Role of cassava CC-type glutaredoxin MeGRXC3 in regulating sensitivity to mannitol-induced osmotic stress dependent on its nuclear activity

We previously identified six drought-inducible CC-type glutaredoxins in cassava cultivars, however, less is known about their potential role in the molecular mechanism by which cassava adapted to abiotic stress. Herein, we investigate one of cassava drought-responsive CC-type glutaredoxins, namely MeGRXC3, that involved in regulation of mannitol-induced inhibition on seed germination and seedling growth in transgenic Arabidopsis. MeGRXC3 overexpression up-regulates several stress-related transcription factor genes, such as PDF1.2, ERF6, ORA59, DREB2A, WRKY40, and WRKY53 in Arabidopsis. Protein interaction assays show that MeGRXC3 interacts with Arabidopsis TGA2 and TGA5 in the nucleus. Eliminated nuclear localization of MeGRXC3 failed to result mannitol-induced inhibition of seed germination and seedling growth in transgenic Arabidopsis. Mutation analysis of MeGRXC3 indicates the importance of conserved motifs for its transactivation activity in yeast. Additionally, these motifs are also indispensable for its functionality in regulating mannitol-induced inhibition of seed germination and enhancement of the stress-related transcription factors in transgenic Arabidopsis. MeGRXC3 overexpression confers mannitol sensitivity in transgenic Arabidopsis possibly through interaction with TGA2/5 in the nucleus, and nuclear activity of MeGRXC3 is required for its function.

