affects plant size, grain weight, and grain size. Therefore, we analyzed the effect of OsTIFY11b and nine other TIFY genes on the growth and yield of corresponding TIFY-FOX lines. Regardless of temperature, grain weight and culm length were enhanced in lines overexpressing TIFY11 subfamily genes, except OsTIFY11e. The TIFY-FOX plants exhibited increased floret number and reduced days to flowering, as well as reduced spikelet fertility, and OsTIFY10b, in particular, enhanced grain yield by minimizing decreases in fertility. We suggest that the enhanced growth of TIFY-transgenic rice is related to regulation of the jasmonate signaling pathway, as in Arabidopsis. Moreover, we discuss the potential application of TIFY overexpression for improving crop yield.

Generation of Wheat Transcription Factor FOX Rice Lines and Systematic Screening for Salt and Osmotic Stress Tolerance.

Transcription factors (TFs) play important roles in plant growth, development, and responses to environmental stress. In this study, we collected 1,455 full-length (FL) cDNAs of TFs, representing 45 families, from wheat and its relatives Triticum urartu, Aegilops speltoides, Aegilops tauschii, Triticum carthlicum, and Triticum aestivum. More than 15,000 T0 TF FOX (Full-length cDNA Over-eXpressing) rice lines were generated; of these, 10,496 lines set seeds. About 14.88% of the T0 plants showed obvious phenotypic changes. T1 lines (5,232 lines) were screened for salt and osmotic stress tolerance using 150 mM NaCl and 20% (v/v) PEG-4000, respectively. Among them, five lines (591, 746, 1647, 1812, and J4065) showed enhanced salt stress tolerance, five lines (591, 746, 898, 1078, and 1647) showed enhanced osmotic stress tolerance, and three lines (591, 746, and 1647) showed both salt and osmotic stress tolerance. Further analysis of the T-DNA flanking sequences showed that line 746 over-expressed TaEREB1, line 898 over-expressed TabZIPD, and lines 1812 and J4065 over-expressed TaOBF1a and TaOBF1b, respectively. The enhanced salt and osmotic stress tolerance of lines 898 and 1812 was confirmed by retransformation of the respective genes. Our results demonstrate that a heterologous FOX system may be used as an alternative genetic resource for the systematic functional analysis of the wheat genome.

CSP41b, a protein identified via FOX hunting using Eutrema salsugineum cDNAs, improves heat and salinity stress tolerance in transgenic Arabidopsis thaliana

Eutrema salsugineum (also known as Thellungiella salsuginea and formerly Thellungiella halophila), a species closely related to Arabidopsis thaliana, shows tolerance not only to salt stress, but also to chilling, freezing, and high temperatures. To identify genes responsible for stress tolerance, we conducted Full-length cDNA Over-eXpressing gene (FOX) hunting among a collection of E. salsugineum cDNAs that were stress-induced according to gene ontology analysis or over-expressed in E. salsugineum compared with A. thaliana. We identified E. salsugineum CSP41b (chloroplast stem-loop-binding protein of 41 kDa; also known as CRB, chloroplast RNA binding; named here as EsCSP41b) as a gene that can confer heat and salinity stress tolerance on A. thaliana. A. thaliana CSP41b is reported to play an important role in the proper functioning of the chloroplast: the atcsp41b mutant is smaller and paler than wild-type plants and shows altered chloroplast morphology and photosynthetic performance. We observed that AtCSP41b-overexpressing transgenic A. thaliana lines also exhibited marked heat tolerance and significant salinity stress tolerance. The EsCSP41b-overexpressing transgenic A. thaliana lines showed significantly higher photosynthesis activity than wild-type plants not only under normal growth conditions but also under heat stress. In wild-type plants, the expression levels of both EsCSP41b and AtCSP41b were significantly reduced under heat or salinity stress. We conclude that maintenance of CSP41b expression under abiotic stresses may alleviate photoinhibition and improve survival under such stresses.

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.

Overexpression of a Novel Component Induces HAK5 and Enhances Growth in Arabidopsis

Plants have developed mechanisms to adapt to the potassium deficient conditions over the years. In Arabidopsis thaliana, expression of a potassium transporter HAK5 is induced in low potassium conditions as an adaptive response to nutrient deficiency. In order to understand the mechanism in which HAK5 is regulated, the full-length cDNA overexpressor gene hunting system was employed as a screening method. Of 40 genes recovered, At4g18280 was found to be dramatically induced in response to potassium-deficiency and salt stress. Plants overexpressing this gene showed higher HAK5 expression and enhanced growth. These plants were also less sensitive to potassium-deficiency in terms of primary root growth. Taken together, these data suggest that this novel component, At4g18280, contributes to regulation of HAK5 and, consequently, tolerance to potassium-deficiency in plants.

