Wild-type Columbia Research Articles

Arabidopsis ABA-Activated Kinase MAPKKK18 is Regulated by Protein Phosphatase 2C ABI1 and the Ubiquitin-Proteasome Pathway.

Phosphorylation and dephosphorylation events play an important role in the transmission of the ABA signal. Although SnRK2 [sucrose non-fermenting1-related kinase2] protein kinases and group A protein phosphatase type 2C (PP2C)-type phosphatases constitute the core ABA pathway, mitogen-activated protein kinase (MAPK) pathways are also involved in plant response to ABA. However, little is known about the interplay between MAPKs and PP2Cs or SnRK2 in the regulation of ABA pathways. In this study, an effort was made to elucidate the role of MAP kinase kinase kinase18 (MKKK18) in relation to ABA signaling and response. The MKKK18 knockout lines showed more vigorous root growth, decreased abaxial stomatal index and increased stomatal aperture under normal growth conditions, compared with the control wild-type Columbia line. In addition to transcriptional regulation of the MKKK18 promoter by ABA, we demonstrated using in vitro and in vivo kinase assays that the kinase activity of MKKK18 was regulated by ABA. Analysis of the cellular localization of MKKK18 showed that the active kinase was targeted specifically to the nucleus. Notably, we identified abscisic acid insensitive 1 (ABI1) PP2C as a MKKK18-interacting protein, and demonstrated that ABI1 inhibited its activity. Using a cell-free degradation assay, we also established that MKKK18 was unstable and was degraded by the proteasome pathway. The rate of MKKK18 degradation was delayed in the ABI1 knockout line. Overall, we provide evidence that ABI1 regulates the activity and promotes proteasomal degradation of MKKK18.

Strigolactones as an auxiliary hormonal defence mechanism against leafy gall syndrome in Arabidopsis thaliana.

Leafy gall syndrome is the consequence of modified plant development in response to a mixture of cytokinins secreted by the biotrophic actinomycete Rhodococcus fascians. The similarity of the induced symptoms with the phenotype of plant mutants defective in strigolactone biosynthesis and signalling prompted an evaluation of the involvement of strigolactones in this pathology. All tested strigolactone-related Arabidopsis thaliana mutants were hypersensitive to R. fascians. Moreover, treatment with the synthetic strigolactone mixture GR24 and with the carotenoid cleavage dioxygenase inhibitor D2 illustrated that strigolactones acted as antagonistic compounds that restricted the morphogenic activity of R. fascians. Transcript profiling of the MORE AXILLARY GROWTH1 (MAX1), MAX2, MAX3, MAX4, and BRANCHED1 (BRC1) genes in the wild-type Columbia-0 accession and in different mutant backgrounds revealed that upregulation of strigolactone biosynthesis genes was triggered indirectly by the bacterial cytokinins via host-derived auxin and led to the activation of BRC1 expression, inhibiting the outgrowth of the newly developing shoots, a typical hallmark of leafy gall syndrome. Taken together, these data support the emerging insight that balances are critical for optimal leafy gall development: the long-lasting biotrophic interaction is possible only because the host activates a set of countermeasures-including the strigolactone response-in reaction to bacterial cytokinins to constrain the activity of R. fascians.

Identification and Characterization of Mutants Related to LRS1 in Arabidopsis thaliana

Lateral root stimulator 1 (LRS1) is a WD40-repeat protein that contains a Damaged DNA Binding (DWD) motif, suggesting an interaction with E3 ubiquitin ligases of the Damaged DNA Binding 1–Cullin 4 (DDB1-CUL4) type. Previous findings indicate LRS1 binds a deubiquitinase, indicating a role in ubiquitin regulation. Plant lines containing Agrobacterium T-DNA insertions in LRS1 have been identified for phenotypic analyses. Initial characterization of one lrs1 line (FS16) showed reduced roots, as previously described for the lrs1-1 allele. Altered leaf, flower, and seed morphologies, in addition to altered hormone responses, have also been observed. Comparison to wild-type sibling segregates (FS15), however, suggested the presence of additional mutations in the T-DNA line under study. To segregate the T-DNA insertion from unrelated mutations, FS16 plants are being back-crossed three times to wild-type Columbia (Col-0) and genotyped using PCR. In addition to these mutants, T-DNA lines mutated in genes encoding potential LRS1-interacting proteins, such as a RING (Really Interesting New Gene) E3 ubiquitin ligase and a histone methyltransferase, are also being analyzed. Characterization of mutant phenotypes will help elucidate the function of LRS1 in vivo and allow the exploration of its role in the regulation of ubiquitination. Funding has been provided by the Salisbury University Henson School of Science Undergraduate Research Grant.

