Detection and analysis of UV-induced mutations in the chromosomal DNA of Arabidopsis.

Under natural conditions, plants are exposed to solar ultraviolet (UV) radiation, which damages chromosomal DNA. Although plant responses to UV-induced DNA damage have recently been elucidated in detail, revealing a set of DNA repair mechanisms and translesion synthesis (TLS), limited information is currently available on UV-induced mutations in plants. We previously reported the development of a supF-based system for the detection of a broad spectrum of mutations in the chromosomal DNA of Arabidopsis. In the present study, we used this system to investigate UV-induced mutations in plants. The irradiation of supF-transgenic plants with UV-C (500 and 1000 J/m2) significantly increased mutation frequencies (26- and 45-fold, respectively). G:C to A:T transitions (43-67% of base substitutions) dominated in the mutation spectrum and were distributed throughout single, tandem, and multiple base substitutions. Most of these mutations became undetectable with the subsequent illumination of UV-irradiated plants with white light for photoreactivation (PR). These results indicated that not only G:C to A:T single base substitutions, but also tandem and multiple base substitutions were caused by two major UV-induced photoproducts, cyclobutane-type pyrimidine dimers (CPDs) and pyrimidine (6-4) pyrimidone photoproducts (6-4 PPs). In contrast, a high proportion of A:T to T:A transversions (56% of base substitutions) was a characteristic feature of the mutation spectrum obtained from photoreactivated plants. These results define the presence of the characteristic feature of UV-induced mutations, and provide insights into DNA repair mechanisms in plants.

Development of a supF-based mutation-detection system in the extreme thermophile Thermus thermophilus HB27.

Thermus thermophilus (T. thermophilus) HB27 is an extreme thermophile that grows optimally at 65-72 °C. Heat-induced DNA lesions are expected to occur at a higher frequency in the genome of T. thermophilus than in those of mesophiles; however, the mechanisms underlying the maintenance of genome integrity at high temperatures remain poorly understood. The study of mutation spectra has become a powerful approach to understanding the molecular mechanisms responsible for DNA repair and mutagenesis in mesophilic species. Therefore, we developed a supF-based system to detect a broad spectrum of mutations in T. thermophilus. This system was validated by measuring spontaneous mutations in the wild type and a udgA, B double mutant deficient in uracil-DNA glycosylase (UDG) activity. We found that the mutation frequency of the udgA, B strain was 4.7-fold higher than that of the wild type and G:C→A:T transitions dominated, which was the most reasonable for the mutator phenotype associated with the loss of UDG function in T. thermophilus. These results show that this system allowed for the rapid analysis of mutations in T. thermophilus, and may be useful for studying the molecular mechanisms responsible for DNA repair and mutagenesis in this extreme thermophile.

Cre/lox-based multiple markerless gene disruption in the genome of the extreme thermophile Thermus thermophilus.

Markerless gene-disruption technology is particularly useful for effective genetic analyses of Thermus thermophilus (T. thermophilus), which have a limited number of selectable markers. In an attempt to develop a novel system for the markerless disruption of genes in T. thermophilus, we applied a Cre/lox system to construct a triple gene disruptant. To achieve this, we constructed two genetic tools, a loxP-htk-loxP cassette and cre-expressing plasmid, pSH-Cre, for gene disruption and removal of the selectable marker by Cre-mediated recombination. We found that the Cre/lox system was compatible with the proliferation of the T. thermophilus HB27 strain at the lowest growth temperature (50 °C), and thus succeeded in establishing a triple gene disruptant, the (∆TTC1454::loxP, ∆TTC1535KpnI::loxP, ∆TTC1576::loxP) strain, without leaving behind a selectable marker. During the process of the sequential disruption of multiple genes, we observed the undesired deletion and inversion of the chromosomal region between multiple loxP sites that were induced by Cre-mediated recombination. Therefore, we examined the effects of a lox66-htk-lox71 cassette by exploiting the mutant lox sites, lox66 and lox71, instead of native loxP sites. We successfully constructed a (∆TTC1535::lox72, ∆TTC1537::lox72) double gene disruptant without inducing the undesired deletion of the 0.7-kbp region between the two directly oriented lox72 sites created by the Cre-mediated recombination of the lox66-htk-lox71 cassette. This is the first demonstration of a Cre/lox system being applicable to extreme thermophiles in a genetic manipulation. Our results indicate that this system is a powerful tool for multiple markerless gene disruption in T. thermophilus.

