Drought is the most serious abiotic stress, which significantly reduces crop productivity. The phytohormone ABA plays a pivotal role in regulating stomatal closing upon drought stress. Here, we ...characterized the physiological function of AtBBD1, which has bifunctional nuclease activity, on drought stress. We found that AtBBD1 localized to the nucleus and cytoplasm, and was expressed strongly in trichomes and stomatal guard cells of leaves, based on promoter:GUS constructs. Expression analyses revealed that
and
are induced early and strongly by ABA and drought, and that
is also strongly responsive to JA. We then compared phenotypes of two
-overexpression lines (
-OX), single knockout
, and double knockout
plants under drought conditions. We did not observe any phenotypic difference among them under normal growth conditions, while OX lines had greatly enhanced drought tolerance, lower transpirational water loss, and higher proline content than the WT and KOs. Moreover, by measuring seed germination rate and the stomatal aperture after ABA treatment, we found that
-OX and
plants showed significantly higher and lower ABA-sensitivity, respectively, than the WT. RNA sequencing analysis of
-OX and
plants under PEG-induced drought stress showed that overexpression of
enhances the expression of key regulatory genes in the ABA-mediated drought signaling cascade, particularly by inducing genes related to ABA biosynthesis, downstream transcription factors, and other regulatory proteins, conferring
-OXs with drought tolerance. Taken together, we suggest that AtBBD1 functions as a novel positive regulator of drought responses by enhancing the expression of ABA- and drought stress-responsive genes as well as by increasing proline content.
Osmotic stress, caused by drought, salinity, or PEG (polyethylene glycol), is one of the most important abiotic factors that hinder plant growth and development. In Arabidopsis, more than 100 ...R2R3-MYB transcription factors (TFs) have been identified, and many of them are involved in the transcriptional regulation of a variety of biological processes related to growth and development, as well as responses to biotic and abiotic stresses. However, the MYB TF involving in both plant development and stress response has rarely been reported. We report here that Arabidopsis AtMYB109, a R2R3-MYB TF, functions as a negative regulator of stomatal closure under osmotic stress as well as of pollen tube elongation. Under PEG-induced osmotic stress, whole leaves of AtMYB109-OXs were intensely wilted, while leaves of the wild-type (WT) and myb109 were weakly affected. Moreover, we confirmed that the wilting in AtMYB109-OXs was more severe than in WT and myb109 under drought conditions, and that after re-watering, WT and myb109 plants promptly recovered, while AtMYB109-OXs failed to survive. In addition, stomatal closure was delayed in the AtMYB109-OXs compared to the WT and myb109. However, proline content and the expression of stress-induced and proline synthesis genes were higher in the overexpression lines than in WT and myb109. Then, we observed that the expression of ICS1, a key gene in SA biosynthesis, was greatly suppressed in AtMYB109-OXs. In addition, we found that AtMYB109 expression gradually increased until the flowers were fully opened and thereafter dramatically decreased during silique development. The pollen tube growth was significantly suppressed in AtMYB109-OXs compared to the WT and myb109. Using EMSA and ChIP-qPCR, we confirmed that AtMYB109 bound to the promoter of RABA4D, a gene encoding a pollen development regulator. Taken together, we suggest the delayed stomatal closing and vulnerable phenotypes in the AtMYB109-OXs under osmotic stress are possibly directly or indirectly associated with a SA-mediated mechanism, and that AtMYB109 suppresses RABA4D that modulates pollen tube growth.
Nucleases are a very diverse group of enzymes that play important roles in many crucial physiological processes in plants. We previously reported that the highly conserved region (HCR), domain of ...unknown function 151 (DUF151) and UV responsive (UVR) domain-containing OmBBD is a novel nuclease that does not share homology with other well-studied plant nucleases. Here, we report that DUF151 domain-containing proteins are present in bacteria, archaea and only Viridiplantae kingdom of eukarya, but not in any other eukaryotes. Two
homologs of OmBBD, AtBBD1 and AtBBD2, shared 43.69% and 44.38% sequence identity and contained all three distinct domains of OmBBD. We confirmed that the recombinant MBP-AtBBD1 and MBP-AtBBD2 exhibited non-substrate-specific DNase and RNase activity, like OmBBD. We also found that a metal cofactor is not necessarily required for DNase activity of AtBBD1 and AtBBD2, but their activities were much enhanced in the presence of Mg
or Mn
. Using a yeast two-hybrid assay, we found that AtBBD1 and AtBBD2 each form a homodimer but not a heterodimer and that the HCR domain is possibly crucial for dimerization.
The basic leucine zipper (bZIP) transcription factors (TFs) function as regulators of many key developmental and physiological processes in all eukaryotes. In this study, we characterized the ...function of Arabidopsis bZIP4, a group S bZIP, whose function was not known. We confirmed that bZIP4 localizes to the nucleus and has DNA-binding affinity. By qRT-PCR and GUS histochemical analysis, we showed that
bZIP4
is specifically expressed in root and that its expression is induced by abiotic stress and ABA. By phenotypic analysis, we demonstrated that the root length and the germination rate of
bZIP4
overexpression (
bZIP4
-Ox) were significantly longer and higher than those of the WT and
bZIP4
-SRDX under higher salt and glucose concentrations, indicating that
bZIP4
-Ox is insensitive and tolerant to abiotic stress. Despite that, we found that
bZIP4
-Ox had enhanced expression of genes encoding protein phosphatases suppressing ABA responsiveness. We also confirmed that bZIP4 interacts with CaM1 and showed that its DNA-binding affinity is inhibited by interaction with CaM1. We propose a model in which the increased cytosolic calcium concentration under stress conditions activates CaM1 to bind bZIP4 to remove it from promoters of genes encoding ABA negative regulators, allowing the plants to operate on a typical ABA signaling pathway.
Sugars Will Eventually be Exported Transporters (SWEETs) are central for sugar allocation in plants. The SWEET family has approximately 20 homologs in most plant genomes, and despite extensive ...research on their structures and molecular functions, it is still unclear how diverse SWEETs recognize different substrates. Previous work using SweetTrac1, a biosensor constructed by the intramolecular fusion of a conformation-sensitive fluorescent protein in the plasma membrane transporter SWEET1 from Arabidopsis thaliana, identified common features in the transporter’s substrates. Here, we report SweetTrac2, a new biosensor based on the Arabidopsis vacuole membrane transporter SWEET2, and use it to explore the substrate specificity of this second protein. Our results show that SWEET1 and SWEET2 recognize similar substrates but some with different affinities. Sequence comparison and mutagenesis analysis support the conclusion that the differences in affinity depend on nonspecific interactions involving previously uncharacterized residues in the substrate-binding pocket. Furthermore, SweetTrac2 can be an effective tool for monitoring sugar transport at vacuolar membranes that would be otherwise challenging to study.
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