plant cell ROS homeostasis under both biotic and abiotic stress [10][11][12]. Overexpression of the ROXY1 strongly increased ROS accumulation and caused higher susceptibility to botrytis in Arabidopsis [6]. On the other hand, La Camera et al. [13] showed that the mutant of GRXS13/ROXY18 possessed increased resistance to botrytis. The roxy18/grxs13 mutant showed a higher basal and photo-oxidative stress induced ROS accumulation and therefore caused sensitivity to methyl viologen (MV) and high light (HL), while overexpression of ROXY18/GRXS13 resulted lower ROS accumulation under MV and HL treatments [14]. These results indicate that CC-type GRXs may play antagonistic roles in ROS homeostasis.
Several CC-type GRXs have shown their potential roles in regulating abiotic stress tolerance. Genetic variation in ZmGRXCC14 shows significant association with drought tolerance at seedling stage [5]. Expression of OsGRX6 changes depending on the level of available nitrate, overexpression of this gene delayed leaf senescence in rice [15]. The expression of OsGRX8 could be induced by auxin and abiotic stresses [16]. Overexpression of OsGRX8 enhanced tolerance to various abiotic stresses such as salinity, osmotic and oxidative stress in transgenic Arabidopsis, while repression of OsGRX8 by RNAi in rice caused a dramatically seed germination inhibition under mannitol treatment [16]. A rice CC-type GRX, OsGRX_C7 plays a positive response in salt induced stress by regulating the expression of transports engaged in Na + homeostasis [17]. Moreover, OsGRX_C7 is also involving in arsenic tolerance by altering the transcript of NIPs [18,19]. Most CC-type GRXs play positively regulator role on abiotic stress tolerance in different plants, on the contrary, cassava CC-type GRX MeGRXC15 negatively regulates drought tolerance in transgenic Arabidopsis [8]. It needs more efforts to unravel functions and molecular mechanisms of cassava CC-type GRXs. Yeast-two-hybrid assay showed that several Arabidopsis CC-type GRXs were able to interact with the bZIP transcription factor TGACG-BINDING FACTOR 2 (TGA2) [20,21]. They play regulatory roles by post-translationally modifying TGA transcription factors in either negative or positive means. For example, ROXY1 regulates petal development by negatively modifying a floral specific TGA transcription factor PAN and positively modifying other TGA transcription factors [12]. ROXY19/GRX480 negatively regulates PDF1.2 and detoxification genes by interaction with TGA2, TGA5, and TGA6 [21,22]. However, a cassava CC-type GRX MeGRXC15 interacted with TGA5, function as a positive regulator of several stress-related transcription factors in transgenic Arabidopsis [8]. ROXY8 and ROXY9 were identified as a regulator in hyponastic growth of Arabidopsis by negatively modifying TGA1 and TGA4 [23]. GRXS25 could trigger metabolism of pesticide residue in tomato plants through activating TGA2 factor by posttranslational redox modification [24]. The interaction between ROXYs and TGA transcription factors dependent on a functionally important conserved amino acid motif, namely ALWL motif at the very C-terminus of ROXYs [21].
Previously works showed that CC-type GRXs are involved in phytohormone signalling pathway by interaction with TGA transcription factors in plants. The ROXY19/GRX480 expression is induced by salicylic acid (SA), and act as a negative regulator in Jasmonic acid (JA)/Ethylene (ET) pathway [21,25], suggesting CC-type GRXs regulates crosstalk between SA and JA/ET pathway. The MeGRXC3 expression is induced by ABA in cassava and regulates several genes which involve in ABA and JA/ET pathway [8], indicating CC-type GRXs also regulates crosstalk between ABA and JA/ET. Overexpression of a rice CC-type GRX OsGRX6 caused endogenous gibberellin acid (GA) increasing [15]. Moreover, another CC-type GRX namely PHS9 regulated seed germination of rice through the integration of ROS signaling and ABA signaling [26]. ROXY8, ROXY9, and ROXY19/GRX480 involve in auxin pathway by regulating auxin-induced and growth-related genes therefore affect hyponastic growth of Arabidopsis [23]. Recently, a tomato CC-type GRX GRXS25 was identified as a regulator in brassinosteroid (BR) pathway [24]. It seemed likely that CCtype GRXs play numerous roles in plant phytohormone signalling.
Previously, we have identified six drought-inducible CC-type GRXs from two cassava cultivars [8]. In this study, we characterized one of these cassava genes to investigate the potential function of them. We found that four cassava drought-responsive CC-type GRXs, including MeGRXC3, MeGRXC7, MeGRXC15, and MeGRXC17 showed transcriptional activation ability in yeast. We produced MeGRXC3, MeGRXC4, MeGRXC15, and MeGRXC18 overexpressed transgenic Arabidopsis. Only MeGRXC3 overexpression caused hypersensitivity to mannitol on seed germination and seedling growth in transgenic Arabidopsis. In addition, expression of several stress-related transcription factors, including PDF1.2, ERF1, ERF6, ORA59, DREB2A, WRKY33, WRKY40, and WRKY53 was dramatically up-regulated by MeGRXC3 overexpression in Arabidopsis. We also identified two Arabidopsis TGA transcription factors, TGA2 and TGA5 that interacted with MeGRXC3 in the nucleus. Further analysis indicates that nuclear activity is required for the function of MeGRXC3 in transgenic Arabidopsis. Mutation of conserved motifs in the nuclear localization restricted MeGRXC3 promoted recovery of seed germination from mannitol treatments and dramatically affected its regulation on the expression of stress-related transcription factor in transgenic Arabidopsis.
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 [27,28]. Therefore, we produced transgenic Arabidopsis that over-expressed MeGRXC3, MeGRXC4, MeGRXC15, and MeGRXC18 respectively (Fig. S1). We selected three homozygous lines for each transgene that exhibited markedly enhanced expression of the CC-type GRX in normal conditions for phenotype analyses (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 [16], here, we used mannitol, a frequently applied compound to induced osmotic stress in transgenic Arabidopsis that overexpressing MeGRXC3, MeGRXC4, MeGRXC15, and MeGRXC18 respectively. We found that 100 mM or 200 mM mannitol treatment severely inhibited seed germination of MeGRXC3-OE Arabidopsis (Fig. 2a,  Fig. S2). However, seed germination of MeGRXC4-OE, MeGRXC15-OE, and MeGRXC18-OE Arabidopsis lines is similar to that of wild type when treated with 100 mM or 200 mM mannitol (Fig. S2). These results indicate that MeGRXC3 may involve in mannitol-induced stress response in transgenic Arabidopsis. 100 mM mannitol treatment dramatically reduced seed germination rate of MeGRXC3-OE transgenic Arabidopsis. Consequently, we used this concentration for subsequent mannitol treatments on transgenic Arabidopsis.

