MeGRXC3 has transcriptional activation ability in yeast and involved in mannitol-induced stress response in transgenic Arabidopsis.
We have identified six CC-type GRX genes, MeGRXC3, C4, C7, C14, C15, and C18 responded to drought in leaves of cassava cultivars . All these six genes were fused to the GAL4 DNA-binding domain (BD) in pGBKT7 (Clontech) respectively, and transformed the constructs into yeast Y187. Yeast cells harboring MeGRXC3:pGBKT7 and other three constructs activated X-α-gal on SD/ -Trp /X-α-gal medium (Fig. 1), suggesting that MeGRXC3, C7, C14, and C15 has transcriptional activation ability.
As transgenic work in cassava is extremely difficult and time-consuming, it was impossible to perform large scale functional identification of drought-responsive genes using transgenic cassava. However, Arabidopsis could be used as model plant for heterologous expression of drought induced cassava genes in gain of function analysis [24, 25]. Therefore, we produced transgenic Arabidopsis that over-expressed MeGRXC3, C4, C15, and C18 respectively. We selected three homozygous lines for each gene that exhibited markedly enhanced expression of CC-type GRXs in normal conditions (Fig. S1). To analyze the abiotic stress tolerance of transgenic Arabidopsis, it is commonly to use in vitro setups in which different growth inhibitory compounds are added to the growth medium. Since CC-type GRX may involve in osmotic induced inhibition on seed germination , here, we used mannitol, a frequently applied compound to induced osmotic stress in transgenic Arabidopsis that overexpressing MeGRXC3, C4, C15, and C18 respectively. We found that 100mM mannitol treatment only severely inhibited seed germination of MeGRXC3-OE Arabidopsis (Table. 1). However, seed germination of MeGRXC4-OE, MeGRXC15-OE, and MeGRXC18-OE Arabidopsis is similar to that of control when treated with 100mM mannitol. These results indicate that MeGRXC3 may involve in mannitol-induced stress response in transgenic Arabidopsis.
Overexpression of MeGRXC3 negatively affects seed germination and seedling growth under mannitol-induced stress
Three MeGRXC3-OE Arabidopsis lines were used for further phenotypic assays. Transgenic Arabidopsis lines that harboring the empty vector were used as control. Seeds were sown on 1/2 MS medium containing with 0mM, 100mM, and 200mM D-mannitol respectively. Effect of mannitol-induced inhibition to seed germination of transgenic Arabidopsis is visible after 14 d of growth (Fig. 2a). The seed germination rate on 100mM mannitol was reduced to less than 64.7% in MeGRXC3-OE lines and to 98.5% in control lines (Fig. 2b). Additionally, the seed germination rate on 200mM mannitol was reduced to less than 26.7% in MeGRXC3-OE lines and to 94.5% in control lines. Thus, seed germination of MeGRXC3-OE lines is hypersensitivity to mannitol, suggesting that MeGRXC3 plays a role in seed germination regulation under mannitol-induced osmotic stress conditions.
To explore whether MeGRXC3 is involved in mannitol-induced growth inhibition in transgenic Arabidopsis, we performed analysis on seedling growth MeGRXC3-OE lines under in vitro stress conditions mediated by different concentrations of mannitol (Fig. 2c). Five-day-old seedlings of transgenic Arabidopsis lines were grown on 1/2 MS medium supplement with 0mM, 100mM, and 200mM D-mannitol respectively. Effect of mannitol-induced inhibition to seedling growth is visible after 14 days grown on the mediums (Fig. 2c). Treatments with 100mM or 200mM mannitol reduced 10.1% or 25.3% biomass of control seedlings. However, biomass of MeGRXC3-OE seedling was reduced by 35.4–59.2% under 100mM mannitol and by 74.6–65.3% under 200mM mannitol (Fig. 2d). It can be concluded that MeGRXC3 overexpression enhanced mannitol-induced growth inhibition in transgenic Arabidopsis.
