Identification and expression pattern of MeSPL9 in cassava
Bioinformatic analysis of the cassava genome identified 21 full-length SPLs, which is greater than the that (16) reported in Arabidopsis (Guo et al. 2008). All of the deduced MeSPL proteins, with lengths ranging from 148 to 1044 amino acids (aa), contained the conserved SBP domain (Table S1). A phylogenetic tree involving 21 MeSPLs and 16 AtSPLs was constructed based on the neighbor-joining (NJ) method. The topology of the tree demonstrated that these SPLs were sparsely distributed in a majority of clades, and clustered into seven distinct groups, namely Group 1 to Group 7 (Fig. 1A). The MeSPLs were named MeSPL1 to MeSPL15 after the corresponding AtSPLs with highest sequence similarity. Remarkably, 10 AtSPLs are post-transcriptionally targeted by miRNA156 (Wu et al. 2009). Their closest homologs in cassava, including MeSPL3a/3b/4/5, MeSPL9/15, MeSPL2a/2b, and SPL6a/6b/6c/13a/13b, are all proved to be cleaved by mes-miR156 (Chen et al. 2015; Li et al. 2020).
To explore the potential function of SPL genes, expression level changes of all 21 SPLs in cassava were analyzed based on our available RNA-Seq data (Li et al. 2017a). The results showed that MeSPL9 was down-regulated especially by drought (Fig. 1B). The spatial expression pattern analysis revealed that MeSPL9 was preferentially expressed in leaves, stems and shoot apex, while lower expression levels could be detected in roots (Fig. 1C). The qRT-PCR was applied to further verify whether the MeSPL9 expression was related to drought stress. The results were overall in line with transcriptome data showing suppressed MeSPL9 expression under drought conditions (Fig. 1D). The above results demonstrate that MeSPLs may play different roles in cassava under drought conditions, and lower MeSPL9 expression is favored in cassava response to drought stress.
Generation and phenotype analysis of rMeSPL9-SRDX transgenic cassava plants
We tried to generate MeSPL9 knockdown mutant plants using the chimeric repressor gene-silencing technology, in which mes-miR156 resistant MeSPL9 (rMeSPL9) was fused with a 12-aa SRDX under the control of constitutive 35S promoter (Fig. 2A) (Hiratsu et al. 2003). Previous studies demonstrated that this chimeric version can productively suppress the target genes (e.g., TCP genes) of various transcription factors (Guo et al. 2010; Koyama et al. 2007). Five independent transgenic cassava plant lines (named rMeSPL9-SRDX #1 to #5) harboring the MeSPL9-SRDX overexpressing cassette were generated by the use of Agrobacterium-mediated transformation of embryonic calli (Zhang et al. 2000). The basic transcriptional level of rMeSPL9-SRDX in each transgenic line was confirmed using qRT-PCR. #2, #3, and #4 lines exhibited an exceptional up-regulation of rMeSPL9-SRDX, approximately 60-fold higher relative to that of WT (Fig. 2B). The positive integration of the 35S::MeSPL9-SRDX T-DNA was confirmed in transgenic lines using Southern blotting. Three lines were verified as positive insertion into the genome by EcoRI digestion (Fig. 2C). At the same time, we also tried to generate rMeSPL9 overexpression transgenic plants using embryonic calli as receptors for transformation. However, the obtained calli failed to regenerate into plants, which is consistent with previous results that extremely high levels of SPL9 may lead to embryonic lethality (Wang et al. 2008). Therefore, #2 and #3, these two lines together with WT were used for subsequent studies. We carefully compared the growth performance of WT and transgenic plants. Generally, expression of rMeSPL9-SRDX had little effect on the development of cassava plant and were not obviously different from WT (Fig. 2D). However, we noticed hyperaccumulation of anthocyanin at the stem-petiole junction and petiole of rMeSPL9-SRDX plants (Fig. 2E). These results are in line with those obtained from the previous reports that SPL9 participates in inhibiting anthocyanin biosynthesis in Arabidopsis (Gou et al. 2011; Wang et al. 2020).