Overexpression of MeGRXC3 negatively affects seed germination and seedling growth under mannitol-induced stress
As MeGRXC3 shows transcriptional activation ability in yeast and overexpression of MeGRXC3 caused mannitol-induced inhibition to seed germination in transgenic Arabidopsis (Table 1), we selected this gene for further functional analysis. Three MeGRXC3-OE Arabidopsis lines, MeGRXC3-OE#3, #10, #15 were used for further phenotypic assays. Seeds were sown on 1/2 MS medium containing with 0 mM and 100 mM mannitol respectively. Effect of mannitol-induced inhibition to seed germination of transgenic Arabidopsis is visible at 14 days after sowing (Fig. 2a). The seed germination rate on 100 mM mannitol was reduced to less than 64.7% in MeGRXC3-OE lines and to 98.5% in wild type (Fig. 2b). 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 mannitolinduced growth inhibition in transgenic Arabidopsis, we performed analysis on seedling growth of MeGRXC3-OE lines under in vitro stress conditions mediated by 100 mM mannitol (Fig. 2c, Fig. S3). Five-day old seedlings of wild type and transgenic Arabidopsis lines were grown on 1/2 MS medium supplement with 100 mM mannitol. Effect of mannitol-induced inhibition to seedling growth is visible after treated by 100 mM mannitol for 14 days (Fig. 2c, Fig. S3). Treatments with 100 mM mannitol reduced 10.1% biomass of wild type seedlings. However, biomass of MeGRXC3-OE seedlings was reduced by 35.4 to 59.2% under 100 mM 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  Table 1 Functional characterization of six cassava droughtresponsive CC-type glutaredoxins a Transcriptional activation activity analysis was performed by using GAL4BD-MeGRX fusions in yeast Y187 b Seeds of three independent homozygote lines of each MeGRX overexpression Arabidopsis were incubated on 1/2 MS medium containing with 100 mM D-mannitol for 14 days

Gene name
Transcriptional activation activity a

Sensitivity for seed germination to D-Mannitol b
MeGRXC3 22:41 Arabidopsis [21], suggesting their gene expression regulation roles in plant. Our previously work also indicated that cassava MeGRXC15 could regulate several stressrelated genes expression in transgenic Arabidopsis [8].
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 [9], and reported mannitol-induced growth inhibition related genes [29], we selected several stress-related genes (PDF1.2, ERF1, ERF6, ORA59, DREB2A, WRKY33, WRKY40, WRKY53, GA2OX6) as candidate genes in this study. The qPCR results show that MeGRXC3 overexpression enhanced the expression of all these selected stress-related genes in transgenic Arabidopsis under normal conditions (Fig. 3). Obviously, MeGRXC3 overexpression dramatically up-regulated expression of ERF6 (more than 23 folds of wild type), which regulates mannitol-induced growth inhibition in Arabidopsis [29]. 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 [11,12,21,25,30,31]. We found that MeGRXC15 could interact with Arabidopsis TGA5 or cassava MeTGA074 in the nucleus [8]. 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 nonfused 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 [8]. 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;  Fig. S4), which evidenced by less than 15.7% seeds of NLS:MeGRXC3 lines were germinated under 100 mM mannitol treatment (Fig. 5d). On the contrary, the restricted localization to the cytoplasm disturbed the mannitol sensitivity of seed germination (Fig. 5c, d; Fig.  S4). Moreover, overexpression of MeGRXC3:3 × GFP did not enhance mannitol-induced growth inhibition in transgenic Arabidopsis (Fig. 5e; Fig. S5), as indicated by reduced biomass of MeGRXC3:3 × GFP transgenic lines is similar to that of control under 100 mM 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 [12,21,30]. The CCMC redox motif and GSH bind motif is required for GRXs redox activity [21]. The L**LL and ALWL motif in CC-type GRXs C terminus are critical for its TGA transcription factors modulation [21,30]. We have found that four cassava drought-responsive CCtype 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, c). Moreover, mutation in the C-terminal L**LL motif (L92N and L93N) also resulted in transcriptional activation ability loss (Fig. 6b, c). However, mutation of the fourth amino acid in the C-terminal ALWL motif (V101G) did not affect transcriptional activation ability (Fig. 6b, c). While mutation of the first amino acid in the C-terminal ALWL motif (A98G) resulted in transcriptional activation ability loss (Fig. 6b, c). 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, c). 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 100 mM mannitol treatment ( Fig.  S6; Fig. 7a), suggesting that MeGRXC3 functions in the nucleus likely dependent on interaction and modification of TGA transcription factors. Redox site (C 21 CMC 24 ) and GSH binding site (P 65 *********G 75 ) are required for the redox activity of CC-type GRX. Substitution mutants of CCMC motif C21ADMC24A and GSH binding motif G75L of MeGRXC3 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. S6; Fig. 7a). These results indicate that redox activity is 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 under normal conditions. Nuclear overexpression of MeGRXC3 (NLS:MeGRXC3) dramatically enhanced the expression of PDF1.2, ERF1, ERF6, ORA59, DREB2A, WRKY33, WRKY40, WRKY53, and GA2OX6 in transgenic Arabidopsis (Fig. 7b-i). 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-i). These results imply these conserved motifs are required for the function of MeGRXC3 in the nucleus.