The NAC Transcription Factor Gene OsY37 (ONAC011) Promotes Leaf Senescence and Accelerates Heading Time in Rice.

Leaf senescence is an important physiological process involving the degradation of a number of metabolites and their remobilization to new reproductive and storage organs. NAC (NAM, ATAF, and CUC) transcription factors are reported as important regulators of the senescence process. Here, we describe the identification and functional characterization of the NAC transcription factor gene, OsY37 (Oryza sativa Yellow37, ONAC011) obtained from Oryza sativa cv. indica, and japonica. We created transgenic plants expressing the OsY37 gene under the control of a strong and constitutive CaMV35S promoter. The resulting transgenic plants overexpressing OsY37 gene showed early heading and precocious senescence phenotype of flag leaves compared with wild-type plants. By contrast, blocking the function of this gene via RNAi (RNA interference) and CRES-T (Chimeric Repressor Silencing Technology) technology, delayed both heading time and leaf senescence. Furthermore, knockdown of OsY37 expression caused dwarfism and high accumulation of chlorophyll during the vegetative phase. Irrespective of early or delayed senescence, transgenic plants showed reduced grain yields. Our results indicate that OsY37 acts as a positive regulator of heading and senescence during the reproductive phase in rice. In addition, OsY37 may be involved in plant development and grain yield.

Induction of a dwarf phenotype with IBH1 may enable increased production of plant-made pharmaceuticals in plant factory conditions.

Year-round production in a contained, environmentally controlled 'plant factory' may provide a cost-effective method to produce pharmaceuticals and other high-value products. However, cost-effective production may require substantial modification of the host plant phenotype; for example, using dwarf plants can enable the growth of more plants in a given volume by allowing more plants per shelf and enabling more shelves to be stacked vertically. We show here that the expression of the chimeric repressor for Arabidopsis AtIBH1 (P35S:AtIBH1SRDX) in transgenic tobacco plants (Nicotiana tabacum) induces a dwarf phenotype, with reduced cell size. We estimate that, in a given volume of cultivation space, we can grow five times more AtIBH1SRDX plants than wild-type plants. Although, the AtIBH1SRDX plants also showed reduced biomass compared with wild-type plants, they produced about four times more biomass per unit of cultivation volume. To test whether the dwarf phenotype affects the production of recombinant proteins, we expressed the genes for anti-hepatitis B virus antibodies (anti-HBs) in tobacco plants and found that the production of anti-HBs per unit fresh weight did not significantly differ between wild-type and AtIBH1SRDX plants. These data indicate that P35S:AtIBH1SRDX plants produced about fourfold more antibody per unit of cultivation volume, compared with wild type. Our results indicate that AtIBH1SRDX provides a useful tool for the modification of plant phenotype for cost-effective production of high-value products by stably transformed plants in plant factory conditions.

Construction of a supF-based system for detection of mutations in the chromosomal DNA of Arabidopsis.

