Increasing evidence has revealed that abscisic acid （ABA） negatively modulates ethylene biosynthesis, although the underlying mechanism remains unclear. To identify the factors involved, we conducted a screen for AB...Increasing evidence has revealed that abscisic acid （ABA） negatively modulates ethylene biosynthesis, although the underlying mechanism remains unclear. To identify the factors involved, we conducted a screen for ABA-insensitive mutants with altered ethylene production in Arabidopsis. A dominant allele of ABI4, abi4-152, which produces a putative protein with a 16-amino-acid truncation at the C-terminus of ABI4, reduces ethylene production. By contrast, two recessive knockout alleles of ABI4, abi4-102 and abi4-103, result in increased ethylene evolution, indicating that ABI4 negatively regulates ethylene produc- tion. Further analyses showed that expression of the ethylene biosynthesis genes ACS4, ACSS, and AC02 was significantly decreased in abi4-152 but increased in the knockout mutants, with partial dependence on ABA. Chromatin immunoprecipitation-quantitative PCR assays showed that ABI4 directly binds the pro- moters of these ethylene biosynthesis genes and that ABA enhances this interaction. A fusion protein containing the truncated ABI4-152 peptide accumulated to higher levels than its full-length counterpart in transgenic plants, suggesting that ABI4 is destabilized by its C terminus. Therefore, our results demon- strate that ABA negatively regulates ethylene production through ABI4-mediated transcriptional repression of the ethylene biosynthesis genes ACS4 and ACS8 in Arabidopsis.展开更多
Fluorescent studies of living plant cells such as confocal microscopy and fluorescence lifetime imaging often suffer from a strong autofluorescent background contribution that significantly reduces the dynamic image c...Fluorescent studies of living plant cells such as confocal microscopy and fluorescence lifetime imaging often suffer from a strong autofluorescent background contribution that significantly reduces the dynamic image contrast and the quantitative access to sub-cellular processes at high spatial resolution. Here, we present a novel technique--fluorescence intensity decay shape analysis microscopy （FIDSAM） to enhance the dynamic contrast of a fluorescence image of at least one order of magnitude. The method is based on the analysis of the shape of the fluorescence intensity decay （fluorescence lifetime curve） and benefits from the fact that the decay patterns of typical fluorescence label dyes strongly differ from emission decay curves of autofluorescent sample areas. Using FIDSAM, we investigated Arabidopsis thaliana hypocotyl cells in their tissue environment, which accumulate an eGFP fusion of the plasma membrane marker protein LTI6b （LTI6b-eGFP） to low level. Whereas in conventional confocal fluorescence images, the membranes of neighboring cells can hardly be optically resolved due to the strong autofluorescence of the cell wall, FIDSAM allows for imaging of single, isolated membranes at high spatial resolution. Thus, FIDSAM will enable the sub-cellular analysis of even low-expressed fluorophoretagged proteins in living plant cells. Furthermore, the combination of FIDSAM with fluorescence lifetime imaging provides the basis to study the local physico-chemical environment of fluorophore-tagged biomolecules in living plant cells.展开更多
Plants contain various factors that transiently interact with subunits or intermediates of the thylakoid multiprotein complexes, promoting their stable association and integration. Hence, assembly factors are essentia...Plants contain various factors that transiently interact with subunits or intermediates of the thylakoid multiprotein complexes, promoting their stable association and integration. Hence, assembly factors are essential for chloroplast development and the transition from heterotrophic to phototrophic growth. Snowy cotyledon 2 （SCO2） is a DNAJ-like protein involved in thylakoid membrane biogenesis and interacts with the light-harvesting chlorophyll-binding protein LHCBI. In Arabidopsis thaliana, SCO2 function was previ- ously reported to be restricted to cotyledons. Here we show that disruption of SC02 in Lotus japonicus results not only in paler cotyledons but also in variegated true leaves. Furthermore, smaller and pale- green true leaves can also be observed in A. thaliana sco2 （atsco2） mutants under short-day conditions. In both species, SCO2 is required for proper accumulation of PSlI-LHCll complexes. In contrast to other variegated mutants, inhibition of chloroplastic translation strongly affects L. japonicus sco2 mutant devel- opment and fails to suppress their variegated phenotype. Moreover, inactivation of the suppressor of variegation AtClpR1 in the atsco2 background results in an additive double-mutant phenotype with variegated true leaves. Taken together, our results indicate that SCO2 plays a distinct role in PSll assembly or repair and constitutes a novel factor involved in leaf variegation.展开更多