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.

Molecular characterization of BrRZFPs genes encoding C3HC4 type RING zinc finger protein under abiotic stress from Chinese cabbage (Brassica rapa L.)

The novel BrRZFPs genes encoding C3HC4-type RING zinc finger protein were identified from FOX (full length cDNA over-expressing) library of Brassica rapa. Ten full-length cDNAs obtained from the library encode zinc-finger protein containing 346 amino acids, designated BrRZFPs. These genes were classified into four groups by phylogenic analysis showing conserved protein sequences at both termini. The tissue distribution of BrRZFPs transcription was examined by qRT-PCR revealing ubiquitous expression pattern. However, each gene was strongly expressed in the specific tissue. Transcriptional analysis showed that those acquired 10 genes were inducible under abiotic stresses. Likewise, the transcript of BrRZFP3 was strongly induced (~12-folds) by exogenous abscisic acid, whereas the transcripts of BrRZFP1, BrRZFP2 and BrRZFP3 were (> 9-folds) induced by cold. We suggest that these BrRZFPs that function as signal or response to abiotic stress are useful for crop improvement.

Development and Identification of Transgenic Rice Lines with Abiotic Stress Tolerance by using a Full-length Overexpressor Gene Hunting System

The latest report on the draft genome of Brassica rapa sequence has been published. To elucidate the functions of these genes and to efficiently search for agriculturally useful genes, a Full-length cDNA Over-eXpressor (FOX) gene hunting system was used. The FOX library from Chinese cabbage was introduced into rice via Agrobacterium-mediated transformation. Approximately 1,150 FOX-rice lines were generated. Genomic PCR analysis indicated that the average length of FL-cDNAs introduced into individual lines was 900~1,200 bp. Basic Local Alignment System Tool (BLAST) analysis of the FL-cDNA genes revealed that 35.5% have unknown function. Most of the randomly selected transgenic rice lines showed overexpression (92%) of these genes relative to the wild-type Gopum. Moreover, 94% of the 850 transgenic rice lines were moderately tolerant (slightly yellow) to cold and 9 lines were tolerant (seedlings were light green). Morphological evaluation of the transgenic rice lines showed minimal phenotypic alteration (12%). Approximately 25.1% and 22% of the plants were significantly ahead in the days to heading and had elevated chlorophyll content, respectively. Other agronomic traits such as filled grains, number of tiller, panicle length, and culm and plant height were relatively less variable among the transgenic lines. These results provide a resource for defining genes that are associated with tolerance in transgenic rice lines.

HsfA1d, a Protein Identified via FOX Hunting Using Thellungiella salsuginea cDNAs Improves Heat Tolerance by Regulating Heat-Stress-Responsive Gene Expression

Thellungiella salsuginea (formerly T. halophila), a species closely related to Arabidopsis (Arabidopsis thaliana), is tolerant not only to high salt levels, but also to chilling, freezing, and ozone. Here, we report that T. salsuginea also shows greater heat tolerance than Arabidopsis. We identified T. salsuginea HsfA1d (TsHsfA1d) as a gene that can confer marked heat tolerance on Arabidopsis. TsHsfA1d was identified via Full-length cDNA Over-eXpressing gene (FOX) hunting from among a collection of heat-stress-related T. salsuginea cDNAs. Transgenic Arabidopsis overexpressing TsHsfA1d showed constitutive up-regulation of many genes in the Arabidopsis AtHsfA1 regulon under normal growth temperature. In Arabidopsis mesophyll protoplasts, TsHsfA1d was localized in both the nucleus and the cytoplasm. TsHsfA1d also interacted with AtHSP90, which negatively regulates AtHsfA1s by forming HsfA1-HSP90 complexes in the cytoplasm. It is likely that the partial nuclear localization of TsHsfA1d induced the expression of the AtHsfA1d regulon in the transgenic plants at normal temperature. We also discovered that transgenic Arabidopsis plants overexpressing AtHsfA1d were more heat-tolerant than wild-type plants and up-regulated the expression of the HsfA1d regulon, as was observed in TsHsfA1d-overexpressing plants. We propose that the products of both TsHsfA1d and AtHsfA1d function as positive regulators of Arabidopsis heat-stress response and would be useful for the improvement of heat-stress tolerance in other plants.

