Xuhui Hong

ABO3, a WRKY transcription factor, mediates plant responses to abscisic acid and drought tolerance in Arabidopsis

The biological functions of WRKY transcription factors in plants have been widely studied, but their roles in abiotic stress are still not well understood. We isolated an ABA overly sensitive mutant, abo3, which is disrupted by a T-DNA insertion in At1g66600 encoding a WRKY transcription factor AtWRKY63. The mutant was hypersensitive to ABA in both seedling establishment and seedling growth. However, stomatal closure was less sensitive to ABA, and the abo3 mutant was less drought tolerant than the wild type. Northern blot analysis indicated that the expression of the ABA-responsive transcription factor ABF2/AREB1 was markedly lower in the abo3 mutant than in the wild type. The abo3 mutation also reduced the expression of stress-inducible genes RD29A and COR47, especially early during ABA treatment. ABO3 is able to bind the W-box in the promoter of ABF2in vitro. These results uncover an important role for a WRKY transcription factor in plant responses to ABA and drought stress.

Overexpression of SOS (Salt Overly Sensitive) Genes Increases Salt Tolerance in Transgenic Arabidopsis

Soil salinity is a major abiotic stress that decreases plant growth and productivity. Recently, it was reported that plants overexpressing AtNHX1 or SOS1 have significantly increased salt tolerance. To test whether overexpression of multiple genes can improve plant salt tolerance even more, we produced six different transgenic Arabidopsis plants that overexpress AtNHX1, SOS3, AtNHX1+SOS3, SOS1, SOS2+SOS3, or SOS1+SOS2+SOS3. Northern blot analyses confirmed the presence of high levels of the relevant gene transcripts in transgenic plants. Transgenic Arabidopsis plants overexpressing AtNHX1 alone did not present any significant increase in salt tolerance, contrary to earlier reports. We found that transgenic plants overexpressing SOS3 exhibit increased salt tolerance similar to plants overexpressing SOS1. Moreover, salt tolerance of transgenic plants overexpressing AtNHX1+SOS3, SOS2+SOS3, or SOS1+SOS2+SOS3, respectively, appeared similar to the tolerance of transgenic plants overexpressing either SOS1 or SOS3 alone.

ABA overly-sensitive 5 (ABO5), encoding a pentatricopeptide repeat protein required for cis-splicing of mitochondrial nad2 intron 3, is involved in the abscisic acid response in Arabidopsis.

To study the molecular mechanism of abscisic acid (ABA) regulation of root development, we screened the root growth of Arabidopsis mutants for sensitivity to ABA. ABA overly-sensitive 5 (ABO5/At1g51965) was identified, and was determined to encode a pentatricopeptide repeat protein required for cis-splicing of mitochondrial nad2 intron 3 (nad2 is one subunit in complex I). Under constant light conditions (24-h light/0-h dark photoperiod), abo5 mutants exhibited various phenotypes and expressed lower transcripts of stress-inducible genes, such as RD29A, COR47 and ABF2, and photosynthesis-related genes proton gradient regulation 5 (PGR5) and PGR5-likephotosynthetic phenotype (PGRL1), but higher levels of nuclear-encoded genes alternative oxidase 1a (AOX1a) and oxidative signal-inducible 1 (OXI1). Prolonged ABA treatment increased the expression of the cox2 gene in complex IV and nad genes in complex I to a higher level than no ABA treatment in the wild type, but only to a moderate level in abo5, probably because abo5 already expressed high levels of mitochondrial-encoded cox2 and nad genes under no ABA treatment. More H(2) O(2) accumulated in the root tips of abo5 than in the wild type, and H(2) O(2) accumulation was further enhanced by ABA treatment. However, these growth phenotypes and gene-expression defects were attenuated by growing abo5 plants under short-day conditions (12-h light/12-h dark photoperiod). Our results indicate that ABO5 is important in the plant response to ABA.

Mutations in ABO1/ELO2, a Subunit of Holo-Elongator, Increase Abscisic Acid Sensitivity and Drought Tolerance in Arabidopsis thaliana

ABSTRACT The phytohormone abscisic acid (ABA) plays an important role in modulating plant growth, development, and stress responses. In a genetic screen for mutants with altered drought stress responses, we identified an ABA-overly sensitive mutant, the abo1 mutant, which showed a drought-resistant phenotype. The abo1 mutation enhances ABA-induced stomatal closing and increases ABA sensitivity in inhibiting seedling growth. abo1 mutants are more resistant to oxidative stress than the wild type and show reduced levels of transcripts of several stress- or ABA-responsive genes. Interestingly, the mutation also differentially modulates the development and growth of adjacent guard cells. Map-based cloning identified ABO1 as a new allele of ELO2, which encodes a homolog of Saccharomyces cerevisiae Iki3/Elp1/Tot1 and human IκB kinase-associated protein. Iki3/Elp1/Tot1 is the largest subunit of Elongator, a multifunctional complex with roles in transcription elongation, secretion, and tRNA modification. Ecotopic expression of plant ABO1/ELO2 in a tot1/elp1Δ yeast Elongator mutant complements resistance to zymocin, a yeast killer toxin complex, indicating that ABO1/ELO2 substitutes for the toxin-relevant function of yeast Elongator subunit Tot1/Elp1. Our results uncover crucial roles for ABO1/ELO2 in modulating ABA and drought responses in Arabidopsis thaliana.