The factors maintaining genomic integrity, which have been studied in detail in other species, have yet to be investigated in plants. Recent progress in gene-silencing technology has made it possible to produce transgenic plants with loss-of-function phenotypes for the effective analysis of these factors, even with the high redundancy of genes in plants. Therefore, a mutation-detection system for plants is necessary to estimate the biological function of a target gene for mutation frequencies and spectra. Here, we reported the development of a novel system to analyze mutations in the chromosomal DNA of plants. The supF gene of E. coli was used as a target for the mutation because it was possible to detect all mutational base changes. Based on the plasmid pTN30, which carries supF, we constructed a binary Ti vector for its introduction to Arabidopsis genomes. The system was validated by measuring mutations in both non-treated and mutagen-treated transgenic plants. DNA fragments including pTN30 were rescued from the plants, and introduced into E. coli KS40/pOF105 to isolate the supF mutant clones conferring both nalidixic acid and streptomycin resistance on transformants. We found that the mutation frequency was approximately three times higher with the ethyl methanesulfonate (EMS) treatment than without it and G:C to A:T transitions dominated, which was the most reasonable mutation induced by EMS. These results show that this system allowed for the rapid analysis of mutations in plants, and may be useful for analyzing plant genes related to the functions of genomic stability and monitoring environmental genotoxic substances.

Aberrant vegetative and reproductive development by overexpression and lethality by silencing of OsHAP3E in rice.

We generated transgenic rice plants overexpressing OsHAP3E which encodes a subunit of a CCAAT-motif binding HAP complex. The OsHAP3E-overexpressing plants showed various abnormal morphologies both in their vegetative and reproductive phases. The OsHAP3E-overexpressing plants were dwarf with erected leaves and similar to brassinosteroid mutants in the vegetative phase. In the reproductive phase, dense panicle was developed, and occasionally successive generation of lateral rachises and formation of double flowers were observed. These phenotypes indicate association of OsHAP3E with determination of floral meristem identity. On the other hand, repression of OsHAP3E by RNAi or by overexpressing chimeric repressor fusion constructs brought about lethality to transformed cells, and almost no transformant was obtained. This suggests that the OsHAP3E function is essential for rice cells. Altogether, our loss-of-function and gain-of-function analyses suggest that OsHAP3E plays important pleiotropic roles in vegetative and reproductive development or basic cellular processes in rice.

The AP2/ERF transcription factor WIND1 controls cell dedifferentiation in Arabidopsis.

Many multicellular organisms have remarkable capability to regenerate new organs after wounding. As a first step of organ regeneration, adult somatic cells often dedifferentiate to reacquire cell proliferation potential, but mechanisms underlying this process remain unknown in plants. Here we show that an AP2/ERF transcription factor, WOUND INDUCED DEDIFFERENTIATION 1 (WIND1), is involved in the control of cell dedifferentiation in Arabidopsis. WIND1 is rapidly induced at the wound site, and it promotes cell dedifferentiation and subsequent cell proliferation to form a mass of pluripotent cells termed callus. We further demonstrate that ectopic overexpression of WIND1 is sufficient to establish and maintain the dedifferentiated status of somatic cells without exogenous auxin and cytokinin, two plant hormones that are normally required for cell dedifferentiation. In vivo imaging of a synthetic cytokinin reporter reveals that wounding upregulates the B-type ARABIDOPSIS RESPONSE REGULATOR (ARR)-mediated cytokinin response and that WIND1 acts via the ARR-dependent signaling pathway to promote cell dedifferentiation. This study provides novel molecular insights into how plants control cell dedifferentiation in response to wounding.

Loss of function of the HSFA9 seed longevity program.

Gain of function approaches that have been published by our laboratory determined that HSFA9 (Heat Shock Factor A9) activates a genetic program contributing to seed longevity and to desiccation tolerance in plant embryos. We now evaluate the role(s) of HSFA9 by loss of function using different modified forms of HaHSFA9 (sunflower HSFA9), which were specifically overexpressed in seeds of transgenic tobacco. We used two inactive forms (M1, M2) with deletion or mutation of the transcription activation domain of HaHSFA9, and a third form (M3) with HaHSFA9 converted to a potent active repressor by fusion of the SRDX motif. The three forms showed similar protein accumulation in transgenic seeds; however, only HaHSFA9-SRDX showed a highly significant reduction of seed longevity, as determined by controlled deterioration tests, a rapid seed ageing procedure. HaHSFA9-SRDX impaired the genetic program controlled by the tobacco HSFA9, with a drastic reduction in the accumulation of seed heat shock proteins (HSPs) including seed-specific small HSP (sHSP) belonging to cytosolic (CI, CII) classes. Despite such effects, the HaHSFA9-SRDX seeds could survive developmental desiccation during embryogenesis and their subsequent germination was not reduced. We infer that the HSFA9 genetic program contributes only partially to seed-desiccation tolerance and longevity.

