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 beta-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.     

Light-induced nuclear translocation of endogenous pea phytochrome A visualized by immunocytochemical procedures

Although the physiological functions of phytochrome A (PhyA) are now known, the distribution of endogenous PhyA has not been examined. We have visualized endogenous PhyA apoprotein (PHYA) by immunolabeling cryosections of pea tissue, using PHYA-deficient mutants as negative controls. By this method, we examined the distribution of PHYA in different tissues and changes in its intracellular distribution in response to light. In apical hook cells of etiolated seedlings, PHYA immunolabeling was distributed diffusely in the cytosol. Exposure to continuous far-red (cFR) light caused a redistribution of the immunolabeling to the nucleus, first detectable after 1.5 hr and greatest at 4.5 hr. During this time, the amounts of spectrally active phytochrome and PHYA did not decline substantially. Exposure to continuous red (cR) light or to a brief pulse of red light also resulted in redistribution of immunolabeling to the nucleus, but this occurred much more rapidly and with a different pattern of intranuclear distribution than it did in response to cFR light. Exposures to cR light resulted in loss of immunolabeling, which was associated with PHYA degradation. These results indicate that the light-induced intracellular location of PHYA is wavelength dependent and imply that this is important for PhyA activity.     

ydk1-D, an auxin-responsive GH3 mutant that is involved in hypocotyl and root elongation

To study the GH3 gene family of Arabidopsis, we investigated a flanking sequence database of Arabidopsis activation-tagged lines. We found a dwarf mutant, named yadokari 1-D (ydk1-D), that had a T-DNA insertion proximal to a GH3 gene. ydk1-D is dominant and has a short hypocotyl not only in light but also in darkness. Moreover, ydk1-D has a short primary root, a reduced lateral root number, and reduced apical dominance. A GH3 gene, named YDK1, was upregulated in ydk1-D, and YDK1 transgenic plants showed the ydk1-D phenotype. YDK1 gene expression was induced by exogenously applied auxin and regulated by auxin-response factor (ARF)7. In addition, YDK1 gene expression was downregulated by blue and far-red (FR) lights. Strong promoter activity of YDK1 was observed in roots and flowers. These results suggest that YDK1 may function as a negative component in auxin signaling by regulating auxin activity.     

PIF4, a phytochrome-interacting bHLH factor,functions as a negative regulator of phytochrome B signaling in Arabidopsis

Plants sense and respond to red and far-red light using the phytochrome (phy) family of photoreceptors. However, the mechanism of light signal transduction is not well defined. Here, we report the identification of a new mutant Arabidopsis locus, srl2 (short under red-light 2), which confers selective hypersensitivity to continuous red, but not far-red, light. This hypersensitivity is eliminated in srl2phyB, but not srl2phyA, double mutants, indicating that this locus functions selectively and negatively in phyB signaling. The SRL2 gene encodes a bHLH factor, designated PIF4 (phytochrome-interacting factor 4), which binds selectively to the biologically active Pfr form of phyB, but has little affinity for phyA. Despite its hypersensitive morphological phenotype, the srl2 mutant displays no perturbation of light-induced expression of marker genes for chloroplast development. These data suggest that PIF4 may function specifically in a branch of the phyB signaling network that regulates a subset of genes involved in cell expansion. Consistent with this proposal, PIF4 localizes to the nucleus and can bind to a G-box DNA sequence motif found in various light-regulated promoters.     

shk1-D, a dwarf Arabidopsis mutant caused by activation of the CYP72C1 gene, has altered brassinosteroid levels

Brassinosteroids (BRs) are plant steroidal hormones that regulate plant growth and development. An Arabidopsis dwarf mutant, shrink1-D (shk1-D), was isolated and the phenotype was shown to be caused by activation of the CYP72C1 gene. CYP72C1 is a member of the cytochrome P450 monooxygenase gene family similar to BAS1/CYP734A1 that regulates BR inactivation. shk1-D has short hypocotyls in both light and dark, and short petioles and siliques. The seeds are also shortened along the longitudinal axis indicating CYP72C1 controls cell elongation. The expression of CPD, TCH4 and BAS1 were altered in CYP72C1 overexpression transgenic lines and endogenous levels of castasterone, 6-deoxocastasterone and 6-deoxotyphasterol were also altered. Unlike BAS1/CYP734A1 the expression of CYP72C1 was not changed by application of exogenous brassinolide. We propose that CYP72C1 controls BR homeostasis by modulating the concentration of BRs.     

