Cassava plants (Manihot esculenta Crantz) resist environmental stresses by shedding leaves in leaf pulvinus abscission zones (AZs), thus leading to adaptation to new environmental conditions. Little is known about the roles of cassava R2R3 MYB factors in regulating AZ separation. Herein, 166 cassava R2R3 MYB genes were identified. Evolutionary analysis indicated that the 166 R2R3 MYB genes could be divided into 11 subfamilies. Transcriptome analysis indicated that 26 R2R3 MYB genes were expressed in AZs across six time points during both ethylene- and water-deficit stress-induced leaf abscission. Comparative expression profile analysis of similar SOTA (Self Organizing Tree Algorithm) clusters demonstrated that 10 R2R3 MYB genes had similar expression patterns at six time points in response to both treatments. GO (Gene Ontology) annotation confirmed that all 10 R2R3 MYB genes participated in the responses to stress and ethylene and auxin stimuli. Analysis of the putative 10 R2R3 MYB promoter regions showed that those genes primarily contained ethylene- and stress-related cis-elements. The expression profiles of the genes acting downstream of the selected MYBs were confirmed to be involved in cassava abscission zone separation. All these results indicated that R2R3 MYB plays an important regulatory role in AZ separation.

Cassava plants (Manihot esculenta Crantz) have obvious abscission zone (AZ) structures in their leaf pulvinus-petioles. Cassava leaf abscission can be triggered by either 17 days of water-deficit stress or 4 days of ethylene treatment. To date, little is known about cassava AP2/ERF factors, and less is known regarding their roles in regulating abscission zone development. Here, the cassava and Arabidopsis AP2/ERF genes were compared, finding that the cassava genome contains approximately 1.54-fold more ERF subfamily than the Arabidopsis genome. Microarray analysis was used to identify the AP2/ERF genes that are expressed in cassava leaf pulvinus-petiole abscission zones by comparing the AP2/ERF gene expression profiles of ethylene- and water-deficit stress-induced leaf abscission. In total, 99 AP2/ERF genes were identified as expressed in AZs across six time points during both ethylene- and water-deficit stress-induced leaf abscission. Comparative expression profile analysis of similar SOTA (Self Organizing Tree Algorithm) clusters at six time points during ethylene- and water-deficit stress-induced leaf abscission demonstrated that 20 ERF subfamily genes had similar expression patterns in response to both treatments. GO (Gene Ontology) annotation confirmed that all 20 ERF subfamily genes participate in ethylene-mediated signalling. Analysis of the putative ERF promoter regions shown that the genes contained primarily ethylene- and stress-related cis-elements. Further analysis of ACC oxidase activity in AZs across six time points during abscission shown increased ethylene production in response to both ethylene and water-deficit stress; however, the difference was more dramatic for water-deficit stress. Finally, the expression ratios of 20 ERF subfamily genes were analysed in two cassava cultivars, ‘KU50’ and ‘SC5’, that exhibit different levels of leaf abscission when challenged with the same water-deficit stress. The analysis indicated that most of the ERF genes were expressed at higher levels in the precocious abscission ‘KU50’ cultivar than in the delayed abscission ‘SC5’ cultivar. Ccomparative analysis of both ethylene- and water-deficit stress-induced leaf abscission shown that the ERF subfamily functions in the regulation of cassava abscission zone development.

The reactive oxygen species (ROS) signal regulates stress-induced leaf abscission in cassava. The relationship between the function of the cassava transcription factor bHLH gene and low temperature-induced leaf abscission is still unclear. Here, we report that MebHLH18, a transcription factor, involved in regulating low temperature-induced leaf abscission in cassava. The expression of the MebHLH18 gene was significantly related to low temperature-induced leaf abscission and POD level. Under low temperatures, the levels of ROS scavengers in different cassava genotypes were significantly different in the low temperature-induced leaf abscission process. Cassava gene transformation showed that MebHLH18 overexpression significantly decreased the low temperature-induced leaf abscission rate. Simultaneously, interference expression increased the rate of leaf abscission under the same conditions. ROS analysis showed a connection between the decrease in the low temperature-induced leaf abscission rate caused by MebHLH18 expression and the increase in antioxidant activity. A Genome-wide association studies analysis showed a relationship between the natural variation of the promoter region of MebHLH18 and low temperature-induced leaf abscission. Furthermore, studies showed that the change in MebHLH18 expression was caused by a single nucleotide polymorphism variation in the promoter region upstream of the gene. The high expression of MebHLH18 led to a significant increase in POD activity. The increased POD activity decreased the accumulation of ROS at low temperatures and the rate of leaf abscission. It indicates that the natural variation in the promoter region of MebHLH18 increases antioxidant levels under low temperatures and slows down low temperature-induced leaf abscission.

Key Message: Natural variation of the MeMYB108 exon was associated with reactive oxygen scavengers led to alleviate leaf abscission under drought in cassava. The reactive oxygen scavengers play important roles in regulating the cassava (Manihot esculenta Crantz) leaf abscission induced by stresses. To date, the relationship between natural variations of MYB genes and reactive oxygen scavengers under drought in cassava genotypes remains unclear. Here, we reported the transcription factor MeMYB108 played an important role in regulating leaf abscission exposed to drought in cassava. The expression levels of MeMYB108 in abscission zones of cassava leaf pulvinus were higher in cassava genotype SC124, which were less easy to shed leaves under stress than cassava genotype SC8 when the leaf abscission induced by the same drought condition. Compared with wild type and interference expression plants, overexpression of MeMYB108 significantly reduced the drought-induced leaf abscission rate under drought. The consecutively 2-year analysis of reactive oxygen scavengers showed significant differences among different cassava genotypes under drought-induced leaf abscission, indicating the relevance between reactive oxygen scavengers and leaf abscission. Correlation analysis revealed the natural variation of the MeMYB108 exon was associated with reactive oxygen scavengers during drought-induced leaf abscission. Association analysis between pairwise LD of DNA polymorphism indicated the MeMYB108 allele enhanced the tolerance of cassava to drought-induced leaf abscission. Complementation transgenic lines containing the elite allele of MeMYB108^SC124 decreased the leaf abscission rate induced by drought conditions, demonstrating natural variation in MeMYB108 contributed to leaf abscission tolerance induced by drought in cassava. Further studies showed MeMYB108 played an active role in the tolerance of cassava to drought-induced leaf abscission by inducing scavenging of reactive oxygen species.