Knock-down of root pvSPS4 does not induce high expression of other SPS homologs
The main methodology of this study, A.rhizogenes mediated transformation, has a highly variable efficiency that depends on the species of interest and the approach that is employed (Estrada-Navarrete et al. 2007; Bandaranayake and Yoder 2018; Xu et al. 2020). To confirm the success of the transformation, following the growth of hairy roots (Fig. 1a) and excision of the primary roots, we have verified the genomic insertion by the visualization of dsRed fluorescent protein that is downstream of a constitutive promoter (Fig. 1b-c). The visualization of fluorescence was performed with both non-destructive (Fig. 1b) and destructive (Fig. 1c-d) methods. The efficiency of the transformation was evaluated with the analysis of 150 separate root tissues of which 123 displayed red fluorescence indicating an 82% transformation rate. As this is in range with the proposed transformation efficiency of the methodology (Estrada-Navarrete et al. 2007) we proceeded to assessment of RNAi efficacy.
RT-qPCR analysis confirmed the knock-down of pvSPS4 gene under treatment conditions in SucPho roots while the Blank and HumFer plants roots have displayed an upregulation pattern for pvSPS4 under salt stress as expected (Fig. 1e) Under control conditions, pvSPS4 expression did not alter for either HumFer or SucPho roots (Fig S1). WB analysis demonstrated stable control and treatment pvSPS4 levels in Blank and SucPho roots. The knock-down caused a 39.24% decrease (p < 0.01) in pvSPS4 protein levels for the SucPho roots under salt treatment conditions. No significant difference was observed for pvSPS4 levels in control conditions among lines (Fig. 1f).
All organisms are able to endure genetic modifications and disruptions and prolong their fitness through genetic robustness. This capability can be due to genetic redundancy which is the compensation for the inadequacy of one gene by an alternative with analogous function (Tautz 1992). Although there are nine SPS homologs in the P.vulgaris genome, only two of them, Phvul.003G170100 (pvSPS1) and Phvul.006G031700 (pvSPS3), possess high amino acid sequence similarity (54% and 57% respectively) with pvSPS4. The transcriptome analysis in our previous study has demonstrated that pvSPS4 and its homolog pvSPS1 have root-specific expression patterns with high transcription rates. However, pvSPS1 presented stable expression under control conditions and salinity stress, unlike pvSPS4. The other SPS homolog, pvSPS3, had very low transcript levels in wild-type root tissues, but a high level of expression in leaves. pvSPS3 also displayed a upregulation in the leaves upon salt stress which indicates a leaf-specific regulation for this gene (Niron et al. 2020).
The effect of pvSPS4 knock-down on the mentioned SPS homologs, in the hairy roots, was first analyzed with RT-qPCR to investigate their potential for compensation of knock-down by an upregulation. Knock-down of pvSPS4 did not induce differential expression of pvSPS1 upon salt treatment. In response to salinity, pvSPS3 demonstrated a significant downregulation (Fig. 2a). Since pvSPS1 is the root-specific homolog, we measured pvSPS1 protein levels with Elisa. The pvSPS1 protein displayed a small but significant increase (approx. 1.3-fold) in SucPho roots in control, but there was no further increase in treatment conditions (Fig. 2b). Also, SucPho roots displayed higher pvSPS1 protein levels under salt stress compared to HumFer but not Blank roots. Anova results for ELISA (Table S1) demonstrated that the increase of pvSPS1 amount in SucPho roots was significantly related to knock-down but not salt treatment.
pvSPS4 knock-down changes the root glucose/sucrose ratio in both control and salt-stress conditions
As SPS genes are associated with the regulation of carbohydrate metabolism and its partitioning, we investigated the effect of pvSPS4 knock-down on root systems glucose and sucrose contents (Fig. 3). The only significant change in glucose levels was a doubling of glucose levels in SucPho roots which decreased to control levels under salt stress. There was no effect on sucrose levels (Fig. 3a). Anova results demonstrated that knock-down, salt treatment and their interaction had significant effects on the divergence in glucose levels (Table S1).
The divergence in glucose amount has reflected on the glucose/sucrose ratio in SucPho roots as it displayed a significant increase in the control and a significant decrease under salt stress compared to Blank and HumFer roots indicated an imbalance of carbohydrate partitioning (Fig. 3b).
