MeMYB108 is associated with drought-induced leaf abscission in cassava
Our previous work confirmed that the leaf abscission of cassava had obvious difference in different cassava genotypes under the same drought condition (Zhao et al. 2015; Liao et al. 2016a; Liao et al. 2016b). In order to figure out the response of MeMYB108 under drought-induced leaf abscission condition in different cassava genotypes, the expression patterns of MeMYB108 were investigated in SC124 and SC8, which represented leaf abscission to a different extent under the same drought condition (Oliveira et al. 2017; Zhao et al. 2015). Specifically, we analyzed the MeMYB108 expression patterns in the samples of the leaf abscission zones, covering no leaf abscission under normal water conditions (NLA), as well as slight leaf abscission (SLA), moderate leaf abscission (MLA) and very serious leaf abscission (VLA) induced by drought with quantitative PCR. The results showed that the expression of MeMYB108 in SC124 was higher under SLA and MLA conditions and more strongly induced under the VLA condition than that in SC8 (Fig. 1a). These results suggested that the expression of MeMYB108 was significantly related to drought-induced leaf abscission in cassava.
MeMYB108 is an MYB family member
The all of MYB gene sequences of cassava genotype AM560-2 were obtained from the JGI cassava genome database (Supplementary Fig. 1a). The MeMYB108 gene region was 1027 bp long, including two introns (148 bp and 98 bp in length, respectively) and three coding exons (160 bp, 130 bp and 517 bp, respectively). The CDS sequence of the MeMYB108 gene was 807 bp in length and encoded 268 amino acids. The CDS sequences were aligned in the conserved domain database (CDD) of NCBI. Two MYB binding domains were discovered in the N-terminus of MeMYB108, indicating the MeMYB108 gene might belong to the R2R3 type MYB family. MeMYB108 was confirmed as a nucleus-localized protein in Nicotiana benthamiana leaves by infiltrating with Agrobacterium tumefaciens (strain GV3101) containing 35S: MeMYB108-GFP (Supplementary Fig. 1b).
Expression profiles of MeMYB108 induced by various leaf abscission-promoting treatments in cassava
To determine the spatiotemporal expression patterns of MeMYB108 under normal growth conditions, we isolated total RNA in three representative tissues (root, petiole, and leaf) from SC8 and SC124 genotypes and performed RT-qPCR analysis (Fig. 1b-h). The data indicate no expression of MeMYB108 in all tested tissues under normal conditions (Fig. 1b); that is, MeMYB108 is an inducible functional gene. Our previous research identified that some functional genes could be induced by environmental factors of leaf abscission promotion, such as ethylene, drought and cold. Moreover, the expression profiles of MeMYB108 in response to those stress-induced leaf abscissions were carried out. In order to specify the relevance of the function of MeMYB108 to leaf abscission, we only focused on its expression pattern in tissues of leaf abscission zones, which were identified to significantly increase in drought and ethylene-induced abscission (Fig. 1c, d) rather than in cold-induced abscission (Fig. 1e). Furthermore, we also found that the expression levels of MeMYB108 in leaf abscission zones were strongly induced by other stresses, including JA (Fig. 1f), NaCl (Fig. 1g) and abscisic acid (Fig. 1h). All those results verified that MeMYB108 could be strongly induced by stresses of leaf abscission promotion, suggesting the pertinence of the function of the MeMYB108 with the stress-induced leaf abscission.
Overexpression of MeMYB108 in cassava decreases the VLA-induced leaf abscission rate
In order to understand whether the biological function of MeMYB108 relates to the stress-induced leaf abscission, MeMYB108 transgenic cassava plants of over-expression and RNA interference were obtained. The transgenic cassava lines were identified by hygromycin screening and real-time RT-PCR (Supplementary Fig. 2). The expression levels of MeMYB108 in overexpression lines (OE1, OE4, OE5) were higher than those in wild-type and MeMYB108-RNA interference lines (RI2, RI5, RI6) (Supplementary Fig. 2). Two transgenic lines (OE1 and OE5) and two RNA interference transgenic lines (RI2 and RI6) were selected for further analysis under drought-induced leaf abscission conditions. Under NLA conditions, no difference was found in growth between transgenic plants and wild plants (Supplementary Fig. 3). However, differences were significant under drought-induced leaf abscission conditions. The drought-induced leaf abscission rates in MeMYB108-OE lines, MeMYB108-RNA interference lines and wild types were measured under 7-day rewatering conditions after 21-day water withholding. Results indicated that only 49.2–74.6% of leaves were shed in MeMYB108-OE plants, while almost all leaves were shed in the wild plants (87.6%-98.3%) and the MeMYB108-RNA interference lines (85.5%-97.8%) (Fig. 2). These results indicated that MeMYB108 regulated the drought-induced leaf abscission in cassava.
