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N H Chua

Publications and source records attributed to N H Chua.

At least 19 recordsLinked to original sources
PubMed2001

LAF1, a MYB transcription activator for phytochrome A signaling.

The photoreceptor phytochrome (phy) A has a well-defined role in regulating gene expression in response to specific light signals. Here, we describe a new Arabidopsis mutant, laf1 (long after far-red light 1) that has an elongated hypocotyl specifically under far-red light. Gene expression studies showed that laf1 has reduced responsiveness to continuous far-red light but retains wild-type responses to other light wavelengths. As far-red light is only perceived by phyA, our results suggest that LAF1 is specifically involved in phyA signal transduction. Further analyses revealed that laf1 is affected in a subset of phyA-dependent responses and the phenotype is more severe at low far-red fluence rates. LAF1 encodes a nuclear protein with strong homology with the R2R3-MYB family of DNA-binding proteins. Experiments using yeast cells identified a transactivation domain in the C-terminal portion of the protein. LAF1 is constitutively targeted to the nucleus by signals in its N-terminal portion, and the full-length protein accumulates in distinct nuclear speckles. This accumulation in speckles is abolished by a point mutation in a lysine residue (K258R), which might serve as a modification site by a small ubiquitin-like protein (SUMO).

Amino Acid Sequence↗
PubMed2001

Inactivation of AtRac1 by abscisic acid is essential for stomatal closure.

Plant water homeostasis is maintained by the phytohormone abscisic acid (ABA), which triggers stomatal pore closure in response to drought stress. We identified the Arabidopsis small guanosine triphosphatase (GTPase) protein AtRac1 as a central component in the ABA-mediated stomatal closure process. ABA treatment induced inactivation of AtRac GTPases and disruption of the guard cell actin cytoskeleton. In contrast, in the ABA-insensitive mutant abi1-1, which is impaired in stomatal closure, neither AtRac inactivation nor actin cytoskeleton disruption was observed on ABA treatment. These observations indicate that AtRac1 inactivation is a limiting step in the ABA-signaling cascade leading to stomatal closure. Consistent with these findings, expression of a dominant-positive mutant of AtRac1 blocked the ABA-mediated effects on actin cytoskeleton and stomatal closure in wild-type plants, whereas expression of a dominant-negative AtRac1 mutant recapitulated the ABA effects in the absence of the hormone. Moreover, the dominant-negative form of AtRac1 could also restore stomatal closure in abi1-1. These results define AtRac1 as a central element for plant adaptation to drought.

Abscisic Acid↗
PubMed2001

A postgermination developmental arrest checkpoint is mediated by abscisic acid and requires the ABI5 transcription factor in Arabidopsis.

Seed dormancy is a trait of considerable adaptive significance because it maximizes seedling survival by preventing premature germination under unfavorable conditions. Understanding how seeds break dormancy and initiate growth is also of great agricultural and biotechnological interest. Abscisic acid (ABA) plays primary regulatory roles in the initiation and maintenance of seed dormancy. Here we report that the basic leucine zipper transcription factor ABI5 confers an enhanced response to exogenous ABA during germination, and seedling establishment, as well as subsequent vegetative growth. These responses correlate with total ABI5 levels. We show that ABI5 expression defines a narrow developmental window following germination, during which plants monitor the environmental osmotic status before initiating vegetative growth. ABI5 is necessary to maintain germinated embryos in a quiescent state thereby protecting plants from drought. As expected for a key player in ABA-triggered processes, ABI5 protein accumulation, phosphorylation, stability, and activity are highly regulated by ABA during germination and early seedling growth.

Abscisic Acid↗
PubMed2001

A plastidic ABC protein involved in intercompartmental communication of light signaling.