拟南芥低盐敏感突变体的筛选

通过对ein3-1功能缺失型突变体种子进行EMS诱变,筛选到47株盐敏感突变体。根据对盐敏感程度的不同将其分为3类,分别为低盐超敏感突变体(low concentration of salt hyper-sensitive mutants,lsh),低盐中等敏感突变体(low con-centration of salt moderate-sensitive mutants,lsm)和低盐弱敏感突变体(lowconcentration of salt slight-sensitive mutants,lss)。以其中一株lss-3为例,进行了深入研究。根据遗传分析和生理试验表明,lss-3是以ein3-1为背景的隐性双突变体,而且具有比Col-0和ein3-1更加敏感的盐表型。三重反应表明,lss-3与ein3-1类似,表现出对ACC不敏感的表型。推测lss-3突变的基因可能与乙烯信号途径组分EIN3有关,也可能与之无关,仅是参与抗盐的一个新基因。     

Distinct roles for light-dependent NADPH:protochlorophyllide oxidoreductases (POR) A and B during greening in higher plants

Light-dependent NADPH:protochlorophyllide oxidoreductase (POR), a nuclear-encoded plastid-localized enzyme, catalyzes the photoreduction of protochlorophyllide (Pchlide) to chlorophyllide in higher plants, algae and cyanobacteria. Angiosperms require light for chlorophyll (Chl) biosynthesis and have recently been shown to contain two POR-encoding genes, PorA and PorB , that are differentially regulated by light and developmental state. PorA expression rapidly becomes undetectable after illumination of etiolated seedlings, whereas PorB expression persists throughout greening and in adult plants. In order to study the in vivo functions of Arabidopsis POR A and POR B we have abolished the expression of PorA through the use of the phytochrome A-mediated far-red high irradiance response. Wild-type seedlings grown in continuous far-red light (cFR) display the morphology of white light (WL)-grown seedlings, but contain only traces of Chl and do not green upon transfer to WL. This cFR-induced greening defect correlates with the absence of PorA mRNA, the putative POR A protein, phototransformable Pchlide-F₆₅₅, and with strongly reduced POR enzymatic activity in plant extracts. In contrast, a cFR-grown phyA mutant expresses the PorA gene, accumulates Chl and visibly greens in WL. Furthermore, constitutive overexpression of POR A in cFR-grown transgenic Arabidopsis wild-type seedlings restores Chl accumulation and WL-induced greening. These data demonstrate that POR A is required for greening and that the availability of POR A limits Chl accumulation during growth in cFR. POR B apparently provides a means to sustain light-dependent Chl biosynthesis in fully greened, mature plants in the absence of phototransformable Pchlide-F₆₅₅.     

Overexpression of light-dependent PORA or PORB in plants depleted of endogenous POR by far-red light enhances seedling survival in white light and protects against photooxidative damage

The structurally related light-dependent protochlorophyllide (Pchlide) oxidoreductases PORA and PORB mediate the only light-requiring step in chlorophyll (Chl) biosynthesis in higher plants. Correlative evidence suggests that some in vivo functions of PORA and PORB may be unique, including a postulated photoprotective role for PORA. For example, wild-type Arabidopsis thaliana seedlings grown in non-photooxidative far-red light (cFR) resemble those grown in white light (WL), but they are yellow and do not green normally thereafter in WL. This defect is accompanied by the absence of detectable PORA and reduced levels of PORB expression. Here, direct evidence is provided that the presence of POR, either as PORA or PORB, can confer photoprotection in plants. In contrast to the wild-type, the plastids of transgenic PORA- or PORB-overexpressing Arabidopsis seedlings grown in cFR possess extensive prolamellar bodies. Upon a subsequent shift to WL, POR-overexpressing seedlings develop thylakoid membranes, accumulate large amounts of Chl and are viable at fluence rates lethal to the wild-type. Intriguingly, the plastid membrane architectures of greening transgenic seedlings seem to depend on whether PORA or PORB has been overproduced. POR-overexpressing seedlings shifted from cFR to WL of fluence rates from 20 to 500 μE m^–2 sec^–1 accumulate substantially higher amounts of Chl than does the wild-type. Furthermore, the WL fluence rate that permits maximal Chl accumulation increases from 8 μE m^–2 sec^–1 in the wild-type to 125 μE m^–2 sec^–1 in transgenic seedlings. POR overexpression during growth in cFR also correlates with a fourfold decrease in the steady-state content of Pchlide, a potentially lethal photosensitizer.     

PIS1, a negative regulator of the action of auxin transport inhibitors in Arabidopsis thaliana

In order to clarify the mechanism underlying the polar auxin transport system, the pis1 mutant in Arabidopsis thaliana that is hypersensitive to N -1-naphthylphthalamic acid (NPA), an auxin transport inhibitor was isolated and characterized. Whereas the pis1 mutant is normally sensitive to phytohormones, auxins, cytokinin and ethylene precursor, this mutant is hypersensitive to NPA over the broad spectrum of its effects such as growth of seedlings, root elongation, root gravitropism, root phototropism and root curling. This result indicates that the pis1 mutant is specifically affected in the polar auxin transport system. This result also defines a genetic factor controlling both gravitropism and phototropism, and strongly indicates the involvement of auxin transport during both tropic responses. NPA, 2,3,5-triiodobenzoic acid (TIBA) and 9-hydroxyfluorene-9-carboxylic acid (HFCA) represent different classes of auxin transport inhibitors. The pis1 mutation conferred hypersensitivity to both NPA and TIBA but not to HFCA. These results show the genetic separation of the actions of NPA/TIBA and of HFCA. The PIS1 gene product might be specifically involved in the response pathway of NPA/TIBA, leading to interference with auxin-efflux carriers, and might act as a negative regulator of the action of NPA/TIBA.     

