AtPUB18 and AtPUB19 are homologous U-box E3 ubiquitin ligases in Arabidopsis (Arabidopsis thaliana). AtPUB19 is a negative regulator of abscisic acid (ABA)-mediated drought responses, whereas the role of AtPUB18 in drought responses is unknown. Here, loss-of-function and overexpression tests identified AtPUB18 as a negative regulator in ABA-mediated stomatal closure and water stress responses. The atpub18-2atpub19-3 double mutant line displayed more sensitivity to ABA and enhanced drought tolerance than each single mutant plant; therefore, AtPUB18 and AtPUB19 are agonistic. Stomatal closure of the atpub18-2atpub19-3 mutant was hypersensitive to hydrogen peroxide (H 2 O 2 ) but not to calcium, suggesting that AtPUB18 and AtPUB19 exert negative effects on the ABA signaling pathway downstream of H 2 O 2 and upstream of calcium. AtPUB22 and AtPUB23 are other U-box E3 negative regulators of drought responses. Although atpub22atpub23 was more tolerant to drought stress relative to wild-type plants, its ABA-mediated stomatal movements were highly similar to those of wild-type plants. The atpub18-2atpub19-3atpub22atpub23 quadruple mutant exhibited enhanced tolerance to drought stress as compared with each atpub18-2atpub19-3 and atpub22atpub23 double mutant progeny; however, its stomatal behavior was almost identical to the atpub18-2atpub19-3 double mutant in the presence of ABA, H 2 O 2 , and calcium. Overexpression of AtPUB18 and AtPUB19 in atpub22atpub23 effectively hindered ABA-dependent stomatal closure, but overexpression of AtPUB22 and AtPUB23 in atpub18-2atpub19-3 did not inhibit ABA-enhanced stomatal closure, highlighting their ABA-independent roles. Overall, these results suggest that AtPUB18 has a linked function with AtPUB19, but is independent from AtPUB22 and AtPUB23, in negative regulation of ABA-mediated drought stress responses.
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The ubiquitin (Ub)-26S proteasome pathway is implicated in various cellular processes in higher plants. AtAIRP1, a C3H2C3-type RING (for Really Interesting New Gene) E3 Ub ligase, is a positive regulator in the Arabidopsis (Arabidopsis thaliana) abscisic acid (ABA)-dependent drought response. Here, the AtAIRP2 (for Arabidopsis ABA-insensitive RING protein 2) gene was identified and characterized. AtAIRP2 encodes a cytosolic C3HC4-type RING E3 Ub ligase whose expression was markedly induced by ABA and dehydration stress. Thus, AtAIRP2 belongs to a different RING subclass than AtAIRP1 with a limited sequence identity. AtAIRP2-overexpressing transgenic (35S:AtAIRP2-sGFP) and atairp2 loss-of-function mutant plants exhibited hypersensitive and hyposensitive phenotypes, respectively, to ABA in terms of seed germination, root growth, and stomatal movement. 35S:AtAIRP2-sGFP plants were highly tolerant to severe drought stress, and atairp2 alleles were more susceptible to water stress than were wild-type plants. Higher levels of drought-induced hydrogen peroxide production were detected in 35S:AtAIRP2-sGFP as compared with atairp2 plants. ABA-inducible drought-related genes were up-regulated in 35S:AtAIRP2-sGFP and down-regulated in atairp2 progeny. The positive effects of AtAIRP2 on ABA-induced stress genes were dependent on SNF1-related protein kinases, key components of the ABA signaling pathway. Therefore, AtAIRP2 is involved in positive regulation of ABA-dependent drought stress responses. To address the functional relationship between AtAIRP1 and AtAIRP2, FLAG-AtAIRP1 and AtAIRP2-sGFP genes were ectopically expressed in atairp2-2 and atairp1 plants, respectively. Constitutive expression of FLAG-AtAIRP1 and AtAIRP2-sGFP in atairp2-2 and atairp1 plants, respectively, reciprocally rescued the loss-of-function ABA-insensitive phenotypes during germination. Additionally, atairp1/35S:AtAIRP2-sGFP and atairp2-2/ 35S:FLAG-AtAIRP1 complementation lines were more tolerant to dehydration stress relative to atairp1 and atairp2-2 single knockout plants. Overall, these results suggest that AtAIRP2 plays combinatory roles with AtAIRP1 in Arabidopsis ABAmediated drought stress responses.
Ethylene at low concentrations inhibits the light-induced opening of the bean hypocotyl hook; auxin inhibits the opening by inducing production of ethylene. Light causes a decrease in ethylene production and an increase in the production of carbon dioxide. Hook opening appears to be a response in which ethylene serves as a natural growth regulator and in which carbon dioxide may be involved also as a growth regulator through its antagonism of the action of ethylene.