An eukaryotic translation initiation factor, AteIF5A-2, affects cadmium accumulation and sensitivity in Arabidopsis.

Cadmium (Cd) is one of the most toxic elements and can be accumulated in plants easily; meanwhile, eIF5A is a highly conserved protein in all eukaryotic organisms. The present work tried to investigate whether eIF5A is involved in Cd accumulation and sensitivity in Arabidopsis (Arabidopsis thaliana L.) by comparing the wild-type Columbia-0 (Col-0) with a knockdown mutant of AteIF5A-2, fbr12-3 under Cd stress conditions. The results showed that the mutant fbr12-3 accumulated more Cd in roots and shoots and had significantly lower chlorophyll content, shorter root length, and smaller biomass, suggesting that downregulation of AteIF5A-2 makes the mutant more Cd sensitive. Real-time polymerase chain reaction revealed that the expressions of metal transporters involved in Cd uptake and translocation including IRT1, ZIP1, AtNramp3, and AtHMA4 were significantly increased but the expressions of PCS1 and PCS2 related to Cd detoxification were decreased notably in fbr12-3 compared with Col-0. As a result, an increase in MDA and H2 O2 content but decrease in root trolox, glutathione and proline content under Cd stress was observed, indicating that a severer oxidative stress occurs in the mutant. All these results demonstrated for the first time that AteIF5A influences Cd sensitivity by affecting Cd uptake, accumulation, and detoxification in Arabidopsis.

A large-scale genetic screen for mutants with altered salicylic acid accumulation in Arabidopsis.

Salicylic acid (SA) is a key defense signal molecule against biotrophic and hemibiotrophic pathogens in plants, but how SA is synthesized in plant cells still remains elusive. Identification of new components involved in pathogen-induced SA accumulation would help address this question. To this end, we performed a large-scale genetic screen for mutants with altered SA accumulation during pathogen infection in Arabidopsis using a bacterial biosensor Acinetobacter sp. ADPWH_lux-based SA quantification method. A total of 35,000 M2 plants in the npr1-3 mutant background have been individually analyzed for the bacterial pathogen Pseudomonas syringae pv. maculicola (Psm) ES4326-induced SA accumulation. Among the mutants isolated, 19 had SA levels lower than npr1 (sln) and two exhibited increased SA accumulation in npr1 (isn). Complementation tests revealed that seven of the sln mutants are new alleles of eds5/sid1, two are sid2/eds16 alleles, one is allelic to pad4, and the remaining seven sln and two isn mutants are new non-allelic SA accumulation mutants. Interestingly, a large group of mutants (in the npr1-3 background), in which Psm ES4326-induced SA levels were similar to those in the wild-type Columbia plants, were identified, suggesting that the signaling network fine-tuning pathogen-induced SA accumulation is complex. We further characterized the sln1 single mutant and found that Psm ES4326-induced defense responses were compromised in this mutant. These defense response defects could be rescued by exogenous SA, suggesting that SLN1 functions upstream of SA. The sln1 mutation was mapped to a region on the north arm of chromosome I, which contains no known genes regulating pathogen-induced SA accumulation, indicating that SLN1 likely encodes a new regulator of SA biosynthesis. Thus, the new sln and isn mutants identified in this genetic screen are valuable for dissecting the molecular mechanisms underlying pathogen-induced SA accumulation in plants.