Changes in physiological parameters resulting from pvSPS4 knock-down
To understand the effects of pvSPS4 knock-down related root-sugar imbalance on carbon fixation and plant physiology, we examined the chlorophyll content (Fig. 4) surface area, water content, and membrane integrity of leaves (Fig. 5).
There was no significant difference between the control levels of chl a, or chl b among the composite lines. Upon salt treatment, only SucPho plants showed a significant content reduction in both pigments, but much higher level in chl b. In treatment conditions SucPho chlorophyll levels were also significantly lower than Blank but not HumFer plants (Fig. 4a-b). Anova results showed that while both pigments were significantly affected by knock-down and salt treatment, but only chl b was affected by the interaction of both (Table S1).
The Blank, HumFer, and SucPho lines presented similar phenotypes in control conditions, with a slight growth reduction observed in SucPho plants (Fig. 5a). A larger impact on growth was seen under salt stress: While the Blank plants appeared relatively healthy, SucPho plants were wilted and displayed signs of tissue necrosis. Performance of HumFer plants under salt stress, on the other hand, appeared to be in between the Blank and SucPho plants with observable indications of stress (Fig. 5a). The reduced growth of SucPho plants was apparent with a 45% reduction in leaf area as well while the loss was only minor for Blank and HumFer plants (Fig. 5b-c). Moreover, SucPho plants have demonstrated a major, 11%, loss in leaf RWC under saline conditions while this loss was about 4% for both Blank and HumFer plants. (Fig. 5d). The effect of knock-down was not significant alone according to Anova results, yet the interaction of it with salt treatment was a significant player for the observed decrease (Table S2).
The leaf area and RWC losses under salt stress in the case of pvSPS4 knock-down suggested high cellular damage for leaf tissues of SucPho plants. Indeed, salt stressed SucPho leaf sections lost significantly more electrolytes on the 4th hour of the sectioning compared to Blank and HumFer leaf sections. On the 8th hour, the only significant difference was observed between Blank and SucPho (Fig. 5e). On the contrary, pvSPS4 knock-down did not produce a high electrolyte leakage in control conditions (Fig S2).
Sugar imbalance had negative effects on the ionic balance of the knock-down plants
Next, we have examined the effects of pvSPS4 knock-down related sugar imbalance on ion uptake, transport, and sodium accumulation by measuring, K+, Ca+2, Mg+2, and Na+ contents of the root (Fig. 6) and leaf tissues (Fig. 7). pvSPS4 knock-down had a major effect on both uptake and transport of the K+: Blank and HumFer root K+ levels acted in concert with previous results for Ispir genotype (Niron et. al. 2020) and dropped significantly under salt stress while SucPho root K+ levels significantly increased (Fig. 6a) as observed in a salt susceptible genotype (Niron et. al. 2020). Moreover, the control K+ content in the roots was significantly higher for Blank and HumFer than SucPho, but it was significantly lower in treatment conditions and interaction of knock-down and treatment was the major source for this variation (Table S3). Leaf tissues of all plants have reacted in a similar fashion with a significant accumulation of K+ in response to salt stress, but the extent of change was different: K+ levels increased by 1.25 and 1.35-fold for Blank and HumFer roots respectively while the increase was 1.94-fold for SucPho plants (Fig. 7a).
In general, pvSPS4 knock-down has caused an imbalance in Ca+2 and Mg+2 contents in the tissues. Salt treated SucPho plants presented a significant decrease in Ca+2 content in both root and leaf tissues but there was no significant difference between the control and the treatment conditions in Blank and HumFer tissues (Fig. 6b and Fig. 7b). Salt treatment has resulted in a 1.97-fold decrease in SucPho roots while there was no significant difference in Blank and Humfer roots. In control conditions, both Blank and HumFer roots displayed significantly lower levels of Ca+2. In treatment conditions, on the other hand, only Blank roots maintained significantly higher levels of Ca+2 (1.59-fold) than SucPho roots (Fig. 6b). In the leaf tissues of SucPho plants, Ca+2 levels were much higher than Blank and Humfer plants (1.81 and 2.16-fold respectively) in control conditions. Salinity elicited a steep decrease that has brought SucPho leaf Ca+2 to similar levels of HumFer, yet Blank leaves had significantly higher Ca+2 content in treatment conditions compared to others (Fig. 7b). For both tissues, interaction of salt treatment and knock-down was the major source of the variation observed (Table S3).