Indoleacetic acid-induced ethylene production and growth in excised segments of etiolated pea shoots (Pisum sativum L. var. Alaska) parallels the free indoleacetic acid level in the tissue which in turn depends upon the rate of indoleacetic acid conjugation and decarboxylation. Both ethylene synthesis and growth require the presence of more than a threshold level of free endogenous indoleacetic acid, but in etiolated tissue the rate of ethylene production saturates at a high concentration and the rate of growth at a lower concentration of indoleacetic acid. Auxin stimulation of ethylene synthesis is not mediated by induction of peroxidase; to the contrary, the products of the auxin action which induce growth and ethylene synthesis are highly labile.It has been suggested that auxin may stimulate ethylene production by inducing the enzymes involved in the formation of the gas (1). This theory is based primarily upon the finding that inhibitors of protein and RNA synthesis prevent auxininduced ethylene production, and also on the observation that stimulation of ethylene production by auxin has a substantial lag period (1,5,7,18,19). Extracts of pea seedlings contain an enzyme that converts methional to ethylene in a cell-free system (20). The enzyme has been identified as a peroxidase (30), a protein which also catalyzes a number of other reactions including oxidation of IAA (13,14,26). The present study presents evidence which shows the peroxidase content not to play a role in the regulation of ethylene biosynthesis in pea seedlings. Growth, ethylene production, the endogenous IAA content, exogenous IAA level, and rate of metabolism of growth hormone were continuously determined and compared in an attempt to determine the mechanism by which auxin induces ethylene production. (29) which is approximately 100% efficient. The radioactivity remaining in the tissue after alcohol extraction was determined after squashing the sections and drying them on a planchet, under which condition self-absorption was negligible. Enzyme Assay for Ethylene Synthesis. Sections of subapical internodes or plumular hooks were homogenized in 50 mm potassium phosphate buffer at pH 7.8 (10 sections/ml), strained through cheesecloth, and centrifuged at lOOOg for 15 min. The preparation could be further purified by dialysis and ammonium sulfate precipitation as described by Ku et al. for the pea enzyme (20), but a variable loss in activity occurred at each step, making the crude extract preferable for quantitative studies. The resulting supernatant (enzyme preparation) was assayed in 5 ml of reaction mixture described by Yang (30), consisting of 10 mM potassium phosphate buffer (pH 7.8), 0.6 mm methional, 3 em manganese sulfate, 80 mm resorcinol, 2 ytM ethylenediaminetetraacetic acid, 80 fM sodium hydrogen sulfite, with 1 ml of enzyme extract. The mixture was incubated at 25 C in a 25-ml Erlenmeyer flask sealed with a vaccine cap, and typically 1-ml samples of air were withdrawn from the flask at 30-min intervals, and ethylene was ...
Ethylene inhibits hook opening in the bean hypocotyl and at high concentrations induces closure of the hook. Indoleacetic acid and 2,4-dichlorophenoxyacetic acid, whose inhibitory effect on hook opening resembles that of ethylene, stimulate ethylene production from the hook tissue, and this ethylene production is physiologically active in inhibiting hook opening. It is concluded that the inhibition of opening by auxin is due at least in a major part to auxin-induced ethylene production by the hook tissue.Carbon dioxide promotes hook opening, apparently by antagonizing the action of endogenous ethylene. The concentration of respiratory CO2 in the internal gas space of the hook tissue is high enough to play a role in the regulation of hook opening.Red light causes a decrease in ethylene production and an increase in CO2 evolution from the hook tissue. These effects are partially reversible by far-red light. It is concluded that both ethylene and CO2 serve as natural growth regulators which mediate the hypocotyl hook-opening response to light in bean seedlings.
Histone H3 variant H3.3, while differing from canonical H3 (H3.1) by only five amino acids, is assembled into nucleosomes, along with histone H4, at genic regions by the histone chaperone HIRA, whereas H3.1 is assembled into nucleosomes in a CAF-1-dependent reaction. Here, we show that phosphorylation of histone H4 Ser 47 (H4S47ph), catalyzed by the PAK2 kinase, promotes nucleosome assembly of H3.3-H4 and inhibits nucleosome assembly of H3.1-H4 by increasing the binding affinity of HIRA to H3.3-H4 and reducing association of CAF-1 with H3.1-H4. These results reveal a mechanism whereby H4S47ph distinctly regulates nucleosome assembly of H3.1 and H3.3.
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