Bacterial auxin signaling: comparative study of growth induction inArabidopsis thaliana and Triticum aestivum

Auxin-producing rhizobacteria were evaluated for the role of their auxin signaling in enhancing the growth of Arabidopsis thaliana (L.) Heynh. and Triticum aestivum L. Arabidopsis thaliana wild type Columbia (Col-0) in comparison with phytohormone mutant lines aux1-7 (insensitive to auxin and ethylene), axr4-1 (insensitive to auxin), and eir1-1 (insensitive to ethylene) was used as a model system. Bacterization of wild type Col-0 recorded significant increases in shoot length (38%) and number of siliqua (180%) with Bacillus subtilis TpP-1 over the controls. Inoculation with aux1-7 and axr4-1 showed statistically comparable results for growth parameters as compared to their respective controls. Treatment of eir1-1 with Pseudomonas sp. AvH-4 and Bacillus subtilis TpP-1 recorded significant increases in shoot length (9%) and number of siliqua (20%), respectively. The pattern of growth response of aux1-7, axr4-1, and eir1-1 indicated that bacterial auxin and ethylene signaling is involved in the growth promotion of A. thaliana. For T. aestivum, significant increases in number of tillers (74%) and seed weight (26%) were recorded with Pseudomonas sp. AvH-4. Results of this study suggested that growth induction by B. subtilis TpP-1, Pseudomonas sp. AvH-4, and Pseudomonas aeruginosa As-17 in A. thaliana is associated with bacterial auxin signaling.

Paired-end analysis of transcription start sites in Arabidopsis reveals plant-specific promoter signatures.

Understanding plant gene promoter architecture has long been a challenge due to the lack of relevant large-scale data sets and analysis methods. Here, we present a publicly available, large-scale transcription start site (TSS) data set in plants using a high-resolution method for analysis of 5' ends of mRNA transcripts. Our data set is produced using the paired-end analysis of transcription start sites (PEAT) protocol, providing millions of TSS locations from wild-type Columbia-0 Arabidopsis thaliana whole root samples. Using this data set, we grouped TSS reads into "TSS tag clusters" and categorized clusters into three spatial initiation patterns: narrow peak, broad with peak, and weak peak. We then designed a machine learning model that predicts the presence of TSS tag clusters with outstanding sensitivity and specificity for all three initiation patterns. We used this model to analyze the transcription factor binding site content of promoters exhibiting these initiation patterns. In contrast to the canonical notions of TATA-containing and more broad "TATA-less" promoters, the model shows that, in plants, the vast majority of transcription start sites are TATA free and are defined by a large compendium of known DNA sequence binding elements. We present results on the usage of these elements and provide our Plant PEAT Peaks (3PEAT) model that predicts the presence of TSSs directly from sequence.

TRICHOME BIREFRINGENCE-LIKE27 affects aluminum sensitivity by modulating the O-acetylation of xyloglucan and aluminum-binding capacity in Arabidopsis.

Xyloglucan (XyG) has been reported to contribute to the aluminum (Al)-binding capacity of the cell wall in Arabidopsis (Arabidopsis thaliana). However, the influence of O-acetylation of XyG, accomplished by the putative O-acetyltransferase TRICHOME BIREFRINGENCE-LIKE27 (TBL27 [AXY4]), on its Al-binding capacity is not known. In this study, we found that the two corresponding TBL27 mutants, axy4-1 and axy4-3, were more Al sensitive than wild-type Columbia-0 plants. TBL27 was expressed in roots as well as in leaves, stems, flowers, and siliques. Upon Al treatment, even within 30 min, TBL27 transcript accumulation was strongly down-regulated. The mutants axy4-1 and axy4-3 accumulated significantly more Al in the root and wall, which could not be correlated with pectin content or pectin methylesterase activity, as no difference in the mutants was observed compared with the wild type when exposed to Al stress. The increased Al accumulation in the wall of the mutants was found to be in the hemicellulose fraction. While the total sugar content of the hemicellulose fraction did not change, the O-acetylation level of XyG was reduced by Al treatment. Taken together, we conclude that modulation of the O-acetylation level of XyG influences the Al sensitivity in Arabidopsis by affecting the Al-binding capacity in the hemicellulose.

Plastid casein kinase 2 knockout reduces abscisic acid (ABA) sensitivity, thermotolerance, and expression of ABA- and heat-stress-responsive nuclear genes.