MeGRXC3 transgenic regulates expression of several stress related transcription factor genes in Arabidopsis
The CC-type GRXs could suppress ORA59 promoter activity by interaction with TGA transcription factors in Arabidopsis , suggesting their gene expression regulation roles in plant. Our previously work also indicated that cassava MeGRXC15 could regulate several stress-related genes expression in transgenic Arabidopsis . Here, to understand the effects of MeGRXC3 overexpression on gene expression regulation, we performed qPCR assays on MeGRXC3-OE Arabidopsis. According to the confirmed or proposed roles of plant GRXs , and reported mannitol-induced growth inhibition related genes , we selected seven stress-related genes (PDF1.2, ERF1, ERF6, WRKY33, WRKY40, WRKY53, GA2OX6) as candidate genes in this study. The qPCR results show that MeGRXC3 overexpression enhanced the expression of all these seven stress-related genes in transgenic Arabidopsis (Fig. 3). Obviously, MeGRXC3 overexpression dramatically up-regulated expression of ERF6 (more than 23 folds of control), which regulate mannitol-induced growth inhibition in Arabidopsis . This suggests that MeGRXC3 affect mannitol stress tolerance in transgenic Arabidopsis probably depends on regulating ERF6 expression.
MeGRXC3 interacts with Arabidopsis TGA2 and TGA5 in the nucleus
Since ROXYs could regulate nuclear gene expression through its interaction with TGA factors [11, 12, 16, 20, 27, 28]. We found that MeGRXC15 could interact with Arabidopsis TGA5 or cassava MeTGA074 in the nucleus . To identify target TGA transcription factor that interact with MeGRXC3, yeast two-hybrid assays was conducted using MeGRXC3 as bait to isolate interaction partners from these TGA factors. The results showed that MeGRXC3 protein was able to interact differentially with TGA factors. It showed a strong affinity for TGA2 and TGA5, but no affinity for TGA1, TGA4, and TGA7, respectively (Fig. 4a).
To further investigate the interactions of MeGRXC3 with TGA factors in planta, the BiFC technique was employed. Nuclear green fluorescence was detected for co-expression of MeGRXC3 and TGA2, or TGA5 (Fig. 4b). As negative controls, co-expression of non-fused YN with one of the YC fusion proteins or non-fused YC with one of the YN fusion proteins failed to reconstitute a fluorescent YFP chromophore (Fig. 4b). As positive controls, green fluorescent protein (GFP) was tagged to the C terminus of TGA factors respectively. Green fluorescence was detected only in the nucleus for transiently expression of TGA2: GFP and TGA5: GFP in tobacco (Fig. 4b). This result suggests the possibility of MeGRXC3 in regulating nuclear gene expression via interaction with TGA factors.
Nucleus localization is required for MeGRXC3 regulating mannitol-induced stress tolerance in transgenic Arabidopsis
The MeGRXC3:GFP fusion protein shows nucleocytoplasmic distribution in Arabidopsis . And BiFC assay show that MeGRXC3 interact with TGA2 and TGA5 in the nucleus. To evaluate whether the nuclear localization is required for function of MeGRXC3 in Arabidopsis, we generated fusion proteins of MeGRXC3 that are either excluded from the nucleus and accumulate in the cytoplasm or only localized in the nucleus (Fig. 5a). Exclusive localization of MeGRXC3 protein in the cytoplasm was achieved by cloning three GFP fragments (3×GFP) in-frame downstream of MeGRXC3, generating a MeGRXC3:3×GFP. Moreover, a nuclear-localized version of MeGRXC3 is created by fusing the nuclear localization signal (NLS) derived from the SV40 large T antigen to the N-terminus of MeGRXC3:GFP, as previously reported for ROXY1 (Li et al., 2009b). We overexpressed these two modified DNA constructs in Arabidopsis under the control of the CaMV 35S promoter for further analyses (Fig. 5b). Indeed, nuclear localization of MeGRXC3 enhanced seed germination sensitivity to mannitol (Fig. 5c), which evidenced by less than 15.7% seeds of NLS:MeGRXC3 lines were germinated (Fig. 5d). On the contrary, the restricted localization to the cytoplasm disturbed the mannitol sensitivity of seed germination (Fig. 5c, d). Moreover, overexpression of MeGRXC3:3×GFP did not enhance mannitol-induced growth inhibition in transgenic Arabidopsis (Fig. 5e), as indicated by reduced biomass of MeGRXC3:3×GFP transgenic lines is similar to that of control under 100mM mannitol treatment (Fig. 5f). These results suggest that nuclear activity of the MeGRXC3 is required and sufficient to regulate response to mannitol-induced osmotic stress in Arabidopsis.
