Cosuppression of AtGELP22 and AtGELP23, two ubiquitinated target proteins of RING E3 ligase AtAIRP5, increases tolerance to drought stress in Arabidopsis.

AtAIRP5 RING E3 ubiquitin ligase was recently identified as a positive regulator of the abscisic acid (ABA)-mediated drought stress response by stimulating the degradation of serine carboxypeptidase-like 1. Here, we identified GDSL-type esterase/lipase 22 (AtGELP22) and AtGELP23 as additional interacting partners of AtAIRP5. Yeast two-hybrid, pull-down, co-immunoprecipitation, and ubiquitination analyses verified that AtGELP22 and AtGELP23 are ubiquitinated target proteins of AtAIRP5. AtGELP22 and AtGELP23 were colocalized with AtAIRP5 to punctate-like structures in the cytosolic fraction, in which PYK10 and NAI2, two ER body marker proteins, are localized. T-DNA insertion atgelp22 and atgelp23 single knockout mutant plants showed phenotypes indistinguishable from those of wild-type plants under ABA treatment. In contrast, RNAi-mediated cosuppression of AtGELP22 and AtGELP23 resulted in hypersensitive ABA-mediated stomatal movements and higher tolerance to drought stress than that of the single mutant and wild-type plants. Taken together, our results suggest that the putative GDSL-type esterases/lipases AtGELP22 and AtGELP23 act as redundant negative regulators of the ABA-mediated drought stress response in Arabidopsis.

Divergent evolution of extreme production of variant plant monounsaturated fatty acids.

Metabolic extremes provide opportunities to understand enzymatic and metabolic plasticity and biotechnological tools for novel biomaterial production. We discovered that seed oils of many Thunbergia species contain up to 92% of the unusual monounsaturated petroselinic acid (18:1Δ6), one of the highest reported levels for a single fatty acid in plants. Supporting the biosynthetic origin of petroselinic acid, we identified a Δ6-stearoyl-acyl carrier protein (18:0-ACP) desaturase from Thunbergia laurifolia, closely related to a previously identified Δ6-palmitoyl-ACP desaturase that produces sapienic acid (16:1Δ6)-rich oils in Thunbergia alata seeds. Guided by a T. laurifolia desaturase crystal structure obtained in this study, enzyme mutagenesis identified key amino acids for functional divergence of Δ6 desaturases from the archetypal Δ9-18:0-ACP desaturase and mutations that result in nonnative enzyme regiospecificity. Furthermore, we demonstrate the utility of the T. laurifolia desaturase for the production of unusual monounsaturated fatty acids in engineered plant and bacterial hosts. Through stepwise metabolic engineering, we provide evidence that divergent evolution of extreme petroselinic acid and sapienic acid production arises from biosynthetic and metabolic functional specialization and enhanced expression of specific enzymes to accommodate metabolism of atypical substrates.

E3 ligase AtAIRP5/GARU regulates drought stress response by stimulating SERINE CARBOXYPEPTIDASE-LIKE1 turnover.

Ubiquitination is a major mechanism of eukaryotic posttranslational protein turnover that has been implicated in abscisic acid (ABA)-mediated drought stress response. Here, we isolated T-DNA insertion mutant lines in which ABA-insensitive RING protein 5 (AtAIRP5) was suppressed, resulting in hyposensitive ABA-mediated germination compared to wild-type Arabidopsis (Arabidopsis thaliana) plants. A homology search revealed that AtAIRP5 is identical to gibberellin (GA) receptor RING E3 ubiquitin (Ub) ligase (GARU), which downregulates GA signaling by degrading the GA receptor GID1, and thus AtAIRP5 was renamed AtAIRP5/GARU. The atairp5/garu knockout progeny were impaired in ABA-dependent stomatal closure and were markedly more susceptible to drought stress than wild-type plants, indicating a positive role for AtAIRP5/GARU in the ABA-mediated drought stress response. Yeast two-hybrid, pull-down, target ubiquitination, and in vitro and in planta degradation assays identified serine carboxypeptidase-like1 (AtSCPL1), which belongs to the clade 1A AtSCPL family, as a ubiquitinated target protein of AtAIRP5/GARU. atscpl1 single and atairp5/garu-1 atscpl1-2 double mutant plants were more tolerant to drought stress than wild-type plants in an ABA-dependent manner, suggesting that AtSCPL1 is genetically downstream of AtAIRP5/GARU. After drought treatment, the endogenous ABA levels in atscpl1 and atairp5/garu-1 atscpl1-2 mutant leaves were higher than those in wild-type and atairp5/garu leaves. Overall, our results suggest that AtAIRP5/GARU RING E3 Ub ligase functions as a positive regulator of the ABA-mediated drought response by promoting the degradation of AtSCPL1.

RGPDB: database of root-associated genes and promoters in maize, soybean, and sorghum.

Root-associated genes play an important role in plants. Despite the fact that there have been studies on root biology, information on genes that are specifically expressed or upregulated in roots is poorly collected. There exist very few databases dedicated to genes and promoters associated with root biology, preventing effective root-related studies. Therefore, we analyzed multiple types of omics data to identify root-associated genes in maize, soybean, and sorghum and constructed a comprehensive online database of these genes and their promoter sequences. This database creates a pivotal platform capable of stimulating and facilitating further studies on manipulating root growth and development.