The Arabidopsis thaliana STYLISH1 protein acts as a transcriptional activator regulating auxin biosynthesis.

The establishment and maintenance of auxin maxima in vascular plants is regulated by auxin biosynthesis and polar intercellular auxin flow. The disruption of normal auxin biosynthesis in mouse-ear cress (Arabidopsis thaliana) leads to severe abnormalities, suggesting that spatiotemporal regulation of auxin biosynthesis is fundamental for normal growth and development. We have shown previously that the induction of the SHORT-INTERNODES/STYLISH (SHI/STY) family member STY1 results in increased transcript levels of the YUCCA (YUC) family member YUC4 and also higher auxin levels and auxin biosynthesis rates in Arabidopsis seedlings. We have also shown previously that SHI/STY family members redundantly affect development of flowers and leaves. Here, we further examine the function of STY1 by analyzing its DNA and protein binding properties. Our results suggest that STY1, and most likely other SHI/STY members, are DNA binding transcriptional activators that target genes encoding proteins mediating auxin biosynthesis. This suggests that the SHI/STY family members are essential regulators of auxin-mediated leaf and flower development. Furthermore, the lack of a shoot apical meristem in seedlings carrying a fusion construct between STY1 and a repressor domain, SRDX, suggests that STY1, and other SHI/STY members, has a role in the formation and/or maintenance of the shoot apical meristem, possibly by regulating auxin levels in the embryo.

Efficient production of male and female sterile plants by expression of a chimeric repressor in Arabidopsis and rice.

Male and female sterile plants are particularly useful for the effective production of commercial hybrid plants and for preventing the diffusion of seeds or pollen grains of genetically modified plants in the open field. In an attempt to create several types of sterile plant by genetic manipulation, we applied our Chimeric REpressor Gene-Silencing Technology (CRES-T) to four transcription factors, namely APETALA3, AGAMOUS, LEAFY and AtMYB26, involved in the regulation of petal and stamen identity, stamen and carpel identity, floral meristem identity and anther dehiscence, respectively, in Arabidopsis. Transgenic plants expressing each chimeric repressor exhibited, at high frequency, a sterile phenotype that resembled the loss-of-function phenotype of each corresponding gene. Furthermore, in the monocotyledonous crop plant 'rice', expression of the chimeric repressor derived from SUPERWOMAN1, the rice orthologue of APETALA3, resulted in the male sterile phenotype with high efficiency. Our results indicate that CRES-T provides a powerful tool for controlling the fertility of both monocots and dicots by exploiting transcription factors that are strongly conserved amongst plants.

AREB1 is a transcription activator of novel ABRE-dependent ABA signaling that enhances drought stress tolerance in Arabidopsis.

ABSCISIC ACID-RESPONSIVE ELEMENT BINDING PROTEIN1 (AREB1) (i.e., ABF2) is a basic domain/leucine zipper transcription factor that binds to the abscisic acid (ABA)-responsive element (ABRE) motif in the promoter region of ABA-inducible genes. Here, we show that expression of the intact AREB1 gene on its own is insufficient to lead to expression of downstream genes under normal growth conditions. To overcome the masked transactivation activity of AREB1, we created an activated form of AREB1 (AREB1DeltaQT). AREB1DeltaQT-overexpressing plants showed ABA hypersensitivity and enhanced drought tolerance, and eight genes with two or more ABRE motifs in the promoter regions in two groups were greatly upregulated: late embryogenesis abundant class genes and ABA- and drought stress-inducible regulatory genes. By contrast, an areb1 null mutant and a dominant loss-of-function mutant of AREB1 (AREB1:RD) with a repression domain exhibited ABA insensitivity. Furthermore, AREB1:RD plants displayed reduced survival under dehydration, and three of the eight greatly upregulated genes were downregulated, including genes for linker histone H1 and AAA ATPase, which govern gene expression and multiple cellular activities through protein folding, respectively. Thus, these data suggest that AREB1 regulates novel ABRE-dependent ABA signaling that enhances drought tolerance in vegetative tissues.

