SPL13 together with SPL9 redundantly regulates wax biosynthesis upon drought stress.

Wax biosynthesis is strictly regulated by many regulators under different environmental conditions. Our previous study showed that the regulation module miR156/SQUAMOSA PROMOTER BINDING PROTEIN-LIKE9 (SPL9)/DEWAX is identified to be involved in the diurnal regulation of wax production, however, it was unknown if other SPLs are also involved in the wax synthesis. Here, we reported that SPL13 regulates drought-induced wax production as well. Moreover, its regulatory role directly or indirectly affects the expression of two wax biosynthesis genes CER1 and CER4. Further study showed that SPL13 together with SPL9 redundantly regulated the wax accumulation upon either normal conditions or drought stress, simultaneous mutation of both genes additively enhanced cuticle permeability and decreased the drought tolerance. However, different from SPL9, SPL13 seemed not to participate in the DEWAX-mediated diurnal regulation of wax production.

Fine-tuning the activities of ß-KETOACYL-COA SYNTHASE 3 (KCS3) and KCS12 in Arabidopsis is essential for maintaining cuticle integrity.

The plant cuticle, consisting of wax and cutin, is involved in adaptations to various environments. ß-Ketoacyl-CoA synthases (KCSs) usually serve as a component of the fatty acid elongation complex that participates in the production of very long-chain fatty acids and provides precursors for the synthesis of various lipids, including wax; however, we recently reported that KCS3 and KCS12 negatively regulate wax biosynthesis. In this current study, we observed that unlike KCS3-overexpressing (OE) lines, KCS12-OE lines had fused floral organs because of abnormal cuticle biosynthesis. This prompted us to compare the functions of KCS3 and KCS12 during cuticle formation. Mutation of KCS3 caused greater effects on wax production, whereas mutation of KCS12 exerted more severe effects on cutin synthesis. The double-mutant kcs3 kcs12 had significantly increased wax and cutin contents compared to either single-mutant, suggesting that KCS12 and KCS3 have additive effects on cuticle biosynthesis. Cuticle permeability was greater for the double-mutant than for the single mutants, which ultimately led to increased susceptibility to drought stress and floral-organ fusion. Taken together, our results demonstrate the regulatory roles of KCS3 and KCS12 during cuticle biosynthesis, and show that maintaining KCS3 and KCS12 expression at certain levels is essential for the formation of a functional cuticle layer.

An ancestral role for 3-KETOACYL-COA SYNTHASE3 as a negative regulator of plant cuticular wax synthesis.

The plant cuticle, a structure primarily composed of wax and cutin, forms a continuous coating over most aerial plant surfaces. The cuticle plays important roles in plant tolerance to environmental stress, including stress imposed by drought. Some members of the 3-KETOACYL-COA SYNTHASE (KCS) family are known to act as metabolic enzymes involved in cuticular wax production. Here we report that Arabidopsis (Arabidopsis thaliana) KCS3, which was previously shown to lack canonical catalytic activity, instead functions as a negative regulator of wax metabolism by reducing the enzymatic activity of KCS6, a key KCS involved in wax production. We demonstrate that the role of KCS3 in regulating KCS6 activity involves physical interactions between specific subunits of the fatty acid elongation complex and is essential for maintaining wax homeostasis. We also show that the role of the KCS3-KCS6 module in regulating wax synthesis is highly conserved across diverse plant taxa from Arabidopsis to the moss Physcomitrium patens, pointing to a critical ancient and basal function of this module in finely regulating wax synthesis.

ArabidopsisKCS5 and KCS6 Play Redundant Roles in Wax Synthesis.

3-ketoacyl-CoA synthases (KCSs), as components of a fatty acid elongase (FAE) complex, play key roles in determining the chain length of very-long-chain fatty acids (VLCFAs). KCS6, taking a predominate role during the elongation from C26 to C28, is well known to play an important role in wax synthesis. KCS5 is one paralog of KCS6 and its role in wax synthesis remains unknown. Wax phenotype analysis showed that in kcs5 mutants, the total amounts of wax components derived from carbon 32 (C32) and C34 were apparently decreased in leaves, and those of C26 to C32 derivatives were obviously decreased in flowers. Heterologous yeast expression analysis showed that KCS5 alone displayed specificity towards C24 to C28 acids, and its coordination with CER2 and CER26 catalyzed the elongation of acids exceeding C28, especially displaying higher activity towards C28 acids than KCS6. BiLC experiments identified that KCS5 physically interacts with CER2 and CER26. Wax phenotype analysis of different organs in kcs5 and kcs6 single or double mutants showed that KCS6 mutation causes greater effects on the wax synthesis than KCS5 mutation in the tested organs, and simultaneous repression of both protein activities caused additive effects, suggesting that during the wax biosynthesis process, KCS5 and KCS6 play redundant roles, among which KCS6 plays a major role. In addition, simultaneous mutations of two genes nearly block drought-induced wax production, indicating that the reactions catalyzed by KCS5 and KCS6 play a critical role in the wax biosynthesis in response to drought.

Molecular Cloning and Functional Analysis of GmLACS2-3 Reveals Its Involvement in Cutin and Suberin Biosynthesis along with Abiotic Stress Tolerance.

Cutin and wax are the main precursors of the cuticle that covers the aerial parts of plants and provide protection against biotic and abiotic stresses. Long-chain acyl-CoA synthetases (LACSs) play diversified roles in the synthesis of cutin, wax, and triacylglycerol (TAG). Most of the information concerned with LACS functions is obtained from model plants, whereas the roles of LACS genes in Glycine max are less known. Here, we have identified 19 LACS genes in Glycine max, an important crop plant, and further focused our attention on 4 LACS2 genes (named as GmLACS2-1, 2, 3, 4, respectively). These GmLACS2 genes display different expression patterns in various organs and also show different responses to abiotic stresses, implying that these genes might play diversified functions during plant growth and against stresses. To further identify the role of GmLACS2-3, greatly induced by abiotic stresses, we transformed a construct containing its full length of coding sequence into Arabidopsis. The expression of GmLACS2-3 in an Arabidopsis atlacs2 mutant greatly suppressed its phenotype, suggesting it plays conserved roles with that of AtLACS2. The overexpression of GmLACS2-3 in wild-type plants significantly increased the amounts of cutin and suberin but had little effect on wax amounts, indicating the specific role of GmLACS2-3 in the synthesis of cutin and suberin. In addition, these GmLACS2-3 overexpressing plants showed enhanced drought tolerance. Taken together, our study deepens our understanding of the functions of LACS genes in different plants and also provides a clue for cultivating crops with strong drought resistance.