The levels of reactive oxygen scavengers vary significantly in different cassava genotypes with drought-induced leaf abscission
Our previous work demonstrated that ROS could regulate cassava leaf abscission under drought conditions. In view of this, we measured the levels of reactive oxygen scavengers (SOD, CAT, POD) in different cassava genotypes with consecutively two-year drought-induced leaf abscission (2014 and 2015). Among 97 cassava genotypes, the SOD content was the highest (33.81) in 2015 and the lowest (0.10) in 2014. The CAT content was the highest (512.81) in 2015 and the lowest (0.00) in 2014. The results showed the differential natural changes of reactive oxygen scavengers in various cassava genotypes with identical drought-induced leaf abscission (Supplementary Table S3). As the statistical results shown in Fig. 1 and Supplementary Table S3, the LAC based on the leaf abscission of various reactive oxygen scavengers is a continuous quantitative character and changes obviously. The greatest coefficient of reactive oxygen scavengers is CAT-R-2014, while the smallest is SOD-R-2014. The largest coefficient of variation is CAT-L-2015 (Fig. 3a; Supplementary Table S3). The results showed that levels of reactive oxygen scavengers varied greatly in different cassava genotypes with drought-induced leaf abscission.
Natural variation of MeMYB108 is associated with reactive oxygen scavengers during drought-induced leaf abscission in cassava
To investigate whether the natural variation of MeMYB108 is associated with reactive oxygen scavengers during leaf abscission induced by drought in cassava, we performed the candidate gene association analysis. The nucleotide diversity analysis of the MeMYB108 gene was carried out in 97 cassava genotypes. The results are shown in Supplementary Table S4. The gene region includes 87 single nucleotide polymorphisms (SNPs) and two insertion/deletion polymorphisms (i.e., Insert/Deletion, Indel). The exon region has a total of 59 SNPs, including 17 synonymous mutations and 42 non-synonymous substitutions; the latter results in 40 amino acid differences. The conserved domain includes 2 synonymous substitutions and 12 non-synonymous substitutions. The DNA nucleotide diversity was analyzed by the sliding window with a window size of 100 bp and a step size of 25 bp using DnaSP5 software. The results are shown in Supplementary Fig. 4. The average nucleotide diversity π value is 0.01322. In detail, the highest acid diversity is discovered in the first intron. A total of 87 SNPs and 2 Indels (including 18 base SSR insertion deletion and 1 single-base insertion deletion) have 38 sites with MAF greater than 5%.
The exon region of the MeMYB108 gene includes 29 haplotypes and 7 major haplotypes (Supplementary Fig. 5). All these haplotypes can be divided into two categories (Supplementary Fig. 6). Three amino acid differences are discovered between the two categories, and positive selection is indicated in the third exon region (Supplementary Fig. 7). Category II may be derived from wild-type cassava W14, which may be introgressed into cassava cultivars during long-term breeding and produced by mutation in different directions. The linkage disequilibrium analysis of cultivars revealed two haplotypes existed in the nucleotide variation of the exon region of the MeMYB108 gene, separated by recombination hotspots (Fig. 3b). The three SNPs that divide the haplotype into two major categories are located in haplotype 2, resulting in two amino acid variations (Fig. 3B).
In order to further understand the relationship between MeMYB108 and drought-induced leaf abscission at the genetic level, we used the Haploview 4.2 software to analyze the missense mutations in related sites of the MeMYB108 gene region. The block was divided into confidence intervals, statistical genes and traits. The haplotype consisting of functional SNP loci was associated with the leaf abscission tolerance coefficient ((LAC)), and the haplotype was correlated with the drought-induced leaf abscission.
The MeMYB108 gene region includes 12 non-synonymous replacement SNPs significantly associated with drought-induced leaf abscission. The haplotype analysis of these 12 functional mutation sites was carried out, among which 9 SNPs constituted 1 monolithic block, including 5 haplotypes (frequency>0.01). The cassava materials were grouped according to the five haplotypes, and a total of 96 haplotype groupings and drought-induced leaf abscission groupings were obtained. According to ANOVA analysis of the LAC of single traits among haplotype groups, MeMYB108, CAT-L-2014 (P=2.0E-03), and POD-R-2015 (P=9.0E-03) was significantly associated (Fig. 3a, Supplementary Table S5).