Auxin Response Factor2 (ARF2) and Its Regulated Homeodomain Gene HB33 Mediate Abscisic Acid Response in Arabidopsis

The phytohormone abscisic acid (ABA) is an important regulator of plant development and response to environmental stresses. In this study, we identified two ABA overly sensitive mutant alleles in a gene encoding Auxin Response Factor2 (ARF2). The expression of ARF2 was induced by ABA treatment. The arf2 mutants showed enhanced ABA sensitivity in seed germination and primary root growth. In contrast, the primary root growth and seed germination of transgenic plants over-expressing ARF2 are less inhibited by ABA than that of the wild type. ARF2 negatively regulates the expression of a homeodomain gene HB33, the expression of which is reduced by ABA. Transgenic plants over-expressing HB33 are more sensitive, while transgenic plants reducing HB33 by RNAi are more resistant to ABA in the seed germination and primary root growth than the wild type. ABA treatment altered auxin distribution in the primary root tips and made the relative, but not absolute, auxin accumulation or auxin signal around quiescent centre cells and their surrounding columella stem cells to other cells stronger in arf2-101 than in the wild type. These results indicate that ARF2 and HB33 are novel regulators in the ABA signal pathway, which has crosstalk with auxin signal pathway in regulating plant growth.

Epigenetic regulation, somatic homologous recombination, and abscisic acid signaling are influenced by DNA polymerase mutation in Arabidopsis.

Based on abscisic acid (ABA) inhibition of seed germination and seedling growth assays, we isolated an ABA overly sensitive mutant (abo4-1) caused by a mutation in the Arabidopsis thaliana POL2a/TILTED1(TIL1) gene encoding a catalytic subunit of DNA polymerase ϵ. The dominant, ABA-insensitive abi1-1 or abi2-1 mutations suppressed the ABA hypersensitivity of the abo4-1 mutant. The abo4/til1 mutation reactivated the expression of the silenced Athila retrotransposon transcriptional silent information (TSI) and the silenced 35S-NPTII in the ros1 mutant and increased the frequency of somatic homologous recombination (HR) ∼60-fold. ABA upregulated the expression of TSI and increased HR in both the wild type and abo4-1. MEIOTIC RECOMBINATION11 and GAMMA RESPONSE1, both of which are required for HR and double-strand DNA break repair, are expressed at higher levels in abo4-1 and are enhanced by ABA, while KU70 was suppressed by ABA. abo4-1 mutant plants are sensitive to UV-B and methyl methanesulfonate and show constitutive expression of the G2/M-specific cyclin CycB1;1 in meristems. The abo4-1 plants were early flowering with lower expression of FLOWER LOCUS C and higher expression of FLOWER LOCUS T and changed histone modifications in the two loci. Our results suggest that ABO4/POL2a/TIL1 is involved in maintaining epigenetic states, HR, and ABA signaling in Arabidopsis.

ROR1/RPA2A, a Putative Replication Protein A2, Functions in Epigenetic Gene Silencing and in Regulation of Meristem Development in Arabidopsis

We screened for suppressors of repressor of silencing1 (ros1) using the silenced 35S promoter-neomycin phosphotransferase II (Pro35S:NPTII) gene as a marker and identified two allelic mutants, ror1-1 and ror1-2 (for suppressor of ros1). Map-based cloning revealed that ROR1 encodes a 31-kD protein similar to DNA replication protein A2 (RPA2A). Mutations in ROR1 reactivate the silenced Pro35S:NPTII gene but not RD29A promoter-luciferase in the ros1 mutant. DNA methylation in rDNA, centromeric DNA, and RD29A promoter regions is not affected by ror1. However, chromatin immunoprecipitation data suggest that histone H3 acetylation is increased and histone H3K9 dimethylation is decreased in the 35S promoter in the ror1 ros1 mutant compared with ros1. These results indicate that release of silenced Pro35S:NPTII by ror1 mutations is independent of DNA methylation. ROR1/RPA2A is strongly expressed in shoot and root meristems. Mutations in ROR1/RPA2A affect cell division in meristems but not final cell sizes. Our work suggests important roles of ROR1/RPA2A in epigenetic gene silencing and in the regulation of plant development.

The protein kinase TOUSLED is required for maintenance of transcriptional gene silencing in Arabidopsis

TOUSLED‐like kinases (TLKs) are highly conserved in plants and animals, but direct evidence linking TLKs and transcriptional gene silencing is lacking. We isolated two new alleles of TOUSLED (TSL). Mutations of TSL in ros1 reactivate the transcriptionally silent 35S‐NPTII transgene and the transcriptionally silent endogenous loci TSI (TRANSCRIPTIONAL SILENCING INFORMATION). Chromatin immunoprecipitation (ChIP) analysis shows that histone H3Lys9 dimethylation is decreased in the reactivated transgene and endogenous TSI loci in the tsl ros1 mutant. However, there is no change in DNA methylation in the affected loci. Western blot and ChIP assay suggest that TSL might not be responsible for histone H3Ser10 phosphorylation. The tsl seedlings were more sensitive to DNA damage reagent methyl methanesulphonate and UV‐B light. Our results provide direct evidence for a crucial role of the TOUSLED protein kinase in the maintenance of transcriptional gene silencing in some genomic regions in a DNA‐methylation‐independent manner in Arabidopsis.