MeSPL9 negatively mediates drought tolerance at the seedling stage
Cassava is established to be tolerant under drought conditions (Okogbenin et al. 2013), and it was of interest to assess whether rMeSPL9-SRDX overexpression could influence drought tolerance of transgenic plants. Therefore, five-week-old WT and rMeSPL9-SRDX plants grown in pots were subjected to drought stress by water depletion. Prior to drought treatment, all plants displayed active growth status (Fig. 3A). After water depletion for three weeks, WT plants exhibited obvious dehydration symptoms including severe wilting and dehydrated leaves. In contrast, rMeSPL9-SRDX lines displayed more green leaves and relatively less severe dehydration. After rewatering, the rMeSPL9-SRDX plants exhibited increased drought tolerance with higher survival rates relative to WT (Fig. 3A, B). Detached leaves were subjected to dehydration to monitor the water loss rate. As can be seen in Fig. 3c, the WT leaves lost more water compared to the rMeSPL9-SRDX leaves during the entire experiment. Furthermore, proline content, a key indicator of plant drought adaptation (Dobra et al. 2011; Sperdouli and Moustakas 2012; Szabados and Savoure 2010), was markedly increased in the rMeSPL9-SRDX lines compared to WT (Fig. 3D). Collectively, the above results suggest that MeSPL9 negatively regulates drought stress adaptation in cassava.
Identification of MeSPL9-regulated genes and related pathways
To elucidate the molecular mechanism underlying the enhanced drought tolerance of rMeSPL9-SRDX lines, we performed RNA-seq to investigate the transcriptomic changes between two genotypes (WT and rMeSPL9-SRDX lines) under two conditions (control and drought). To this end, the whole transcriptome profiles of rMeSPL9-SRDX and WT plants under control (designated #2C, #3C for rMeSPL9-SRDX lines and WTC for wild-type) and drought treatment conditions (designated #2D, #3D for rMeSPL9-SRDX lines and WTD for wild-type) were compared. In WT, a total of 1,668 DEGs were detected under drought conditions. Among them, 76% of the genes (1,263/1,668) were upregulated and 24% of the genes (405/1,668) were downregulated (Fig. 4A, Table S2). In contrast, 1,940 and 2,385 drought-responsive DEGs were identified in rMeSPL9-SRDX line #2 and #3, respectively, among which approximately 71% of the genes were upregulated. These data suggested that there were larger shifts in gene expression under drought stress in rMeSPL9-SRDX lines. MeSPL9-regulated DEGs were determined by performing pairwise comparisons between rMeSPL9-SRDX lines and WT (#2C vs. WTC, #3C vs. WTC, #2D vs. WTD and #3D vs. WTD). As a result, 644 and 308 DEGs were detected in the #2 and #3 plants, respectively, under control conditions compared to WT. Similarly, 550 and 345 DEGs were found in these two lines, respectively, during drought treatment relative to WT (Fig. 4A, Table S2). We observed that the number of up-regulated DEGs was much greater than that of down-regulated DEGs in rMeSPL9-SRDX plants, suggesting that MeSPL9 may inhibit some of the drought-responsive genes.
Further investigation demonstrated that the expression of 15 and 30 genes were promoted and suppressed in rMeSPL9-SRDX lines in response to drought stress, respectively (Fig. 4B, C). Gene ontology (GO) enrichment analysis revealed that the DEGs belonging to ‘response to abiotic/biotic stress’ were overrepresented both in the control- and drought-treated rMeSPL9-SRDX plants (Fig. 4D, E). To further elucidate the roles of the MeSPL9-regulated DEGs, KEGG enrichment analysis was performed. Anthocyanin biosynthesis and proline metabolism were significantly up‐regulated in rMeSPL9-SRDX lines under control condition. However, the majority of genes related to monoterpenoid biosynthesis were strongly up‐regulated after drought treatment. It is noteworthy that genes involved in alpha-linolenic acid metabolism were notably overrepresented under both control and drought conditions (Fig. 4F, G). The biosynthetic process of hormone JA using alpha-linolenic acid as substrates in chloroplast has been extensively studied (Han 2017; Wasternack and Hause 2013). The above results indicated that the DEGs involved in plant metabolism and JA signaling pathways may play key roles during drought stress.