A chimeric AtMYB23 repressor induces hairy roots, elongation of leaves and stems, and inhibition of the deposition of mucilage on seed coats in Arabidopsis.

We reported previously that a chimeric repressor, in which a transcription factor was fused to the EAR motif repression domain, acted as a dominant repressor and suppressed the expression of target genes, such that resultant phenotypes were similar to those associated with loss-of-function alleles. We report here that expression of the chimeric AtMYB23 repressor induced a variety of morphological changes, namely the ectopic formation of root hairs, a short primary root, elongation of leaves and of inflorescence stems, and absence of the accumulation of mucilage on seed coats, in addition to disruption of the development of trichomes. The short primary root and the elongation of leaves and stems appeared to be due to the reduced and enhanced lengthwise expansion, respectively, of epidermal cells. Expression of the GL2 gene, which is involved in the formation of root hairs and the accumulation of mucilage, was suppressed in both the roots and siliques of the transgenic plants. In contrast, the expression of genes related to cell elongation, such as DWF1, SAUR, AQP, AGP15, DET3 and XET-1, was enhanced in leaves of the transgenic plants. Results suggest that the AtMYB23 transcription factor has the molecular function of regulating the development of epidermal cells not only in leaves but also in stems, roots and seeds. We describe that this type of chimeric repressor can be exploited as a useful tool for the functional analysis of redundant transcription factors.

Identification of the minimal repression domain of SUPERMAN shows that the DLELRL hexapeptide is both necessary and sufficient for repression of transcription in Arabidopsis.

We reported previously that the carboxy-terminal 30 amino acids of SUPERMAN (SUPRD) function as a repression domain in Arabidopsis. In this study, we identified the peptide sequences in SUPRD that is both necessary and sufficient for repression of transcription. To our surprise, the hexapeptide DLELRL was sufficient, by itself, to confer the ability to repress transcription on a DNA-binding domain. A database search revealed that there are 32 TFIIIA-type zinc finger proteins in the Arabidopsis genome that contain a hexapeptide sequence similar or identical to that of DLELRL. These peptides acted as repression domains, suggesting that these zinc finger proteins might function as active repressors. Further mutational analysis within DLELRL revealed that an amphiphilic motif composed of six amino acids (XLxLXL) with preferences at the first and fifth positions is necessary and sufficient for strong repression. An assay of positional effects suggested that GAL4DB-DLELRL might function as a short-range repressor. A possible mechanism of the DLELRL-mediated repression is discussed.

A dehydration-induced NAC protein, RD26, is involved in a novel ABA-dependent stress-signaling pathway.

Arabidopsis thaliana RD26 cDNA, isolated from dehydrated plants, encodes a NAC protein. Expression of the RD26 gene was induced not only by drought but also by abscisic acid (ABA) and high salinity. The RD26 protein is localized in the nucleus and its C terminal has transcriptional activity. Transgenic plants overexpressing RD26 were highly sensitive to ABA, while RD26-repressed plants were insensitive. The results of microarray analysis showed that ABA- and stress-inducible genes are upregulated in the RD26-overexpressed plants and repressed in the RD26-repressed plants. Furthermore, RD26 activated a promoter of its target gene in Arabidopsis protoplasts. These results indicate that RD26 functions as a transcriptional activator in ABA-inducible gene expression under abiotic stress in plants.

Circularized chromosome with a large palindromic structure in Streptomyces griseus mutants.