The correlation analyses between candidate gene MeMYB108 and the drought-induced leaf abscission were conducted with the rare allelic variation filtered out under the condition of MAF>0.05. The results are shown in Fig. 3b. A total of 38 SNP/Indel marker sites in the MeMYB108 gene region are involved in the leaf abscission induced by drought-related phenotypic analysis. MeMYB108 is significantly correlated with the drought tolerance coefficient of drought-induced leaf abscission of CAT, SOD, and POD under the threshold conditions (P<0.05, FDR<0.05). When P<0.01, MeMYB108 was significantly correlated with CAT. When P<0.05, a total of 30 SNP loci and 1 intron SSR marker were significantly associated with drought-induced leaf abscission, 21 of which were located in exons, including 9 synonymous mutations and 12 non-synonymous mutations. The average interpretation rate of SNPs for phenotypic variation was 10.13%, ranging from 3.99–39.14%. Non-synonymous mutations were significantly associated with the drought tolerance coefficient of the drought-induced leaf abscission, such as CAT and SOD (Fig. 3b, Supplementary Table S6). Two groups of three SNPs with high linkage disequilibrium were indicated in the third exon region, located in the haplotype 2 interval. The first three SNPs are SNP1_31465734, SNP1_31465773 and SNP1_31465774, significantly associated with the drought tolerance coefficient of CAT and SOD. The second group of 3 SNPs was SNP1_31465762, SNP1_31465765 and SNP1_31465804, significantly relevant to CAT (Fig. 3b, Supplementary Table S6). The results showed that natural variations in MeMYB108 had close ties to CAT with drought-induced leaf abscission.
Genetic complementation test transferring the gene fragment of MeMYB108 from SC124 to SC8 alleviates leaf abscission under the VLA condition
Six SNPs, i.e., SNP1_31465734, SNP1_31465773, SNP1_31465774, SNP1_31465762, SNP1_31465765 and SNP1_31465804, were significantly associated with CAT with drought-induced leaf abscission (Fig. 3b, Fig. 4a). The NJ cluster tree of 97 cassava genotypes/lines was constructed based on the six SNPs, and 97 cassava accessions were divided into two groups (Supplementary Fig. 8). Significant linkage disequilibrium can be found among the six SNPs, and three haplotypes exist with frequencies of 0.853, 0.134 and 0.013, respectively (Fig. 4b). In order to confirm the genetic effect of different alleles of MeMYB108 on drought-induced leaf abscission of cassava, we selected the genotypes SC5 and SC205 (with identical alleles to SC124) and the genotypes SC9 and SC7 (with same alleles as SC8) at these six SNPs (Fig. 4c). We examined whether any difference exists in leaf abscission rates among these genotypes with the same drought-induced leaf abscission. The results indicated the leaf abscission rates of genotypes with the same allele as SC124 (SC5 and SC205) were significantly lower than those with the same allele as SC8 (SC9 and SC7) under VLA conditions (Fig. 4c, d). Only 46.7–63.2% of the plants with the same allele as SC124 wild-type shed their leaves, while almost 86.8–97.5% of the plants with the same allele as SC8 plants shed their leaves under VLA conditions (Fig. 4d).
In order to verify whether MeMYB108 had the same allele as SC124 with drought-induced delayed leaf abscission in cassava, the genetic complementarity test of the MeMYB108 gene fragment was conducted from SC124 to SC8 by transforming the fragment of MeMYB108 of SC124 to SC8. We selected 2 independent lines expressing MeMYB108 (MeMYB108-124). The results showed that under VLA conditions (28 days without watering and 7 days rewatering), the leaf abscission rate of MeMYB108-124 was lower than that of wild type SC8 (Fig. 4e). Only 57.9–73.5% of the MeMYB108-124 plants and almost 87.3–98.9% of the wild-type plants shed their leaves under VLA conditions (Fig. 4f). These results supported that MeMYB108 with the same allele as SC124 might help delay drought-induced leaf abscission in cassava.
MeMYB108-124 genetic complementation plants accumulate greater amounts of CAT in cassava under VLA conditions
The above study confirmed the relationship between the natural variation of MeMYB108 and CAT. In order to further study whether that relation existed in the complementary line MeMYB108-124, the CAT level of MeMYB108-124 was analyzed. We firstly analyzed the gene expression patterns of MeMYB108-124 and SC8 by qRT-PCR, covering the periods of drought-induced leaf abscission. In the three transgenic lines, the expression levels of MeCAT in MeMYB108-124 were significantly higher than that of wild type under VLA conditions and also than the wild type SC8 under MLA and VLA conditions (Fig. 5a). This indicated that the natural variations of MeMYB108 were obviously related to CAT levels in transgenic plant MeMYB108-124. Moreover, the CAT activities in wild-type SC8 and transgenic plant MeMYB108-124 were also analyzed. The total cellular CAT activities in wild-type and transgenic plant dramatically increased under VLA conditions compared with SLA conditions and remained at a high level under MLA conditions (Fig. 5b). The CAT activities were 0.224 and 0.66 under normal and MLA conditions, respectively, in the wild type but were 0.218 and 1.10, respectively, in the transgenic line. The differential CAT activities in the SC8 and transgenic line confirmed the important role of ROS scavengers in modulating drought-induced leaf abscission.