*3.5. GhSAMS2 Gene Silencing Significantly Increased Sensitivity to Drought and Salt Stress*

To verify the function of *GhSAMS2* in response to abiotic stresses, it was knocked down through VIGS in cotton seedlings, and the plants' morphological and physiological characteristics were analyzed under drought and salt stress conditions. The phenotypes of the cotton seedlings grown in hydroponics under various conditions are shown in Figure S3. The plants infiltrated with pTRV2: PDS exhibited photo-bleached leaves after 14 days of post-inoculation (Figure S3a). The WT- and TRV2:00-infected seedlings had rapid growth and, morphologically, looked much healthier after three weeks of inoculation (Figure S3b,c). To confirm that *GhSAMS2* was effectively silenced, we analyzed its expression in the

different cotton seedlings via RT-qPCR assays (Figure S3e). The results confirmed that the expression of *GhSAMS2* in WT was significantly higher than that in TRV2:*GhSAMS2* VIGS plants. Figure S4 presents the phenotypes of the WT, TRV2:00, and TRV2:*GhSAMS2* plants under the drought and salt stresses.


We investigated various morphological and physiological parameters under stress conditions. We found minimal differences in the plant heights and root lengths between the VIGS plants and the controls (Figure 4A,C). The control plants had slightly longer roots compared to the treated ones. The root fresh weight and shoot fresh weight of WT were significantly higher than those of the silenced *Gh\_A08G1067* plants after stress treatment (Figure 4B,D). The TRV2:*GhSAMS2* plants showed a significant reduction in leaves' RLWC (relative water content) and chlorophyll content compared to the controls (Figure 4E,F). As expected, the *Gh\_SAMS2*-infiltrated leaves exhibited a significantly increased ELWL (excised leaf water loss) and ion leakage compared to the WT and TRV2:00 plants (Figure 4G,H), indicating the deterioration of biological membranes.

We further analyzed biochemical parameters, including malondialdehyde (MDA) and H2O2 contents and the activity of antioxidant enzyme peroxidase (POD) and catalase (CAT). The contents of MDA and H2O2 in the TRV2:*GhSAMS2* plants were significantly higher than those in the WT under the drought and salt stress conditions (Figure 5c,d). Supportively, the antioxidative activities of POD and CAT were significantly lower in the VIGS plants compared to those in the WT under the conditions of drought and salt stress (Figure 5a,b).

**Figure 4.** VIGS and WT plants' physiological and morphological traits analyzed under the conditions of drought and salt stress. (**A**) Plant height. (**B**) Shoot fresh weight. (**C**) Root length. (**D**) Root fresh weight. (**E**) Relative leaf water content. (**F**) Leaves' chlorophyll content. (**G**) Excised leaf water loss. (**H**) Ion leakage in the leaf. TRV2:00, Positive control; WT, Wild type; and TRV2:*GhSAMS2*, VIGS plants. Different letters above the bars indicate statistically significant differences at *p* < 0.05.

**Figure 5.** Oxidant and antioxidant enzyme biochemical assays in the leaves of WT and VIGs plants after 24 h post-stress-exposure. (**a**) Determination of catalase quantity. (**b**) Determination of peroxidase quantity. (**c**) Determination of hydrogen peroxide quantity. (**d**) Determination of Malondialdehyde quantity. TRV2:00, Positive control; WT, Wild type; and TRV2:*GhSAMS2*, VIGS plants. Different letters above the bars indicate statistically significant differences at *p* < 0.05.