JA participates in regulation of stomatal closure in rMeSPL9-SRDX plants
Our KEGG analysis revealed that MeSPL9 may repress JA biosynthesis and signaling in cassava plants. Indeed, previous reports have demonstrated that SPLs can attenuate JA responses by stabilizing JAZ proteins (Mao et al. 2017), the repressor of JA signaling pathway. As we know, JA function as an important regulator in plant development and response to abiotic stimuli, including drought, as well as biotic stimuli (Ahmad et al. 2016; Wasternack and Strnad 2016). In this study, we further found that a number of drought-induced DEGs involved in JA biosynthesis, catabolism, and signaling processes were significantly up-regulated in rMeSPL9-SRDX plants under both control and drought conditions compared with WT according to the transcriptional data (Fig. 5A, B). To validate the results obtained from the transcriptomic analysis, we analyzed the expression of various JA biosynthesis-related genes by qRT-PCR. The expression of MeLOXs, MeAOS, MeAOC, and MeOPRs in rMeSPL9-SRDX lines was continuously induced before and after drought treatment, which is mostly consistent with the RNA-seq results (Fig. S1A-F). These findings prompt us to evaluate the endogenous JA level in transgenic cassava plants. As shown in Fig. 5c, no significant differences in SA and ABA contents were observed between transgenic and WT plants, while JA content was markedly induced in rMeSPL9-SRDX lines compared to WT, indicating that MeSPL9 may negatively regulate the synthesis of endogenous JA.
Previous reports have shown that JA promotes drought resistance of plants by moderating stomatal closure (Xing et al. 2020). We therefore postulate that JA accumulation may confer enhanced drought resistance in rMeSPL9-SRDX plants. To test this postulation, exogenous JA was applied to wild-type cassava seedlings before drought treatment. As a result, seedlings sprayed exogenous MeJA exhibited higher survival rate after drought treatment compared with the control (Fig. 5D, F), indicating that JA was able to increase drought resistance in cassava. Meanwhile we examined the stomatal aperture of both WT and rMeSPL9-SRDX leaves treated with PEG and JA, respectively. As shown in Fig. 5e, smaller stomatal aperture was observed in rMeSPL9-SRDX leaves compared with WT under normal conditions, which corresponded to the lower water loss rates as mentioned above. Stomatal aperture assays also revealed that both exogenous JA and PEG treatment can inhibit stomatal opening in WT and transgenic leaves, whereas rMeSPL9-SRDX conferred a relative lower stomatal closing capacity than WT and showed hyposensitivity to exogenous JA (Fig. 5E, G). Taken together, these results suggested that the elevated JA levels, especially in rMeSPL9-SRDX plants, are responsible for stomatal closure in order to prevent water loss, thereby leading to drought resistance.
rMeSPL9-SRDX plants contain higher levels of soluble sugars
To gain additional insight into the mechanism underlying drought resistance conferred by rMeSPL9-SRDX overexpression, we analyzed soluble sugar levels in PEG treated leaves. By comparison, the sugar contents in rMeSPL9-SRDX leaves, such as glucose, fructose and galactose were significantly higher to the WT under control condition, and were elevated even further as a result of drought treatment (Fig. 6A-C). However, no difference in sucrose levels was detected between WT and rMeSPL9-SRDX plants (Fig. 6D). This result suggested that accumulation of soluble sugar contents could serve as another indicator for evaluating the drought response in rMeSPL9-SRDX leaves. When combined with the transcriptome data presented above, the genes related to carbon metabolism and plant glycolysis pathway appear to be positively associated with drought response. Subsequently, we observed the expression of genes encoding enolase 1 (ENO1), glucose 6‐phosphate/phosphate translocator 2 (GPT2), pyruvate kinase (PK), and vacuolar invertase (INVA) in the starch and sucrose metabolism pathways was remarkably up-regulated in rMeSPL9-SRDX plants (Fig. 6E). Glyceraldehyde-3-phosphate dehydrogenases (GAPCs) are ubiquitous proteins that function in the glycolytic pathway and are involved in stress response (Guo et al. 2012; Li et al. 2019b). In rMeSPL9-SRDX plants, the expression level of MeGAPC was also significantly increased. Moreover, we also discovered down-regulated genes by overexpressing rMeSPL9-SRDX, such as sucrose-phosphate synthase (SPS), which serves as one of the rate-limiting steps in sucrose synthesis in plants (Fig. 6E). Additionally, the expression of MeENO1, MeGPT2, MePK, and MeGAPC was notably induced under drought stress. However, no particular difference in the expression of MeINVA and MeSPS was observed between control and drought conditions.