Discussion
CC-type GRX is a land plant-specific GRX subgroup that participates in organ development and stress responses through interaction with TGA transcription factors. Recently, several CC-type GRXs have been intensively studied for their role in plant abiotic stress response and phytohormone signalling [5,15,16,32,33]. We have found that six CC-type GRXs, including MeGRXC3, MeGRXC4, MeGRXC7, MeGRXC14, MeGRXC15, and MeGRXC18 were induced by drought stress and exogenous ABA treatments in leaves of cassava cultivars [8]. This suggesting that CC-type GRXs regulated drought response probably in an ABAdependent pathway. However, it is difficult to analyze all these drought-responsive CC-type GRXs in transgenic cassava. We need criteria for choosing candidate genes that should be further investigated. Therefore, we characterized these cassava genes in yeast and Arabidopsis to investigate the potential regulatory roles of them. Cassava is very different from Arabidopsis, the results cannot be used to determine biological function of cassava CC-type GRX, but it provides clues for further analysis of these genes in transgenic cassava. Fusion of Arabidopsis ROXYs to GAL4 BD shows no autonomous transactivation in yeast [12,21,30]. By contrast, in our study, BD-MeGRXC3, BD-MeGRXC7, BD-MeGRXC14, and BD-MeGRXC15 exhibited strong autonomous transactivation activity in yeast (Fig. 1), indicating that MeGRXC3 could recruit transcription factor in yeast nucleus and generate a complex protein like GAL4BD-MeGRX-TF (Activation Domain). Thus, the recombination structure was able to function as a transcription factor promoting the transcription of reporter gene in yeast strain Y187. However, MeGRXC4 and MeGRXC18 did not show autonomous transactivation activity in yeast (Fig. 1). These results suggest these CCtype GRXs may play different roles in cassava drought response. Therefore, we produced transgenic Arabidopsis that overexpressing MeGRXC3, MeGRXC4, MeGRXC15, and MeGRXC18 respectively, to identify whether they have different functions in plant.
To evaluate abiotic stress tolerance of transgenic plants, researchers commonly use in vitro setups in which different inhibitory supplements are added to the culture medium. For example, mannitol and polyethylene glycol (PEG) are frequently applied supplements to induce stress to the plant. Our data showed that mannitol treatment dramatically inhibited seed germination of MeGRXC3-OE transgenic Arabidopsis, but did not affect that of MeGRXC4-OE, MeGRXC15-OE, and MeGRXC18-OE transgenic Arabidopsis (Fig. 2; Fig.  S1; Table 1). In parallel, overexpression of MeGRXC3 enhanced mannitol-induced seedling growth inhibition in transgenic Arabidopsis (Fig. 2). These indicating that cassava drought-responsive CC-type GRXs really have different functions in plant. Mannitol can result in activation of stress-responsive genes, such as several ETHYLENE RESPONSE FACTORs including ERF1, ERF6 in Arabidopsis [29,34]. Here, MeGRXC3 overexpression resulted in a significant up-regulation of ERF6 in transgenic Arabidopsis (Fig. 3). In Arabidopsis, Overexpression of ERF6 caused extreme mannitol-induced growth inhibition, which directly activates many stressresponsive and transcriptional regulation genes such as WRKY33 and GA2OX6 [35]. Here, these two genes were also up-regulated by overexpression of MeGRXC3 in transgenic Arabidopsis (Fig. 3). Therefore, we propose the hypothesis that MeGRXC3 negatively regulates mannitol tolerance by up-regulating ERF6 in transgenic Arabidopsis. ERF6 could regulate expression of ROS-induced gene such as RBOHs, PRX, APX4, and CATs, play an important role in ROS signaling pathway in Arabidopsis [36][37][38][39][40]. ROS is the most essential signaling of drought induced leaf abscission, which might be regulated by several ERF transcription factors in cassava cultivars [41,42]. Compared with wild type, MeGRXC3-OE transgenic Arabidopsis seedlings accumulated more ROS (Fig. S7). Together, it implies the possibility of MeGRXC3 involving drought induced leaf abscission in cassava through regulating ERF transcription factors. Expression of ERF6 could be induced by exogenous ROS treatment [39], however, it is not clear whether the endogenous ROS increment up-regulate ERF6 expression in MeGRXC3-OE transgenic Arabidopsis.
