Hsu-Liang Hsieh

Drought and salt stress tolerance of an Arabidopsis glutathione S-transferase U17 knockout mutant are attributed to the combined effect of glutathione and abscisic acid.

Although glutathione S-transferases (GSTs) are thought to play major roles in oxidative stress metabolism, little is known about the regulatory functions of GSTs. We have reported that Arabidopsis (Arabidopsis thaliana) GLUTATHIONE S-TRANSFERASE U17 (AtGSTU17; At1g10370) participates in light signaling and might modulate various aspects of development by affecting glutathione (GSH) pools via a coordinated regulation with phytochrome A. Here, we provide further evidence to support a negative role of AtGSTU17 in drought and salt stress tolerance. When AtGSTU17 was mutated, plants were more tolerant to drought and salt stresses compared with wild-type plants. In addition, atgstu17 accumulated higher levels of GSH and abscisic acid (ABA) and exhibited hyposensitivity to ABA during seed germination, smaller stomatal apertures, a lower transpiration rate, better development of primary and lateral root systems, and longer vegetative growth. To explore how atgstu17 accumulated higher ABA content, we grew wild-type plants in the solution containing GSH and found that they accumulated ABA to a higher extent than plants grown in the absence of GSH, and they also exhibited the atgstu17 phenotypes. Wild-type plants treated with GSH also demonstrated more tolerance to drought and salt stresses. Furthermore, the effect of GSH on root patterning and drought tolerance was confirmed by growing the atgstu17 in solution containing l-buthionine-(S,R)-sulfoximine, a specific inhibitor of GSH biosynthesis. In conclusion, the atgstu17 phenotype can be explained by the combined effect of GSH and ABA. We propose a role of AtGSTU17 in adaptive responses to drought and salt stresses by functioning as a negative component of stress-mediated signal transduction pathways.

PHYTOCHROME INTERACTING FACTOR3 Associates with the Histone Deacetylase HDA15 in Repression of Chlorophyll Biosynthesis and Photosynthesis in Etiolated Arabidopsis Seedlings

PIF3 recruits HDA15 to repress chlorophyll biosynthesis and photosynthesis gene expression in etiolated seedlings, suggesting that epigenetic regulation is critical for phytochrome- and PIF3-regulated light signal transduction. This study provides insight into the molecular mechanism underlying photomorphogenesis in plants. PHYTOCHROME INTERACTING FACTOR3 (PIF3) is a key basic helix-loop-helix transcription factor of Arabidopsis thaliana that negatively regulates light responses, repressing chlorophyll biosynthesis, photosynthesis, and photomorphogenesis in the dark. However, the mechanism for the PIF3-mediated transcription regulation remains largely unknown. In this study, we found that the REDUCED POTASSIUM DEPENDENCY3/HISTONE DEACETYLASE1-type histone deacetylase HDA15 directly interacted with PIF3 in vivo and in vitro. Genome-wide transcriptome analysis revealed that HDA15 acts mainly as a transcriptional repressor and negatively regulates chlorophyll biosynthesis and photosynthesis gene expression in etiolated seedlings. HDA15 and PIF3 cotarget to the genes involved in chlorophyll biosynthesis and photosynthesis in the dark and repress gene expression by decreasing the acetylation levels and RNA Polymerase II–associated transcription. The binding of HDA15 to the target genes depends on the presence of PIF3. In addition, PIF3 and HDA15 are dissociated from the target genes upon exposure to red light. Taken together, our results indicate that PIF3 associates with HDA15 to repress chlorophyll biosynthetic and photosynthetic genes in etiolated seedlings.

Light-modulated abundance of an mRNA encoding a calmodulin-regulated, chromatin-associated NTPase in pea

A cDNA encoding a 47 kDa nucleoside triphosphatase (NTPase) that is associated with the chromatin of pea nuclei has been cloned and sequenced. The translated sequence of the cDNA includes several domains predicted by known biochemical properties of the enzyme, including five motifs characteristic of the ATP-binding domain of many proteins, several potential casein kinase II phosphorylation sites, a helix-turn-helix region characteristic of DNA-binding proteins, and a potential calmodulin-binding domain. The deduced primary structure also includes an N-terminal sequence that is a predicted signal peptide and an internal sequence that could serve as a bipartite-type nuclear localization signal. Both in situ immunocytochemistry of pea plumules and immunoblots of purified cell fractions indicate that most of the immunodetectable NTPase is within the nucleus, a compartment proteins typically reach through nuclear pores rather than through the endoplasmic reticulum pathway. The translated sequence has some similarity to that of human lamin C, but not high enough to account for the earlier observation that IgG against human lamin C binds to the NTPase in immunoblots. Northern blot analysis shows that the NTPase mRNA is strongly expressed in etiolated plumules, but only poorly or not at all in the leaf and stem tissues of light-grown plants. Accumulation of NTPase mRNA in etiolated seedlings is stimulated by brief treatments with both red and far-red light, as is characteristic of very low-fluence phytochrome responses. Southern blotting with pea genomic DNA indicates the NTPase is likely to be encoded by a single gene.