Streptomyces linear chromosomes display various types of rearrangements after telomere deletion, including circularization, arm replacement, and amplification. We analyzed the new chromosomal deletion mutants Streptomyces griseus 301-22-L and 301-22-M. In these mutants, chromosomal arm replacement resulted in long terminal inverted repeats (TIRs) at both ends; different sizes were deleted again and recombined inside the TIRs, resulting in a circular chromosome with an extremely large palindrome. Short palindromic sequences were found in parent strain 2247, and these sequences might have played a role in the formation of this unique structure. Dynamic structural changes of Streptomyces linear chromosomes shown by this and previous studies revealed extraordinary strategies of members of this genus to keep a functional chromosome, even if it is linear or circular.

Chromosomal circularization in Streptomyces griseus by nonhomologous recombination of deletion ends.

Streptomyces linear chromosomes frequently cause deletions at both ends spontaneously or by various mutagenic treatments, and concomitantly display dynamic structural changes such as circularization and arm replacement. We have cloned and sequenced the fusion junctions of circularized chromosomes in two deletion mutants of Streptomyces griseus. No homology and a 1-bp overlap were found between the deletion ends of the mutant chromosomes. Taking this together with previous results, we concluded that chromosomal circularization in Streptomyces occurs by nonhomologous recombination between deletion ends.

Dominant repression of target genes by chimeric repressors that include the EAR motif, a repression domain, in Arabidopsis.

The redundancy of genes for plant transcription factors often interferes with efforts to identify the biologic functions of such factors. We show here that four different transcription factors fused to the EAR motif, a repression domain of only 12 amino acids, act as dominant repressors in transgenic Arabidopsis and suppress the expression of specific target genes, even in the presence of the redundant transcription factors, with resultant dominant loss-of-function phenotypes. Chimeric EIN3, CUC1, PAP1, and AtMYB23 repressors that included the EAR motif dominantly suppressed the expression of their target genes and caused insensitivity to ethylene, cup-shaped cotyledons, reduction in the accumulation of anthocyanin, and absence of trichomes, respectively. This chimeric repressor silencing technology (CRES-T), exploiting the EAR-motif repression domain, is simple and effective and can overcome genetic redundancy. Thus, it should be useful not only for the rapid analysis of the functions of redundant plant transcription factors but also for the manipulation of plant traits via the suppression of gene expression that is regulated by specific transcription factors.

The large linear plasmid pSLA2-L of Streptomyces rochei has an unusually condensed gene organization for secondary metabolism.

The complete nucleotide sequence of the large linear plasmid pSLA2-L in Streptomyces rochei strain 7434AN4 has been determined. pSLA2-L was found to be 210 614 bp long with a GC content of 72.8% and carries 143 open reading frames. It is especially noteworthy that three-quarters of the pSLA2-L DNA is occupied by secondary metabolism-related genes, namely two type I polyketide synthase (PKS) gene clusters for lankacidin and lankamycin, a mithramycin synthase-like type II PKS gene cluster, a carotenoid biosynthetic gene cluster and many regulatory genes. In particular, the lankacidin PKS is unique, because it may be a mixture of modular- and iterative-type PKSs and carries a fusion protein of non-ribosomal peptide synthetase and PKS. It is also interesting that all the homologues of the afsA, arpA, adpA and strR genes in the A-factor regulatory cascade in Streptomyces griseus were found on pSLA2-L, and disruption of the afsA homologue caused non-production of both lankacidin and lankamycin. These results, together with the finding of three possible replication origins at 50-63 kb from the right end, suggest that the present form of pSLA2-L might have been generated by a series of insertions of the biosynthetic gene clusters into the left side of the original plasmid.

Cloning and analysis of the telomere and terminal inverted repeat of the linear chromosome of Streptomyces griseus.

Cloning and sequencing of the telomere of Streptomyces griseus revealed five palindromic sequences in the terminal 116 nucleotides, all of which can make a hairpin loop structure. However, the end sequence cannot form the foldback secondary structure that is common in Streptomyces telomeres and is suggested to be necessary for terminal replication. Both inside ends of the terminal inverted repeat (TIR) were also cloned and sequenced. The results confirmed the size of the TIR to be 24 kb and identified two almost identical open reading frames that might have been involved in the formation of the TIR.