In Arabidopsis, ROXY19/GRX480 repressing the JA/ ET pathway by negatively modified TGA2 [21]. TGA2 is a bZIP transcription factor, could recognize TGACG element in the promoter of many stress-inducible genes [25,43]. Ectopically expressed ROXY19/GRX480 repressed many genes which contained TGACG element in the promoter [22]. However, overexpression of MeGRXC3 in Arabidopsis enhanced the expression of several transcription factors involved in JA/ET pathway, such as PDF1.2, ERF6, ORA59, WRKY33, and WRKY53 (Fig. 3). Expression of PDF1.2 is regulated by ethylene responsive transcription factor ORA59, ROXY19/GRX480 suppressed ORA59 promoter activity through negatively modifying TGA2 therefore repressed expression of PDF1.2 [21]. On the contrary, MeGRXC3 overexpression dramatically upregulated ORA59 and PDF1.2 (Fig. 3). It indicating that MeGRXC3 may positively modify TGA2 in transgenic Arabidopsis. This result is consistent with MeGRXC3 showing transcription activation ability in yeast (Fig. 1). In addition, we have found that MeGRXC3 was induced by exogenous ABA application in cassava cultivars [8]. Like MeGRXC15, MeGRXC3 also interacted with Arabidopsis TGA2 and TGA5 in the nucleus (Fig. 4). Together, it can be concluded that MeGRXC3 involving crosstalk between ABA and JA/ET signalling pathways through positively modifying TGA2.
The nuclear interaction with TGA factors is required for ROXY1 function in petal development of Arabidopsis [12]. Likewise, eliminated nuclear localization of MeGRXC3 failed to result mannitol-induced germination and growth inhibition in transgenic Arabidopsis (Fig. 5). This indicates that nuclear localization is required for function of MeGRXC3 in transgenic Arabidopsis under mannitol stress. Additionally, the redox site is required for disulfide reductase activity of CC-type GRXs and GSH is the cofactor for the reduction reaction. Substitution mutants in redox site (C 21 DMC 24 ) and GSH (P 65 *********G 75 ) binding site of MeGRXC3 caused autonomous transactivation activity loss in yeast (Fig. 6), and abolished mannitol hypersensitivity in transgenic Arabidopsis (Fig. 7a). Furthermore, these two substitution mutants significantly altered the regulation of MeGRXC3 on expression of ERF6 and ORA59 (Fig. 7b,  d, h). This suggests that the redox activity of MeGRXC3 is essential for the regulation of the target transcription factor. The C-terminal L**LL and ALWL motif in CCtype GRXs are necessary for their interaction with TGA transcription factors [12,21,30]. And the ALWL motif is required for ROXY19/GRX480 repressing the expression of PDF1.2 and ORA59 by interaction with TGA2 in Arabidopsis [21]. Mutations or deletion of the C-terminal L**LL motif (LGPL 92 L 93 ) or ALWL motif (A 98 IWV) of MeGRXC3 also resulted in alterations of autonomous transactivation activity in yeast (Fig. 6), abolishment of mannitol hypersensitivity in transgenic Arabidopsis (Fig. 7a), and alterations of stress-related genes regulation in nucleus (Fig. 7b-i), indicating that the interaction with TGA transcription factor is required for the functions of MeGRXC3. Together, our data implying that MeGRXC3 is able to recruit and positively modified a TGA transcription factor in plant.