Plants perceive light via specialized photoreceptors of which the phytochromes (phyA-E), absorbing far-red (FR) and red light (R) are best understood. Several nuclear and cytoplasmic proteins have been characterized whose deficiencies lead to changes in light-dependent morphological responses and gene expression. However, no plastid protein has yet been identified to play a role in phytochrome signal transduction. We have isolated a new Arabidopsis mutant, laf (long after FR) 6, with reduced responsiveness preferentially toward continuous FR light. The disrupted gene in laf6 encodes a novel plant ATP-binding-cassette (atABC1) protein of 557 amino acids with high homology to ABC-like proteins from lower eukaryotes. In contrast to lower eukaryotic ABCs, however, atABC1 contains an N-terminal transit peptide, which targets it to chloroplasts. atABC1 deficiency in laf6 results in an accumulation of the chlorophyll precursor protoporphyrin IX and in attenuation of FR-regulated gene expression. The long hypocotyl phenotype of laf6 and the accumulation of protoporphyrin IX in the mutant can be recapitulated by treating wild-type (WT) seedlings with flumioxazin, a protoporphyrinogen IX oxidase (PPO) inhibitor. Moreover, protoporphyrin IX accumulation in flumioxazin-treated WT seedlings can be reduced by overexpression of atABC1. Consistent with the notion that ABC proteins are involved in transport, these observations suggest that functional atABC1 is required for the transport and correct distribution of protoporphyrin IX, which may act as a light-specific signaling factor involved in coordinating intercompartmental communication between plastids and the nucleus.

ATP-Binding Cassette Transporters↗
PubMed2001

Molecular identification and characterization of the Arabidopsis AtADF1, AtADFS and AtADF6 genes.

Actin depolymerizing factor (ADF) is a key regulator of the organization of the actin cytoskeleton during various cellular activities. We found that ADF genes in Arabidopsis form a large family consisting of at least nine members, four of which were cloned and sequenced in this study. Comparison of genomic and cDNA sequences showed that the AtADF1, AtADF5, and AtADF6 genes all contain two introns at conserved positions. Analysis of transgenic Arabidopsis plants carrying promoter-GUS fusion constructs revealed that AtADF1 and AtADF6 are expressed in the vascular tissues of all organs, whereas expression of AtADF5 is restricted to the root tip meristem. GFP-AtADFI, GFP-AtADF5, and GFP-AtADF6 fusion proteins were found to bind to actin filaments in vivo, and to reorganize the actin cytoskeleton when transiently expressed in plant cells.

Actin Depolymerizing Factors↗
PubMed2001

Chemical-regulated, site-specific DNA excision in transgenic plants.

We have developed a chemical-inducible, site-specific DNA excision system in transgenic Arabidopsis plants mediated by the Cre/loxP DNA recombination system. Expression of the Cre recombinase was tightly controlled by an estrogen receptor-based fusion transactivator XVE. Upon induction by beta-estradiol, sequences encoding the selectable marker, Cre, and XVE sandwiched by two loxP sites were excised from the Arabidopsis genome, leading to activation of the downstream GFP (green fluorescent protein) reporter gene. Genetic and molecular analyses indicated that the system is tightly controlled, showing high-efficiency inducible DNA excision in all 19 transgenic events tested with either single or multiple T-DNA insertions. The system provides a highly reliable method to generate marker-free transgenic plants after transformation through either organogenesis or somatic embryogenesis.

Arabidopsis↗
PubMed2001

Reduced expression of alpha-tubulin genes in Arabidopsis thaliana specifically affects root growth and morphology, root hair development and root gravitropism.

Different alpha-tubulin cDNA sequences fused in an antisense orientation to a CaMV 35S promoter were introduced into Arabidopsis thaliana plants. Several independent transgenic lines that showed a moderate but clear reduction of alpha-tubulin gene expression (TUA6/AS lines) were obtained and phenotypically characterized. Although no apparent abnormalities were detected in the aerial parts of TUA6/AS plants, root development was severely affected. Cells in TUA6/AS root tips were found to contain aberrant microtubular structures, to expand abnormally and to be unable to undergo regular cell division. These cellular defects caused a dramatic radial expansion of the root tip and inhibited root elongation. In addition, TUA6/AS roots displayed ectopic formation of root hairs, root hair branching and a reduced ability to respond to gravitropic challenges. Our results contribute to an improved understanding of the different roles microtubules play during root development and demonstrate that reverse genetics is a powerful tool to analyze cytoskeletal functions during plant organogenesis.