Overexpression of PRE1 and its homologous genes activates Gibberellin-dependent responses in Arabidopsis thaliana

Gibberellins control various aspects of growth and development. Here, we identified a gene, designated paclobutrazol resistance1 (PRE1), by screening Arabidopsis activation-tagged lines. PRE1 encodes a helix-loop-helix protein and belongs to a small gene family. Physiological and genetic analysis indicated that overexpression of PRE1 altered various aspects of gibberellin-dependent responses such as germination, elongation of hypocotyl/petiole, floral induction and fruit development, and suppressed gibberellin-deficient phenotypes of the ga2 mutant. Expression of some gibberellin-responsive genes was also affected by PRE1. Expression of PRE1 was shown to be early gibberellin inducible in the wild-type plants and under control of SPY and GAI, upstream negative regulators of gibberellin signaling. The shortened hypocotyl length phenotype of the gai-1 mutant was suppressed by PRE1 overexpression. Ectopic overexpression of each of the four PRE1-related genes conferred pleiotropic phenotypes similar to PRE1 overexpression, indicative of overlapping functions among the PRE gene family. Our results of gain-of-function studies suggest that PRE genes may have a regulatory role in gibberellin-dependent development in Arabidopsis thaliana.     

A gain-of-function mutation in the Arabidopsis pleiotropic drug resistance transporter PDR9 confers resistance to auxinic herbicides

Arabidopsis (Arabidopsis thaliana) contains 15 genes encoding members of the pleiotropic drug resistance (PDR) family of ATP-binding cassette transporters. These proteins have been speculated to be involved in the detoxification of xenobiotics, however, little experimental support of this hypothesis has been obtained to date. Here we report our characterization of the Arabidopsis PDR9 gene. We isolated a semidominant, gain-of-function mutant, designated pdr9-1, that exhibits increased tolerance to the auxinic herbicide 2,4-dichlorophenoxyacetic acid (2,4-D). Reciprocally, loss-of-function mutations in PDR9 confer 2,4-D hypersensitivity. This altered auxin sensitivity defect of pdr9 mutants is specific for 2,4-D and closely related compounds as these mutants respond normally to the endogenous auxins indole-3-acetic acid and indole-butyric acid. We demonstrate that 2,4-D, but not indole-3-acetic acid transport is affected by mutations in pdr9, suggesting that the PDR9 transporter specifically effluxes 2,4-D out of plant cells without affecting endogenous auxin transport. The semidominant pdr9-1 mutation affects an extremely highly conserved domain present in all known plant PDR transporters. The single amino acid change results in increased PDR9 abundance and provides a novel approach for elucidating the function of plant PDR proteins.     

Characterization of the primary immunity region of the Escherichia coli linear plasmid prophage N15.

N15 is the only bacteriophage of Escherichia coli known to lysogenize as a linear plasmid. Clear-plaque mutations lie in at least two regions of the 46-kb genome. We have cloned, sequenced, and characterized the primary immunity region, immB. This region contains a gene, cB, whose product shows homology to lambdoid phage repressors. The cB3 mutation confers thermoinducibility on N15 lysogens, consistent with CB being the primary repressor of N15. Downstream of cB lies the locus of N15 plasmid replication. Upstream of cB lies an operon predicted to encode two products: one homologous to the late repressor of P22 (Cro), the other homologous to the late antiterminator of phi 82 (Q). The Q-like protein is essential for phage development. We show that CB protein regulates the expression of genes that flank the cB gene by binding to DNA at symmetric 16-bp sites. Three sites are clustered upstream of cB and overlap a predicted promoter of the cro and Q-like genes as well as two predicted promoters of cB itself. Two sites downstream of cB overlap a predicted promoter of a plasmid replication gene, repA, consistent with the higher copy number of the mutant, N15cB3. The leader region of repA contains terminators in both orientations and a putative promoter. The organization of these regulatory elements suggests that N15 plasmid replication is controlled not only by CB but also by an antisense RNA and by a balance between termination and antitermination.     

A mutation in the Arabidopsis DET3 gene uncouples photoregulated leaf development from gene expression and chloroplast biogenesis

The genetic and phenotypic characterization of a new Arabidopsis mutant, de-etiolated -3, ( det 3), involved in light-regulated seedling development is described. A recessive mutation in the DET 3 gene uncouples light signals from a subset of light-dependent processes. The det 3 mutation causes dark-grown Arabidopsis thaliana seedlings to have short hypocotyls, expanded cotyledons, and differentiated leaves, traits characteristic of light-grown seedlings. Despite these morphological changes, however, the det 3 mutant does not develop chloroplasts or show elevated expression of nuclear- and chloroplast-encoded light-regulated mRNAs. The det 3 mutation thus uncovers a downstream branch of the light transduction pathways that separates leaf development from chloroplast differentiation and light-regulated gene expression. In addition, light-grown det 3 plants have reduced stature and apical dominance, suggesting that DET3 functions during growth in normal light conditions as well. The genetic interactions between mutations in det 1, det 2, and det 3 are described. The phenotypes of doubly mutant strains suggest that there are at least two parallel pathways controlling light-mediated development in Arabidopsis .