High-throughput analysis of rice genes by means of the heterologous full-length cDNA overexpressor (FOX)-hunting system

Mutant populations are indispensable tools for investigating plant gene functions. Gain-of-function technology is one of the approaches used for the systematic production of mutant resources and activation tagging is a well-established method to generate gain-of-function mutants in plants. As an alternative approach for the systematic generation of a gain-of-function mutant population, we developed the Full-length cDNA OvereXpressor (FOX)-hunting system in which full-length cDNAs (fl-cDNAs) are overexpressed in plants to quickly identify candidate genes. The FOX-hunting system was used for high-throughput analysis of rice (Oryza sativa) genes heterologously expressed in Arabidopsis thaliana (rice FOX Arabidopsis lines). A large screening to identify and characterize rice genes with rice FOX Arabidopsis lines revealed that one of the isolated genes, BROAD-SPECTRUM RESISTANCE 1 (BSR1) conferred multiple or broad-spectrum disease resistance in both a dicotyledonous and monocotyledonous plant. We found that expression of rice fl-cDNAs without a homolog in Arabidopsis affected morphological traits. In addition, overexpression of homologous genes of rice and Arabidopsis led to a similar phenotype. Thus, we conclude that the FOX-hunting system is an excellent heterologous system and offers a new tool with which to explore gene function in rice.

BAT1, a putative acyltransferase, modulates brassinosteroid levels in Arabidopsis

Brassinosteroids (BRs) are essential for various aspects of plant development. Cellular BR homeostasis is critical for proper growth and development of plants; however, its regulatory mechanism remains largely unknown. BAT1 (BR-related acyltransferase 1), a gene encoding a putative acyltransferase, was found to be involved in vascular bundle development in a full-length cDNA over-expressor (FOX) screen. Over-expression of BAT1 resulted in typical BR-deficient phenotypes, which were rescued by exogenously applied castasterone and brassinolide. Analyses of BR profiles demonstrated that BAT1 alters levels of several brassinolide biosynthetic intermediates, including 6-deoxotyphasterol, typhasterol and 6-deoxocastasterone. BAT1 is mainly localized in the endoplasmic reticulum. BAT1 is highly expressed in young tissues and vascular bundles, and its expression is induced by auxin. These data suggest that BAT1 is involved in BR homeostasis, probably by conversion of brassinolide intermediates into acylated BR conjugates.

Overexpression of a Rice TIFY Gene Increases Grain Size through Enhanced Accumulation of Carbohydrates in the Stem

Screening of rice full-length cDNA overexpressing (FOX) lines allowed the identification of a TIFY gene, TIFY11b, as a growth-promoting gene whose overexpression increased plant height and seed size. The grains of TIFY11b-overexpressing plants exceeded those of non-transformants in length, width and thickness, resulting in 9-21% increases in grain weight. The increase was achieved by overexpressing the gene in the whole plant body, but not by seed-restricted expression, indicating that seed enlargement is attributable to overexpression in vegetative organs such as the leaf. The whole-body overexpressing plants developed longer leaves along with higher levels of starch and sucrose in the leaf sheath and culm at the heading stage than the non-transformants. Although overexpression of TIFY11b did not alter the photosynthetic rate per leaf area before and after heading, it caused an accumulation of higher levels of the carbohydrate assimilate, probably due to increased photosynthesis per plant, suggesting that the increase in grain size and weight is attained by enhanced accumulation and translocation of the carbohydrate in the culms and leaf sheaths of the transgenic plants. Thus, TIFY11b is a novel grain-size increasing gene.

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.

Tilling and Fox-Hunting: New Methods for Functional Analysis of Genes

Summary Theoretical and experimental bases of TILLING and FOX-hunting, new tools for precise identification of genes in functional studies are reviewed. TILLING (Targeting Induced Local Lesions IN Genomes) is a technique combining chemical mutagenesis with its sensitive and precise detection. The method involves PCR amplification of DNA samples pooled following extraction from a collection of chemically-treated organisms and a subsequent screening for mutations using Cel1 endonuclease, detecting mismatches in heteroduplexes [52]. FOX-hunting (Full-length cDNA Over-eXpressing gene hunting system) is a new method of plant gene overexpression, which enables a quick gene isolation and sequencing, paralelly with functional studies.