Arabidopsis↗
PubMed2001

The phytochrome A specific signaling component PAT3 is a positive regulator of Arabidopsis photomorphogenesis.

Phytochrome A plays a major role in early seedling development by triggering the transition from etiolated growth to greening. Seedlings germinated under constant far-red (FR) light show a partially de-etiolated phenotype that is not seen in phyA mutants. This phytochrome A specific response was used to screen a population of T-DNA mutagenized Arabidopsis seedlings. One mutant line, pat3 (phytochrome A signal transduction3), which showed no inhibition of hypocotyl elongation under FR light conditions and no FR-induced killing response, contained a T-DNA insertion in a 609-bp ORF. The recessive mutation co-segregated with the T-DNA resistance marker and could be allelic to fhy1. A 2,248-bp genomic fragment of the PAT3 locus can complement the pat3 mutant phenotype. PAT3 transcript peaked 3 d after germination and was downregulated by light. PAT3 has no significant homology to any known protein and shows no preferential cellular localization. The protein can activate transcription in yeast when fused to the GAL4 DNA-binding domain. Our results show that PAT3 is a positive regulator of phytochrome A signal transduction.

Alleles↗
PubMed2001

Overexpression of Arabidopsis ESR1 induces initiation of shoot regeneration.

Functional screening of an Arabidopsis cDNA library enabled the identification of a novel cDNA, ESR1 (for Enhancer of Shoot Regeneration), that can confer cytokinin-independent shoot formation when overexpressed in Arabidopsis root explants. Neither callus induction nor root formation was affected by ESR1 overexpression. ESR1 encodes a putative transcription factor with an AP2/EREBP domain. Surprisingly, ESR1 overexpression also greatly increased the efficiency of shoot regeneration from root explants in the presence of cytokinin, with a shift in the optimal cytokinin concentration required for this process. The effects of ESR1 overexpression on shoot regeneration are synergistic with those of cytokinin. Overexpression of ESR1 cannot induce callus formation or root formation, suggesting that its effects are specific to shoot formation. In wild-type Arabidopsis plants, ESR1 expression was induced by cytokinin. ESR1 transcript levels also increased transiently during shoot regeneration from root explants, most probably in response to cytokinin in the shoot-inducing medium. This transient increase occurred after the acquisition of competence for regeneration and before shoot formation, which is consistent with the physiological effects of ESR1 overexpression. Our results suggest that ESR1 may regulate the induction of shoot regeneration after the acquisition of competence for organogenesis.

Amino Acid Sequence↗
PubMed2001

Arabidopsis PLC1 is required for secondary responses to abscisic acid signals.

The role of inositol 1,4,5-trisphosphate (Ins[1,4,5]P3) in transducing the abscisic acid (ABA) signal during seed germination and in the stress responses of mature plants is poorly understood. We have considered the contributions of the phospholipase C1 (encoded by AtPLC1) and an Ins(1,4,5)P3 5-phosphatase (encoded by AtIP5PII) to ABA signaling by using a modified version of the glucocorticoid-inducible system to regulate transgene expression. In the presence of the dexamethasone (Dex) inducer, transgenic lines expressing the AtPLC1 antisense and AtIP5PII sense transgenes showed no inhibition of germination and growth by ABA, whereas in the absence of the inducer they were sensitive. In the presence of Dex, these lines accumulated lower Ins(1,4,5)P3 levels upon ABA treatment compared with that of the control transgenic lines. RNA gel blot analysis revealed a decrease in the induction of the ABA-responsive genes RD29a, KIN2, and RD22 but not COR47 in the Dex-induced transgenic plants. In transgenic lines expressing the inducible AtPLC1 sense transgene, an increase in AtPLC1 expression was not sufficient to activate the expression of ABA-responsive genes in vegetative tissues. In vitro experiments demonstrated the induced PLC1 expression when extracts were assayed in the presence of calcium, but no increase in Ins(1,4,5)P3 levels in vivo was detected, suggesting that the PLC1 enzyme was latent. Our results indicate that although an increase in PLC1 activity and increased Ins(1,4,5)P3 levels are necessary for maximal gene induction by ABA, overexpression of AtPLC1 itself is not sufficient to trigger the expression of ABA-responsive genes. We propose that AtPLC1 plays a role in secondary ABA responses.