Conclusions
CC-type GRXs play important roles with TGA transcription factors in the regulation of organ development, seed germination, defense pathway, nitrate metabolism, and abiotic stress responses. We have identified six droughtresponsive CC-type GRXs from cassava cultivars, however, the molecular functions of these genes are still unclear. This study demonstrates that a cassava CC-type GRX, namely MeGRXC3, regulates mannitol-induced osmotic stress tolerance by nuclear interaction with TGA transcription factors and positively regulating several stress-related transcription factors including ERF6 and ORA59.

Plant materials
Seeds of Arabidopsis thaliana ecotype Columbia-0 (Col-0, ABRC stock number CS60000) was obtained from ABRC and kept in our lab (Institute of Tropical Biosciences and Biotechnology, Chinese Academy of Tropical Agricultural Sciences, Haikou, China). Experimental research on all plants complied with institutional and national guidelines. Arabidopsis and tobacco plants were grown in greenhouse at the Institute of Tropical Biosciences and Biotechnology (Haikou, China). The plants were grown under 12 h light/12 h dark at 20-23 °C until the primary inflorescence was 5-15 cm tall and a secondary inflorescence appeared at the rosette. Arabidopsis transformation was achieved using the floral dip method [44] with A. tumefaciens strain LBA4404 carrying the appropriate DNA constructs.

Transactivation analysis in yeast
The MeGRXC3, MeGRXC4, MeGRXC7, MeGRXC14, MeGRXC15, and MeGRXC18 were in frame fused to the GAL4 binding domain (BD) in pGBKT7 (Clontech) respectively. The stop-codon-less coding sequences of MeGRXC3 mutants were also fused in-frame to the DNA binding domain of GAL4 BD in pGBKT7. The resulted constructs were confirmed by sequencing and transferred into yeast strain Y187 (Clontech). Yeast cells were selected on SD/−Trp medium and positive colonies were checked by PCR using gene specific primers. Three yeast colonies harboring the indicated plasmid were incubated at 30 °C on SD/−Trp medium containing 20 μg/mL X-αgal until blue colonies were formed.

Seed germination assays of transgenic Arabidopsis
For the germination assays, seeds of each line were surface sterilized, sown on solid agar medium plates (1/2 MS, pH 5.7, and 0.7% phytagel) with D-mannitol (0 mM, 100 mM, or 200 mM). Seeds were incubated in the dark at 4 °C for 48 h, and then incubated in 8 h/16 h light/dark growing chamber at 22 °C. Germination was judged by the protrusion of the radicle and the germination rate was scored as the percentage at 14 days after sowing. For each germination assay, the offspring of three independent homozygous lines were used, and at least three biological replicate experiments were performed.

Mannitol tolerance assays of transgenic Arabidopsis seedling
To study the response of transgenic Arabidopsis seedling to mannitol stress, seeds were sown on 1/2 MS medium, then postgermination seedlings were transferred to 1/2 MS medium containing with 0 mM or 100mMD-mannitol at 7 days after sowing. Seedlings were incubated in 8 h/16 h light/dark growing chamber at 22 °C for 14 days. Biomass of treating seedlings was measured as total dry weight. The results were shown as percentage, which biomass of wild type seedlings on 1/2 MS medium containing with 0 mM D-mannitol was indicated as 100%.

