Diurnal Regulation of Plant Epidermal Wax Synthesis through Antagonistic Roles of the Transcription Factors SPL9 and DEWAX

Abstract

Plant surface waxes form an outer barrier that protects the plant from many forms of environmental stress. The deposition of cuticular waxes on the plant surface is regulated by external environmental changes, including light and dark cycles. However, the underlying molecular mechanisms controlling light regulation of wax production are still poorly understood, especially at the posttranscriptional level. In this paper, we report the regulation of cuticular wax production by the miR156-SPL9 (SQUAMOSA PROMOTER BINDING PROTEIN-LIKE 9) module in Arabidopsis (Arabidopsis thaliana). When compared with wild-type plants, miR156 and SPL9 mutants showed significantly altered cuticular wax amounts in both stems and leaves. Furthermore, it was found that SPL9 positively regulates gene expression of the alkane-forming enzyme ECERIFERUM1 (CER1), as well as the primary (1-) alcohol-forming enzyme ECERIFERUM4 (CER4), to enhance alkane and 1alcohol synthesis, respectively. Our results indicate that complex formation of SPL9 with a negative regulator of wax synthesis, DEWAX, will hamper SPL9 DNA binding ability, possibly by interfering with SPL9 homodimerization. Combined with their diurnal gene and protein expressions, this dynamic repression-activation transcriptional module defines a dynamic mechanism that may allow plants to optimize wax synthesis during daily cycles. These findings provide a regulatory framework for environmental signal integration in the regulation of wax synthesis.

Characterization of the Role of SPL9 in Drought Stress Tolerance in Medicago sativa

“…The role of miR156 in plant growth and development has been well documented [ 19 , 24 , 25 , 26 , 27 ], but functional characterization of its target SPL genes is still lagging, especially in major crops, such as alfalfa. In this study, we found MsSPL9 to play a role in determining plant height and stem thickness in alfalfa.…”

“…MicroRNAs have been garnering attention for their roles in regulating various aspects of development and stress response in plants [ 18 , 19 , 20 , 21 , 22 , 23 , 24 , 25 , 26 , 27 ]. Specifically, studies have shown that alfalfa yield as well as tolerance to heat, salt, and drought stress are improved or maintained in miR156 overexpression (miR156OE) plants [ 18 , 19 , 20 , 21 , 22 , 23 ].…”

“…miR156 functions by silencing downstream genes encoding members of the Squamosa Promoter Binding Protein-Like (SPL) family [ 19 , 20 , 21 , 22 , 23 , 24 , 25 , 26 , 27 , 28 ]. SPLs, like miR156, regulate other downstream genes resulting in specific impacts on phenotypes and traits [ 21 , 22 , 26 , 27 ]. In alfalfa, at least seven MsSPL genes have been reported to be regulated by miR156 through transcript cleavage, including MsSPL9 [ 28 ].…”

“…Previous investigations in alfalfa found that MsSPL9 is downregulated in plants overexpressing miR156 [ 28 ]. While the full functional characterization of each of the seven MsSPLs has not been completed, a few have been studied in a number of other plant species [ 18 , 21 , 22 , 23 , 24 , 25 , 26 , 27 ]. A phylogenetic comparison of the Squamosal Promoter Binding Domain (SBP) of SPL s analysed in Arabidopsis thaliana , Glycine max , Medicago sativa , and Medicago truncatula revealed that MsSPL9 is more closely related to MtSPL9 , AtSPL9 / AtSPL15 , and GmSPL9 / GmSPL17 than any other SPL [ 28 ].…”

“…A phylogenetic comparison of the Squamosal Promoter Binding Domain (SBP) of SPL s analysed in Arabidopsis thaliana , Glycine max , Medicago sativa , and Medicago truncatula revealed that MsSPL9 is more closely related to MtSPL9 , AtSPL9 / AtSPL15 , and GmSPL9 / GmSPL17 than any other SPL [ 28 ]. AtSPL9 was shown to control vegetative phase transition [ 26 ] and epidermal wax synthesis [ 27 ], which are also regulated by miR156. Furthermore, enhanced expression of AtSPL9 reduced anthocyanin accumulation and overexpression of miR156 increased expression of the dihydroflavonol 4-reductase ( DFR) gene, leading to improved drought tolerance [ 21 , 29 ].…”