Blank and HumFer roots did not show a significant change in Mg+2 levels under saline conditions, but SucPho roots presented a significant decline (2.77-fold). Anova results displayed knock-down as the main source of variation here (Table S3). And, even though control Mg+2 levels were similar for roots of all composite lines, Blank roots have accumulated significantly more Mg+2 compared to both HumFer and SucPho roots (Fig. 6c). Leaf Mg+2 contents displayed a similar response as Ca+2: Blank and HumFer leaf Mg+2 levels did not show a significant change upon salt stress. Although SucPho leaf Mg+2 content in control was lower than both Blank and Humfer to begin with, when exposed to stress conditions, it displayed a steep 1.69-fold increase. The SucPho leaf Mg+2 control level was significantly lower compared to Blank and HumFer leaves while it was significantly higher in treatment conditions (Fig. 7c). In contrast to roots, interaction of knock-down with salt treatment was the main source of variation for leaf Mg+2 levels (Table S3).
All plants presented a majorly significant Na+ upsurge in the case of salt treatment and there was no significant difference between the control or treatment levels of tissues (Fig. 6d and Fig. 7d). On the other hand, roots displayed a meaningful difference for salt-related Na+ uptake regulation in the case of pvSPS4 knock-down: SucPho roots accumulated significantly higher Na+ compared to Blank and HumFer roots under saline conditions. Interestingly HumFer roots accumulated even lower amount of Na+ than Blank roots (Fig. 6d).
Osmoprotection and antioxidant capacity have dropped for SucPho plants
To interpret the physiological consequences of root pvSPS4 knock-down on the tissues, we examined the changes in osmoprotection and antioxidant capacities of the composite lines (Fig. 8). Both systems are known to be highly salt stress-responsive, and a decrease in capacity signifies the increase in sensitivity to stress (reviewed in Parida and Das 2005). Content analysis of proline, a key osmoprotectant (Hayat et al. 2012), in leaves and roots helped us infer the osmoprotection capability of the composite lines in response to salt stress. Curiously, leaf tissues of all lines accumulated significantly different levels of proline from each other in control conditions. Upon salt stress, all composite lines managed to significantly increase their proline content, but to different extents. Blank leaf proline levels have increased by 17.95-fold, while HumFer leaf proline levels have displayed a 3.51-fold upsurge; for SucPho leaves the increase was only 1.63-fold (Fig. 8a).
For root tissues in control conditions, Blank roots accumulated a significantly higher amount of proline compared to HumFer and SucPho roots. Nonetheless, the root proline levels of Blank and HumFer plants displayed a moderate but significant increase (1.91 and 2.01-fold respectively) as a salt stress response, but such increase was absent in SucPho roots (Fig. 8b).
A perturbed antioxidant removal capacity for leaves of the SucPho plants was observed under salt stress (Fig. 8c, d). In control conditions, pvSPS4 knock-down did not cause a significant change in leaf APX activity but SucPho leaves could not manage to increase APX activity under stress conditions as well. On the other hand, APX activity of Blank and HumFer leaves increased by 4.77 and 7.95-fold respectively (Fig. 8c). Similarly, SucPho leaves did not demonstrate any escalation in CAT activity in saline conditions in contrast to Blank leaves which presented more than a 1.5-fold increase, but such response was also absent in HumFer plants as well.
The correlation of responses was higher for Blank and HumFer than SucPho
Although most of the physiological and molecular responses of Blank and HumFer plants to salt stress were similar to each other and deviated from the responses of SucPho plants, there were noteworthy differences between them in control and treatment conditions when compared separately. The adopted methodology introduced another variable, separate from the salt stress application, with the activation of RNAi machinery. Thus, we mapped all the results for the salt stress response of the lines together in the log2 scale to visualize the variation among them (Fig. 9a). Responses of the Blank (black) and HumFer (yellow) plants presented a highly overlapping pattern compared to SucPho (red) in the map. Moreover, we performed a correlation analysis to measure the resemblance of the response patterns of the lines (Fig. 9b). Responses of Blank and HumFer plants displayed very high correlation with Spearman r-value of 0.95. On the other hand, root pvSPS4 knock-down caused a major decrease in correlation level. These results demonstrated the extent of the knock-down of the gene over the influence of RNAi machinery on the stress response.