Conserved motifs are required for MeGRXC3 transcriptional activation ability in yeast
The ability of modulating TGA transcription factors is indispensable for CC-type GRXs function in Arabidopsis [12, 16, 27]. The CCMC redox motif and GSH bind motif is required for GRXs redox activity . The L**LL and ALWL motif in CC-type GRXs C terminus are critical for its TGA transcription factors modulation [16, 27]. We have found that four cassava drought-responsive CC-type GRXs including MeGRXC3 show transcriptional activation ability in yeast (Fig. 1). According to conserved motifs within MeGRXC3 (Fig. 6a), we performed mutant on each motif and created a series of MeGRXC3 mutants, which were fused to GAL4 DNA binding domain, and transformed into yeast strain Y187 respectively. When the GSH binding motif has been mutated (P65L or G75L) caused loss of transcriptional activation ability (Fig. 6b). Moreover, mutation in the C-terminal L**LL motif (L92N and L93N) also resulted in transcriptional activation ability loss (Fig. 6b). However, mutation of the fourth amino acid in the C-terminal ALWL motif (V101G) did not affect transcriptional activation ability (Fig. 6b). While mutation of the first amino acid in the C-terminal ALWL motif (A98G) resulted in transcriptional activation ability loss (Fig. 6b). Furthermore, the CCMC motif of CC-type GRXs is required for its redox activity. Mutation of this motif (C21ADMC24A) also resulted in loss of transcriptional activation ability (Fig. 6b). Together, the results suggest that all the conserved motifs are required for the transcriptional activation ability of MeGRXC3 in yeast.
Conserved motifs are indispensable for MeGRXC3 function in the nucleus
To truly understand the nuclear contribution of MeGRXC3 function in planta, we expressed a series of NLS:MeGRXC3:GFP mutant constructs, driven by the 35S promoter in Arabidopsis. Herein, mutation of A98G in the C-terminal ALWL motif and mutation of L92NL93N in the L**LL motif in NLS:MeGRXC3:GFP fusion protein resulted a dramatic recovery in seed germination under mannitol treatment (Fig. 7a), suggesting that MeGRXC3 functions in the nucleus likely dependent on interaction and regulation of TGA transcription factors. Substitution mutants of CCMC motif C21ADMC24A and GSH binding motif G75L were fused to NLS at N-terminus and GFP at C-terminus respectively. Likewise, substitution mutants of these two motifs also caused a striking recovery in seed germination under mannitol treatment (Fig. 7a). These results indicate that conserved motifs are indispensable for MeGRXC3 function in regulating mannitol-induced osmotic stress response in transgenic Arabidopsis.
We therefore analyzed the gene expression alteration by mutation of MeGRXC3 conserved motifs in the abovementioned transgenic Arabidopsis plants. Nuclear overexpression of MeGRXC3 (NLS:MeGRXC3) dramatically enhanced the expression of PDF1.2, ERF1, ERF6, WRKY33, WRKY40, WRKY53, and GA2OX6 in transgenic Arabidopsis (Fig. 7b-h). However, NLS:MeGRXC3 induced expression enhancement of these seven gene was obviously reduced by substitution mutations in MeGRXC3 conserved motifs, especially by L92NL93N and A98G mutations (Fig. 7b-h). These results imply that MeGRXC3 regulates gene expression likely by positively modulating TGA transcription factors in the nucleus.