Abscisic Acid↗
PubMed2001

ADF proteins are involved in the control of flowering and regulate F-actin organization, cell expansion, and organ growth in Arabidopsis.

Based mostly on the results of in vitro experiments, ADF (actin-depolymerizing factor) proteins are thought to be key modulators of the dynamic organization of the actin cytoskeleton. The few studies concerned with the in vivo function of ADF proteins that have been reported to date were performed almost exclusively using single-cell systems and have failed to produce consistent results. To investigate ADF functions in vivo and during the development of multicellular organs, we generated transgenic Arabidopsis plants that express a cDNA encoding an ADF protein (AtADF1) in the sense or the antisense orientation under the control of a strong constitutively active promoter. Selected lines with significantly altered levels of AtADF protein expression were characterized phenotypically. Overexpression of AtADF1 resulted in the disappearance of thick actin cables in different cell types, caused irregular cellular and tissue morphogenesis, and reduced the growth of cells and organs. In contrast, reduced AtADF expression promoted the formation of actin cables, resulted in a delay in flowering, and stimulated cell expansion as well as organ growth. These results are consistent with the molecular functions of ADF as predicted by in vitro studies, support the global roles of ADF proteins during the development of a multicellular organism, and demonstrate that these proteins are key regulators of F-actin organization, flowering, and cell and organ expansion in Arabidopsis.

Actin Depolymerizing Factors↗
PubMed2000

An alternative cytokinin biosynthesis pathway.

Studies of de novo cytokinin biosynthesis in isopentenyltransferase (ipt)-transformed Arabidopsis thaliana, involving in vivo deuterium labeling and mass spectrometry, showed that the biosynthetic rate of zeatinriboside-5'-monophosphate was around 66-fold higher than that of isopentenyladenosine-5'-monophosphate (iPMP), the proposed primary product of the Agrobacterium ipt. Double tracer analysis, using [(2)H(6)] isopentenyladenosine and deuterium oxide, provided evidence for an alternative, iPMP-independent, biosynthetic pathway for zeatin-type cytokinins, present in both ipt-expressing and wild-type Arabidopsis thaliana. Reduction of the biosynthetic flux in the alternative pathway by use of mevastatin, an inhibitor for 3-hydroxy-3-methylglutaryl CoA reductase, indicated a terpenoid origin for the side-chain precursor of the iPMP independent pathway.

Alkyl and Aryl Transferases↗
PubMed2000

Arabidopsis NAC1 transduces auxin signal downstream of TIR1 to promote lateral root development.

Auxin plays a key role in lateral root formation, but the signaling pathway for this process is poorly understood. We show here that NAC1, a new member of the NAC family, is induced by auxin and mediates auxin signaling to promote lateral root development. NAC1 is a transcription activator consisting of an N-terminal conserved NAC-domain that binds to DNA and a C-terminal activation domain. This factor activates the expression of two downstream auxin-responsive genes, DBP and AIR3. Transgenic plants expressing sense or antisense NAC1 cDNA show an increase or reduction of lateral roots, respectively. Finally, TIR1-induced lateral root development is blocked by expression of antisense NAC1 cDNA, and NAC1 overexpression can restore lateral root formation in the auxin-response mutant tir1, indicating that NAC1 acts downstream of TIR1.

Amino Acid Sequence↗
PubMed2000

A comparative structural analysis of the ADF/cofilin family.