Glutathione S-Transferase Interacting with Far-Red Insensitive 219 Is Involved in Phytochrome A-Mediated Signaling in Arabidopsis

Far-red (FR) insensitive 219 (FIN219) was previously shown to be involved in phytochrome A-mediated FR light signaling. To further understand its function and regulatory relation with other light-signaling components, a yeast two-hybrid approach was used to isolate FIN219-interacting partners. Here, we demonstrate that FIN219-interacting protein 1 (FIP1) interacts with FIN219 in vitro and in vivo and is composed of 217 amino acids that belong to the tau class of the large glutathione S-transferase gene family. FIP1 was further shown to have glutathione S-transferase activity. The gain of function and partial loss of function of FIP1 resulted in a hyposensitive hypocotyl phenotype under continuous FR (cFR) light and a delayed flowering phenotype under long-day conditions, which suggests that FIP1 may exist in a complex to function in the regulation of Arabidopsis (Arabidopsis thaliana) development. In addition, FIP1 mRNA was down-regulated in the suppressor of phytochrome A-105 1 mutant and differentially expressed in constitutive photomorphogenic 1-4 (cop1-4) and cop1-5 mutants under cFR. Intriguingly, FIP1 expression was up-regulated in the fin219 mutant under all light conditions, except cFR. Furthermore, promoter activity assays revealed that FIP1 expression was light dependent, mainly associated with vascular tissues, and developmentally regulated. Subcellular localization studies revealed that the β-glucuronidase-FIP1 fusion protein was localized in the nucleus and cytoplasm. Taken together, these data indicate that FIP1 may interact with FIN219 to regulate cell elongation and flowering in response to light.

A Glutathione S-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.

Regulation of a recombinant pea nuclear apyrase by calmodulin and casein kinase II

A cDNA encoding a pea nuclear apyrase was previously cloned. Overexpressions of a full-length and a truncated cDNA have been successfully expressed in Escherichia coli BL21(DE3). The resulting fusion proteins, apyrase and the C-terminus (residues 315-453) of apyrase, were used for calmodulin (CaM) binding and phosphorylation studies. Fusion protein apyrase but not the C-terminus of apyrase can be recognized by polyclonal antibody pc480. This suggested that the motif recognized by pc480 was located in the N-terminal region of apyrase. The recombinant apyrase protein also showed an activity 70 times higher than that of endogenous apyrase using ATP as a substrate. The recombinant apyrase has a preference for ATP more than other nucleoside triphosphate substrates. CaM can bind to recombinant apyrase, but not to the C-terminus of apyrase. This implies that the CaM-binding domain must be in the first 315 amino acids of the N-terminal region of apyrase. We found that one segment from residue 293 to 308 was a good candidate for the CaM-binding domain. This segment 293 FNKCKNTIRKALKLNY 308 has a basic amphiphilic-helical structure, which shows the predominance of basic residues on one side and hydrophobic residues on the other when displayed on a helical wheel plot. Using the gel mobility shift binding assay, this synthetic peptide was shown to bind to CaM, indicating that it is the CaM-binding domain. Both recombinant apyrase and the C-terminus of apyrase can be phosphorylated by a recombinant human protein kinase CKII. Phosphorylation does not affect CaM binding to recombinant apyrase. However, CaM does inhibit CKII phosphorylation of recombinant apyrase and this inhibition can be blocked by 5 mM EGTA.