Quantitative real-time PCR (qPCR) analysis
Total RNA was isolated from Arabidopsis leaves using an RNAprep Pure Plant Kit (TIANGEN). cDNA synthesis was performed using FastQuant RT Kits (TIANGEN). Gene expression analysis in Arabidopsis was performed by qPCR with gene-specific primers (Table S1). All qPCR reactions were carried out in triplicate, with SYBR ® Premix Ex Taq ™ II Kit (Takara) on a StepOne ™ Real-Time PCR system (Applied Biosystems). The comparative ΔΔCT method was employed to evaluate amplified product quantities in the samples.

Protein subcellular localization
Leaves from 4-week-old Nicothiana benthamiana plants were transformed by infiltration using a 5-mL syringe (without needle) to transfer Agrobacterium cells (OD 600 = 1.2) harboring appropriate DNA constructs. After 3 days, infiltrated N. benthamiana leaves were examined for reconstitution of GFP fluorescence by a confocal laser scanning microscope (Olympus FluoView FV1100).

Yeast two-hybrid assay
For screen the interaction proteins of MeGRXC3, a yeast two-hybrid assay has been performed in yeast strain Y2HGold based on the Matchmaker ® Gold Yeast Two-Hybrid System User Manual (Clontech). DNA construct of MeGRXC3P65L:pGBKT7 was used as bait. The cDNA sequences of Arabidopsis TGA1, TGA2, TGA4, TGA5, and TGA7 were introduced into the pGADT7, in frame fused to GAL4 activate domain (AD). All constructs were pairwise co-transformed into yeast strain Y2HGold. The presence of transgenes was confirmed by growth on DDO (SD/−Leu/−Trp) plates. Interactions between two proteins were confirmed by growth on QDO/X/A (SD/− Ade/−His/−Leu/−Trp with 40 μg/mL X-alpha-Gal and 200 ng/mL Aureobasidin A).

Bimolecular fluorescence complementation
To confirm the interactions between MeGRXC3 and TGA factors, a bimolecular fluorescence complementation assay has been performed by tobacco transient system as previously report [45]. The full-length coding sequence without stop-codon of MeGRXC3 was in frame fused to N-or C-terminus of the yellow fluorescent protein (YFP) fragment (YN/YC) respectively to produce 35S:MeGRXC3:YN:pBiFC and 35S:MeGRXC3:YC:pBiFC. The full-length coding sequence without stop-codon of TGA2 and TGA5 were in frame fused to YC or YN respectively to produce 35S:TGA2:YC:pBiFC, 35S:TGA5:YN:pBiFC, 35S: TGA2:YC:pBiFC, and 35S: TGA5:YN:pBiFC. The resulting constructs were then introduced into A. tumefaciens LBA4404 strains. Then the assays were performed as the method of protein subcellular localization described.

Mutation of MeGRXC3
Nuclear localization signal sequence (PKKKRKV) from the SV40 large T antigen was fused to the N-terminus of MeGRXC3:GFP by PCR method to create NLS:MeGRXC3:GFP as described in reference [12]. Three folds of GFP (3 × GFP) DNA was synthesized and fused to C-terminus of MeGRXC3 to create MeGRXC3:3 × GFP as described in reference [12]. Conserved motifs in MeGRXC3 were mutated by sitedirected mutation method to make NLS:MeGRXC3 mutants. The C 21 DMC 24 motif was modified to A 21 DMA 24 . The P 65 was replaced by L 65 as well as G 75 was replaced by L 75 in GHS binding motif respectively. The C-terminal LGPL 92 L 93 motif was replaced by LGPN 92 N 93 . The very C-terminal motif A 98 IWV 101 motif was replaced by G 98 IWI and AIWV 101 , respectively. β-galactosidase quantitative analysis was performed in Y187 strain that harboring respective MeGRXC3mutant:pGBKT7 construct according to Matchmarker ® Gold Yeast Two-Hybrid System User Manual (Clontech).