Actin-depolymerizing factor (ADF) and cofilin define a family of actin-binding proteins essential for the rapid turnover of filamentous actin in vivo. Here we present the 2.0 A crystal structure of Arabidopsis thaliana ADF1 (AtADF1), the first plant crystal structure from the ADF/cofilin (AC) family. Superposition of the four AC isoform structures permits an accurate sequence alignment that differs from previously reported data for the location of vertebrate-specific inserts and reveals a contiguous, vertebrate-specific surface opposite the putative actin-binding surface. Extending the structure-based sequence alignment to include 30 additional isoforms indicates three major groups: vertebrates, plants, and "other eukaryotes." Within these groups, several structurally conserved residues that are not conserved throughout the entire AC family have been identified. Residues that are highly conserved among all isoforms tend to cluster around the tryptophan at position 90 and a structurally conserved kink in alpha-helix 3. Analysis of surface character shows the presence of a hydrophobic patch and a highly conserved acidic cluster, both of which include several residues previously implicated in actin binding.

Actin Depolymerizing Factors↗
PubMed2000

Root hair formation: F-actin-dependent tip growth is initiated by local assembly of profilin-supported F-actin meshworks accumulated within expansin-enriched bulges.

Plant root hair formation is initiated when specialized elongating root epidermis cells (trichoblasts) assemble distinct domains at the plasma membrane/cell wall cell periphery complexes facing the root surface. These localities show accumulation of expansin and progressively transform into tip-growing root hair apices. Experimentation showed that trichoblasts made devoid of microtubules (MTs) were unaffected in root hair formation, whereas those depleted of F-actin by the G-actin sequestering agent latrunculin B had their root hair formation blocked after the bulge formation stage. In accordance with this, MTs are naturally depleted from early outgrowing bulges in which dense F-actin meshworks accumulate. These F-actin caps remain associated with tips of emerging and growing root hairs. Constitutive expression of the GFP-mouse talin fusion protein in transgenic Arabidopsis, which visualizes all classes of F-actin in a noninvasive mode, allowed in vivo confirmation of the presence of distinct F-actin meshworks within outgrowing bulges and at tips of young root hairs. Profilin accumulates, at both the protein and the mRNA levels, within F-actin-enriched bulges and at tips of emerging hairs. ER-based calreticulin and HDEL proteins also accumulate within outgrowing bulges and remain enriched at tips of emerging hairs. All this suggests that installation of the actin-based tip growth machinery takes place only after expansin-associated bulge formation and requires assembly of profilin-supported dynamic F-actin meshworks.

Actins↗
PubMed2000

PAT1, a new member of the GRAS family, is involved in phytochrome A signal transduction.

Light signaling via the phytochrome A (phyA) photoreceptor controls basic plant developmental processes including de-etiolation and hypocotyl elongation. We have identified a new Arabidopsis mutant, pat (phytochrome A signal transduction)1-1, which shows strongly reduced responses in continuous far-red light. Physiological and molecular data indicate that this mutant is disrupted at an early step of phyA signal transduction. The PAT1 gene encodes a cytoplasmic protein of 490 amino acids with sequence homologies to the plant-specific GRAS regulatory protein family. In the pat1-1 mutant, a T-DNA insertion introduces a premature stop codon, which likely results in the production of a truncated PAT1 protein of 341 amino acids. The semidominant phenotype of this mutant can be recapitulated by overexpression of an appropriately truncated PAT1 gene in the wild type. The results indicate that the truncated PAT1 protein acts in a dominant-negative fashion to inhibit phyA signaling.

Amino Acid Motifs↗
PubMed2000

Chemical-inducible systems for regulated expression of plant genes.

Chemical regulation of transgene expression presents a powerful tool for basic research in plant biology and biotechnological applications. Various chemical-inducible systems based on de-repression, activation and inactivation of the target gene have been described. The utility of inducible promoters has been successfully demonstrated by the development of a marker-free transformation system and large-scale gene profiling. In addition, field applications appear to be promising through the use of registered agrochemicals (e.g. RH5992) as inducers.

Chimera↗