Plastid casein kinase 2 (CK2) is a major Ser/Thr-specific enzyme for protein phosphorylation in the chloroplast stroma and its kinase activity is regulated by redox signals. To understand the role of CK2 phosphorylation of chloroplast proteins in abiotic stress signalling, an Arabidopsis plastid CK2 (CKA4) knockout mutant was investigated in terms of the plant response to abscisic acid (ABA) and heat stress. CKA4 expression was upregulated by ABA and heat treatment. The cka4 mutant showed reduced sensitivity to ABA during seed germination and seedling growth, and increased stomatal aperture and leaf water loss with a slightly reduced leaf ABA level. The cka4 mutant was more sensitive to heat stress than the wild-type Columbia-0. The expression levels of a number of genes in the ABA regulatory network were reduced in the cka4 mutant. Many heat-upregulated genes (heat-shock factors and heat-shock proteins) were also reduced in the cka4 mutant. The cka4 mutant showed reduced expression levels of plastid-encoded RNA polymerase target genes (atpB and psbA). CKA4 knockout mutation also resulted in a reduction in expression of some critical genes (PTM, ABI4, and PRS1) involved in retrograde signalling from the chloroplast to the nucleus. Similar results were observed in mutant plants with the knockout mutation in both CKA4 and CKA3, which encodes a nuclear CK2 α3 subunit. CKA3 expression was not responsive to ABA and heat stress. These results suggest that CKA4 is an enhancing factor in abiotic stress signalling through modulating the expression of some molecular players in retrograde signalling.

Fine Scale Genetic Mapping of the TT9 Locus of Arabidopsis thaliana

Numerous transparent testa (tt) or tannin deficient seed (tds) mutants have been isolated in the model plant Arabidopsis thaliana. These mutants are characterized by a reduction or lack of proanthocyanidins in the testa, resulting in pale brown or yellow seeds. Among the tt mutants, all the corresponding genes have been cloned except for tt9 and 13. As a first step towards determining the identity of the tt9 locus, fine scale genetic mapping was carried out using a mapping population resulting from a cross between tt9 plants in the Landsberg erecta (Ler) ecotype background with wild-type Columbia (Col) plants. Several simple sequence length polymorphism markers were developed, and a large population of plants was analyzed. Extensive analysis of this mapping population suggests the tt9 locus is found in a 140,000 base pair region on chromosome three. According to the TAIR 10 genome release for Arabidopsis, there are thirty-four genes in this region. Several approaches were utilized in an effort to identify which of these genes is the TT9 locus, including sequencing of candidate genes, comparison of steady-state mRNA levels of candidate genes in the parental line and the tt9 mutant, and analysis of T-DNA tagged lines. However, the TT9 locus has not yet been identified based on any of these analyses. This suggests that the mutation of interest may lie in a non-coding region of a candidate gene, in a micro-RNA, or other as-yet-unannotated gene in the mapping interval. The fine-scale mapping described here will facilitate future efforts to identify a definitive causative mutation in this elusive locus, for example, by whole genome sequencing.

Growth stimulation in inflorescences of an Arabidopsis tubulin mutant under microgravity conditions in space

Cortical microtubules are involved in plant resistance to hypergravity, but their roles in resistance to 1g gravity are still uncertain. To clarify this point, we cultivated an Arabidopsis α-tubulin 6 mutant (tua6) in the Cell Biology Experiment Facility on the Kibo Module of the International Space Station, and analyzed growth and cell wall mechanical properties of inflorescences. Growth of inflorescence stems was stimulated under microgravity conditions, as compared with ground and on-orbit 1g conditions. The stems were 10-45% longer and their growth rate 15-55% higher under microgravity conditions than those under both 1g conditions. The degree of growth stimulation tended to be higher in the tua6 mutant than the wild-type Columbia. Under microgravity conditions, the cell wall extensibility in elongating regions of inflorescences was significantly higher than the controls, suggesting that growth stimulation was caused by cell wall modifications. No clear differences were detected in any growth or cell wall property between ground and on-orbit 1g controls. These results support the hypothesis that cortical microtubules generally play an important role in plant resistance to the gravitational force.

Theglabra1mutation affects the stomatal patterning ofArabidopsis thalianarosette leaves

Abstract. In Arabidopsis thaliana, the GLABRA 1 (GL1) gene encodes a R2R3-MYB transcription factor that regulates epidermal genes necessary to promote trichome initiation. To determine whether the GL1 gene is also involved in the stomatal patterning of rosette leaves, the epidermal cell pattern of a glabrous mutant (gl1-1) was compared to that of the wild type Columbia ecotype. The gl1-1 mutant was found to have lower stomatal densities and greater distances between stomata as compared to the Columbia ecotype. These findings suggest that stomatal patterning and trichome developmental processes share genetic components to specify cell fate and identity.