文摘Increasing evidence has revealed that abscisic acid （ABA） negatively modulates ethylene biosynthesis, although the underlying mechanism remains unclear. To identify the factors involved, we conducted a screen for ABA-insensitive mutants with altered ethylene production in Arabidopsis. A dominant allele of ABI4, abi4-152, which produces a putative protein with a 16-amino-acid truncation at the C-terminus of ABI4, reduces ethylene production. By contrast, two recessive knockout alleles of ABI4, abi4-102 and abi4-103, result in increased ethylene evolution, indicating that ABI4 negatively regulates ethylene produc- tion. Further analyses showed that expression of the ethylene biosynthesis genes ACS4, ACSS, and AC02 was significantly decreased in abi4-152 but increased in the knockout mutants, with partial dependence on ABA. Chromatin immunoprecipitation-quantitative PCR assays showed that ABI4 directly binds the pro- moters of these ethylene biosynthesis genes and that ABA enhances this interaction. A fusion protein containing the truncated ABI4-152 peptide accumulated to higher levels than its full-length counterpart in transgenic plants, suggesting that ABI4 is destabilized by its C terminus. Therefore, our results demon- strate that ABA negatively regulates ethylene production through ABI4-mediated transcriptional repression of the ethylene biosynthesis genes ACS4 and ACS8 in Arabidopsis.
文摘Fluorescent studies of living plant cells such as confocal microscopy and fluorescence lifetime imaging often suffer from a strong autofluorescent background contribution that significantly reduces the dynamic image contrast and the quantitative access to sub-cellular processes at high spatial resolution. Here, we present a novel technique--fluorescence intensity decay shape analysis microscopy （FIDSAM） to enhance the dynamic contrast of a fluorescence image of at least one order of magnitude. The method is based on the analysis of the shape of the fluorescence intensity decay （fluorescence lifetime curve） and benefits from the fact that the decay patterns of typical fluorescence label dyes strongly differ from emission decay curves of autofluorescent sample areas. Using FIDSAM, we investigated Arabidopsis thaliana hypocotyl cells in their tissue environment, which accumulate an eGFP fusion of the plasma membrane marker protein LTI6b （LTI6b-eGFP） to low level. Whereas in conventional confocal fluorescence images, the membranes of neighboring cells can hardly be optically resolved due to the strong autofluorescence of the cell wall, FIDSAM allows for imaging of single, isolated membranes at high spatial resolution. Thus, FIDSAM will enable the sub-cellular analysis of even low-expressed fluorophoretagged proteins in living plant cells. Furthermore, the combination of FIDSAM with fluorescence lifetime imaging provides the basis to study the local physico-chemical environment of fluorophore-tagged biomolecules in living plant cells.
文摘Plants contain various factors that transiently interact with subunits or intermediates of the thylakoid multiprotein complexes, promoting their stable association and integration. Hence, assembly factors are essential for chloroplast development and the transition from heterotrophic to phototrophic growth. Snowy cotyledon 2 （SCO2） is a DNAJ-like protein involved in thylakoid membrane biogenesis and interacts with the light-harvesting chlorophyll-binding protein LHCBI. In Arabidopsis thaliana, SCO2 function was previ- ously reported to be restricted to cotyledons. Here we show that disruption of SC02 in Lotus japonicus results not only in paler cotyledons but also in variegated true leaves. Furthermore, smaller and pale- green true leaves can also be observed in A. thaliana sco2 （atsco2） mutants under short-day conditions. In both species, SCO2 is required for proper accumulation of PSlI-LHCll complexes. In contrast to other variegated mutants, inhibition of chloroplastic translation strongly affects L. japonicus sco2 mutant devel- opment and fails to suppress their variegated phenotype. Moreover, inactivation of the suppressor of variegation AtClpR1 in the atsco2 background results in an additive double-mutant phenotype with variegated true leaves. Taken together, our results indicate that SCO2 plays a distinct role in PSll assembly or repair and constitutes a novel factor involved in leaf variegation.