Light Regulation of the Abundance of mRNA Encoding a Nucleolin-Like Protein Localized in the Nucleoli of Pea Nuclei

A cDNA encoding a nucleolar protein was selected from a pea (Pisum sativum) plumule library, cloned, and sequenced. The translated sequence of the cDNA has significant percent identity to Xenopus laevis nucleolin (31%), the alfalfa (Medicago sativa) nucleolin homolog (66%), and the yeast (Saccharomyces cerevisiae) nucleolin homolog (NSR1) (28%). It also has sequence patterns in its primary structure that are characteristic of all nucleolins, including an N-terminal acidic motif, RNA recognition motifs, and a C-terminal Gly- and Arg-rich domain. By immunoblot analysis, the polyclonal antibodies used to select the cDNA bind selectively to a 90kD protein in purified pea nuclei and nucleoli and to an 88-kD protein in extracts of Escherichia coli expressing the cDNA. In immunolocalization assays of pea plumule cells, the antibodies stained primarily a region surrounding the fibrillar center of nucleoli, where animal nucleolins are typically found. Southern analysis indicated that the pea nucleolin-like protein is encoded by a single gene, and northern analysis showed that the labeled cDNA binds to a single band of RNA, approximately the same size as the cDNA. After irradiation of etiolated pea seedlings by red light, the mRNA level in plumules decreased during the 1st hour and then increased to a peak of six times the 0-h level at 12 h. Far-red light reversed this effect of red light, and the mRNA accumulation from red/far-red light irradiation was equal to that found in the dark control. This indicates that phytochrome may regulate the expression of this gene.

Arabidopsis mTERF15 Is Required for Mitochondrial nad2 Intron 3 Splicing and Functional Complex I Activity

Mitochondria play a pivotal role in most eukaryotic cells, as they are responsible for the generation of energy and diverse metabolic intermediates for many cellular events. During endosymbiosis, approximately 99% of the genes encoded by the mitochondrial genome were transferred into the host nucleus, and mitochondria import more than 1000 nuclear-encoded proteins from the cytosol to maintain structural integrity and fundamental functions, including DNA replication, mRNA transcription and RNA metabolism of dozens of mitochondrial genes. In metazoans, a family of nuclear-encoded proteins called the mitochondrial transcription termination factors (mTERFs) regulates mitochondrial transcription, including transcriptional termination and initiation, via their DNA-binding activities, and the dysfunction of individual mTERF members causes severe developmental defects. Arabidopsis thaliana and Oryza sativa contain 35 and 48 mTERFs, respectively, but the biological functions of only a few of these proteins have been explored. Here, we investigated the biological role and molecular mechanism of Arabidopsis mTERF15 in plant organelle metabolism using molecular genetics, cytological and biochemical approaches. The null homozygous T-DNA mutant of mTERF15, mterf15, was found to result in substantial retardation of both vegetative and reproductive development, which was fully complemented by the wild-type genomic sequence. Surprisingly, mitochondria-localized mTERF15 lacks obvious DNA-binding activity but processes mitochondrial nad2 intron 3 splicing through its RNA-binding ability. Impairment of this splicing event not only disrupted mitochondrial structure but also abolished the activity of mitochondrial respiratory chain complex I. These effects are in agreement with the severe phenotype of the mterf15 homozygous mutant. Our study suggests that Arabidopsis mTERF15 functions as a splicing factor for nad2 intron 3 splicing in mitochondria, which is essential for normal plant growth and development.

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.

Arabidopsis bZIP16 Transcription Factor Integrates Light and Hormone Signaling Pathways to Regulate Early Seedling Development

This work identifies of bZIP16 as a transcriptional repressor regulating the expression of PIL5, RGL2, and genes influencing light, GA, and ABA signals. It indicates that bZIP16 is a newly identified G-box binding transcription factor integrating light and hormone pathways to promote seed germination and hypocotyl elongation during the early stages of Arabidopsis seedling development. Transcriptomic adjustment plays an important role in Arabidopsis thaliana seed germination and deetiolation in response to environmental light signals. The G-box cis-element is commonly present in promoters of genes that respond positively or negatively to the light signal. In pursuing additional transcriptional regulators that modulate light-mediated transcriptome changes, we identified bZIP16, a basic region/Leu zipper motif transcription factor, by G-box DNA affinity chromatography. We confirmed that bZIP16 has G-box–specific binding activity. Analysis of bzip16 mutants revealed that bZIP16 is a negative regulator in light-mediated inhibition of cell elongation but a positive regulator in light-regulated seed germination. Transcriptome analysis supported that bZIP16 is primarily a transcriptional repressor regulating light-, gibberellic acid (GA)–, and abscisic acid (ABA)–responsive genes. Chromatin immunoprecipitation analysis revealed that bZIP16 could directly target ABA-responsive genes and RGA-LIKE2, a DELLA gene in the GA signaling pathway. bZIP16 could also indirectly repress the expression of PHYTOCHROME INTERACTING FACTOR3-LIKE5, which encodes a basic helix-loop-helix protein coordinating hormone responses during seed germination. By repressing the expression of these genes, bZIP16 functions to promote seed germination and hypocotyl elongation during the early stages of Arabidopsis seedling development.