Physiological and Mechanical Role of 14-3-3 Lambda in Arabidopsis thaliana during Drought Stress

Climate changes this century due to global warming compel a greater understanding of molecular mechanisms for plant resistance to drought conditions. Plants undergo changes in their anatomy, morphology, mechanical properties and also in their gene expression in response to environmental stress. Using reverse genetics, anatomy and molecular biology approaches, the role of a 14-3-3 protein isoform in the presence of simulated drought conditions in the model organism Arabidopsis thaliana was investigated. The14-3-3 proteins serve the function of signal transduction in eukaryotes and their role in several abiotic and biotic stress conditions have been previously reported. In this study we used 12 isoforms of 14-3-3 that are expressed and well characterized in A. thaliana to determine which isoform of 14-3-3 contributed to drought stress resistance. A phenotypic assay for drought was conducted by growing all the mutants of the isoforms in media with Abscisic acid (ABA). The 14-3-3 lambda (Lambda) mutants showed deficiency in lateral root growth. Further investigations revealed significant differences in ion leakage, absolute water content, water potential and tensile strength in mutants deficient in 14-3-3Lambda versus wild-type (Columbia-0). Leaf anatomy of the 14-3-3Lambda mutants demonstrated a greater cell density and less intracellular airspace compared to Col-0 in well-hydrated and drought conditions. Roots of the 14-3-3Lambda mutants exhibited collapsed epidermal, endodermal and cortical cells in both wet and dry conditions. We thus conclude that 14-3-3Lambda is involved in drought tolerance and contributes in the development of roots and leaves crucial for drought resistance.

The Ethylene Signaling Pathway is Needed to Restrict Root Gall Growth in Arabidopsis after Infection with the Obligate Biotrophic Protist Plasmodiophora brassicae

A possible role for ethylene in the interaction of Arabidopsis thaliana and the obligate biotrophic protist Plasmodiophora brassicae, causing clubroot disease, has been investigated. The ethylene (ET) precursor 1-aminocyclopropane-1-carboxylic acid (ACC) was lower in infected compared to control roots during early time points of the interaction. We therefore analyzed the transcription of selected genes encoding proteins involved in ET synthesis. One gene for ACC oxidase was consistently upregulated in infected roots, whereas transcriptional regulation of other ET biosynthesis genes varied according to the time point during infection. The ET-overproducing mutant eto2 was slightly less susceptible to the clubroot pathogen. The analysis of mutants within the ET signaling pathway showed that they were more susceptible to the clubroot pathogen than wild-type Columbia, that is, they formed larger galls. The findings are discussed in relation to a potential effect of ET on Arabidopsis root infection by P. brassicae.

Fission Yeast HMT1 Lowers Seed Cadmium through Phytochelatin-Dependent Vacuolar Sequestration in Arabidopsis

Much of our dietary uptake of heavy metals is through the consumption of plants. A long-sought strategy to reduce chronic exposure to heavy metals is to develop plant varieties with reduced accumulation in edible tissues. Here, we describe that the fission yeast (Schizosaccharomyces pombe) phytochelatin (PC)-cadmium (Cd) transporter SpHMT1 produced in Arabidopsis (Arabidopsis thaliana) was localized to tonoplast, and enhanced tolerance to and accumulation of Cd2+, copper, arsenic, and zinc. The action of SpHMT1 requires PC substrates, and failed to confer Cd2+ tolerance and accumulation when glutathione and PC synthesis was blocked by L-buthionine sulfoximine, or only PC synthesis is blocked in the cad1-3 mutant, which is deficient in PC synthase. SpHMT1 expression enhanced vacuolar Cd2+ accumulation in wild-type Columbia-0, but not in cad1-3, where only approximately 35% of the Cd2+ in protoplasts was localized in vacuoles, in contrast to the near 100% found in wild-type vacuoles and approximately 25% in those of cad2-1 that synthesizes very low amounts of glutathione and PCs. Interestingly, constitutive SpHMT1 expression delayed root-to-shoot metal transport, and root-targeted expression confirmed that roots can serve as a sink to reduce metal contents in shoots and seeds. These findings suggest that SpHMT1 function requires PCs in Arabidopsis, and it is feasible to promote food safety by engineering plants using SpHMT1 to decrease metal accumulation in edible tissues.

Expression of enzymes involved in chlorophyll catabolism in Arabidopsis is light controlled.

We found that the levels of mRNA of two enzymes involved in chlorophyll catabolism in Arabidopsis (Arabidopsis thaliana), products of two chlorophyllase genes, AtCLH1 and AtCLH2, dramatically increase (by almost 100- and 10-fold, respectively) upon illumination with white light. The measurements of photosystem II quantum efficiency in 3-(3,4-dichlorophenyl)-1,1-dimethylurea-inhibited leaves show that their expression is not related to photosynthesis but mediated by photoreceptors. To identify the photoreceptors involved, we used various light treatments and Arabidopsis photoreceptor mutants (cry1, cry2, cry1cry2, phot1, phot2, phot1phot2, phyA phyB, phyAphyB). In wild-type Columbia, the amount of transcripts of both genes increase after white-light irradiation but their expression profile and the extent of regulation differ considerably. Blue and red light is active in the case of AtCLH1, whereas only blue light raises the AtCLH2 mRNA level. The fundamental difference is the extent of up-regulation, higher by one order of magnitude in AtCLH1. Both blue and red light is active in the induction of AtCLH1 expression in all mutants, pointing to a complex control network and redundancy between photoreceptors. The blue-specific up-regulation of the AtCLH2 transcript is mediated by cryptochromes and modulated by phototropin1 and phytochromes. Individually darkened leaves were used to test the effects of senescence on the expression of AtCLH1 and AtCLH2. The expression profile of AtCLH1 remains similar to that found in nonsenescing leaves up to 5 d after darkening. In contrast, the light induction of AtCLH2 mRNA declines during dark treatment. These results demonstrate that the expression of enzymes involved in chlorophyll catabolism is light controlled.

FAR-RED INSENSITIVE219 Modulates CONSTITUTIVE PHOTOMORPHOGENIC1 Activity via Physical Interaction to Regulate Hypocotyl Elongation in Arabidopsis

FAR-RED INSENSITIVE219 (FIN219) in Arabidopsis (Arabidopsis thaliana) is involved in phytochrome A-mediated far-red (FR) light signaling. Previous genetic studies revealed that FIN219 acts as an extragenic suppressor of CONSTITUTIVE PHOTOMORPHOGENIC1 (COP1). However, the molecular mechanism underlying the suppression of COP1 remains unknown. Here, we used a transgenic approach to study the regulation of COP1 by FIN219. Transgenic seedlings containing ectopic expression of the FIN219 amino (N)-terminal domain in wild-type Columbia (named NCox for the expression of the N-terminal coiled-coil domain and NTox for the N-terminal 300-amino acid region) exhibited a dominant-negative long-hypocotyl phenotype under FR light, reflected as reduced photomorphogenic responses and altered levels of COP1 and ELONGATED HYPOCOTYL5 (HY5). Yeast two-hybrid, pull-down, and bimolecular fluorescence complementation assays revealed that FIN219 could interact with the WD-40 domain of COP1 and with its N-terminal coiled-coil domain through its carboxyl-terminal domain. Further in vivo coimmunoprecipitation study confirms that FIN219 interacts with COP1 under continuous FR light. Studies of the double mutant fin219-2/cop1-6 indicated that HY5 stability requires FIN219 under darkness and FR light. Moreover, FIN219 levels positively regulated by phytochrome A can modulate the subcellular location of COP1 and are differentially regulated by various fluence rates of FR light. We conclude that the dominant-negative long-hypocotyl phenotype conferred by NCox and NTox in a wild-type background was caused by the misregulation of COP1 binding with the carboxyl terminus of FIN219. Our data provide a critical mechanism controlling the key repressor COP1 in response to FR light.

Ratio-Based Analysis of Differential mRNA Processing and Expression of a Polyadenylation Factor Mutant pcfs4 Using Arabidopsis Tiling Microarray

BackgroundAlternative polyadenylation as a mechanism in gene expression regulation has been widely recognized in recent years. Arabidopsis polyadenylation factor PCFS4 was shown to function in leaf development and in flowering time control. The function of PCFS4 in controlling flowering time was correlated with the alternative polyadenylation of FCA, a flowering time regulator. However, genetic evidence suggested additional targets of PCFS4 that may mediate its function in both flowering time and leaf development.Methodology/Principal FindingsTo identify further targets, we investigated the whole transcriptome of a PCFS4 mutant using Affymetrix Arabidopsis genomic tiling 1.0R array and developed a data analysis pipeline, termed RADPRE (Ratio-based Analysis of Differential mRNA Processing and Expression). In RADPRE, ratios of normalized probe intensities between wild type Columbia and a pcfs4 mutant were first generated. By doing so, one of the major problems of tiling array data—variations caused by differential probe affinity—was significantly alleviated. With the probe ratios as inputs, a hierarchy of statistical tests was carried out to identify differentially processed genes (DPG) and differentially expressed genes (DEG). The false discovery rate (FDR) of this analysis was estimated by using the balanced random combinations of Col/pcfs4 and pcfs4/Col ratios as inputs. Gene Ontology (GO) analysis of the DPGs and DEGs revealed potential new roles of PCFS4 in stress responses besides flowering time regulation.Conclusion/SignificanceWe identified 68 DPGs and 114 DEGs with FDR at 1% and 2%, respectively. Most of the 68 DPGs were subjected to alternative polyadenylation, splicing or transcription initiation. Quantitative PCR analysis of a set of DPGs confirmed that most of these genes were truly differentially processed in pcfs4 mutant plants. The enriched GO term “regulation of flower development” among PCFS4 targets further indicated the efficacy of the RADPRE pipeline. This simple but effective program is available upon request.

CESTA, a positive regulator of brassinosteroid biosynthesis

Brassinosteroids (BRs) are steroid hormones that are essential for the development of plants. A tight control of BR homeostasis is vital for modulating their impact on growth responses. Although it is recognized that the rapid adaptation of de novo synthesis has a key role in adjusting required BR levels, our knowledge of the mechanisms governing feedback control is limited. In this study, we identify the transcription factor CESTA as a regulator of BR biosynthesis. ces-D was isolated in a screen of Arabidopsis mutants by BR over-accumulation phenotypes. Loss-of-function analysis and the use of a dominant repressor version revealed functional overlap among CESTA and its homologues and confirmed the role of CESTA in the positive control of BR-biosynthetic gene expression. We provide evidence that CESTA interacts with its homologue BEE1 and can directly bind to a G-box motif in the promoter of the BR biosynthesis gene CPD. Moreover, we show that CESTA subnuclear localization is BR regulated and discuss a model, in which CESTA interplays with BEE1 to control BR biosynthesis and other BR responses.

A GlutathioneS-Transferase Regulated by Light and Hormones Participates in the Modulation of Arabidopsis Seedling Development

Glutathione S-transferases (GSTs) have been well documented to be involved in diverse aspects of biotic and abiotic stresses, especially detoxification processes. Whether they regulate plant development remains unclear. Here, we report on our isolation by reverse transcription-polymerase chain reaction of a plant GST, AtGSTU17, from Arabidopsis (Arabidopsis thaliana) and demonstrate that its expression is regulated by multiple photoreceptors, especially phytochrome A (phyA) under all light conditions. Further physiological studies indicated that AtGSTU17 participates in various aspects of seedling development, including hypocotyl elongation, anthocyanin accumulation, and far-red light-mediated inhibition of greening with a requirement of functional phyA. The loss-of-function mutant of AtGSTU17 (atgstu17) resulted in reduced biomass of seedlings and number of lateral roots in the presence of auxin, as well as insensitivity to abscisic acid (ABA)-mediated inhibition of root elongation, with similarity to different phyA mutant alleles. Moreover, the root phenotype conferred by atgstu17 was reflected by histochemical β-glucuronidase staining of AtGSTU17 promoter activity with the addition of auxin or ABA. Further microarray analysis of wild-type Columbia and atgstu17 seedlings treated with far-red irradiation or ABA revealed that AtGSTU17 might modulate hypocotyl elongation by positively regulating some light-signaling components and negatively regulating a group of auxin-responsive genes and modulate root development by negatively controlling an auxin transport protein in the presence of ABA. Therefore, our data reveal that AtGSTU17 participates in light signaling and might modulate various aspects of Arabidopsis development by affecting glutathione pools via a coordinated regulation with phyA and phytohormones.