#### **4. Discussion**

Crop production is negatively affected by salinity and alkalinity in semi-arid and arid regions. It is estimated that 831 million hectares of soils in the world are affected by excessive salinity and alkalinity, of which 397 million hectares are saline soils compared to 434 million hectares of alkaline soils [51]. Hence, propagating cultivars that are salt-tolerant to utilize saline soils is of absolute urgency [52]. Previous studies in cotton have pointed out that cotton may have thousands of putative functional genes, but since it is labor-intensive and ineffective to carry out the stable genetic transformation of cotton, the majority of these genes have not yet been characterized [1]. The *SAMS2* gene from previous studies has been found to play a crucial role in plant development regulation, metabolism, and abiotic and biotic stress tolerance mechanisms [12]. The SAMS gene family has been well studied in many different dicot and monocot plants such as tomato, *Arabidopsis*, sunflower, eggplant, soybean, *Medicago truncatula*, barley, sorghum, *Triticum urartu*, and rice [11]. This study identified and functionally characterized the cotton *SAMS2* gene for the targeted enhancement of multiple abiotic stresses tolerance in *G. hirsutum*.

According to the prediction of the subcellular localization, cytoplasm and cytoskeleton are the key sites where *GhSAMS* genes are localized. Besides the cytoplasm, cytosol and chloroplasts also contain substantial SAMS proteins, as previously reported [53]. Interestingly, all the sixteen cotton SAMS genes lacked introns in their gene structure. It is reported that, in eukaryotic organisms, many genes are intronless [54]. Additionally, intronless genes are enriched in plant species such as Populus, *Arabidopsis*, and rice [6]. Therefore, the datasets provided by intronless genes have great potential for comparative genomics and evolutionary studies in eukaryotic organisms. However, studies within a phylogenetic framework on intronless genes are limited to few species, based on the previous evolutionary studies that have been carried out [55]. The lack of intron in the *GhSAMS* genes suggests that they might play important roles in biotic and abiotic stress acclimation mechanisms [56]. The results of the cis-acting regulatory elements analysis support this statement. Key abiotic stress responsiveness cis-elements were detected within the promoter regions of the *GhSAMS* genes [57]. The specific function of each *GhSAMS* gene could be predicted through the phylogenetic relationships.

The *GhSAMS* proteins recorded negative GRAVY values, indicating that they are hydrophilic [33]. Hydrophilic proteins have been highly linked to plant protection through antioxidants and membrane stabilizers during water stress conditions [58]. Furthermore, they prevent the collapse of cells in deficient water conditions by acting as space fillers [59]. Additionally, the presence of hydrophilic proteins in certain plants, invertebrates, and microorganisms has been highly associated with their adaptations to water-scarce ecological conditions [60]. *GhSAMS* genes are stable proteins, as shown by the instability index

values, a property that allows cellular biochemical reactions to proceed despite unfavorable environmental conditions. Enzymes' stability within cells is often shown by the instability index of various proteins involved in multiple reactions for a particular time [61]. Moreover, the proteins encoded by *GhSAMSs* have significantly higher thermal stability, as recorded in high aliphatic index values [62].

Gene expression analysis revealed that most of the *GhSAMS* genes are significantly induced by abiotic stresses. These findings are consistent with previous reports on SAMS genes in various crops such as tomato, *Arabidopsis*, rice, and soybean [63]. Among upland cotton SAMS genes, *GhSAMS2* exhibited the highest expression under both salt and drought stress conditions, suggesting its pivotal role in the plant's adaptation to unfavorable environmental conditions. Supportively, *GhSAMS2* exhibited the highest stability index and interaction frequency with the *GhCBL10* bait protein. The gene's function has been previously studied in many plant species via knocking down using the VIGS tool [64]. The down-regulation of *GhSAM2* via VIGS and the post-exposure of VIGS plants to drought and salt stress confirmed the key role of this gene in moderating abiotic stress tolerance. The TRV2:*GhSAMS2* plants showed growth and biomass accumulation defects compared to the controls under drought and salt stress conditions. Their leaves contained less chlorophyll and exhibited higher ion leakage, indicating the high sensitivity of VIGS-*GhSAM2* plants to abiotic stresses. In general, plants exhibit wilting behaviors when exposed to drought and salt stress [65]. The disruption of stress tolerance mechanisms by abiotic stress in plants often exacerbates the transpiration rate, biological membrane deterioration, and cells' function perturbation [66]. Damage to the phospholipid membrane structure due to oxidation is mainly induced by drought and salt stresses. Hydrogen peroxide and Malondialdehyde contents are the biochemical parameters usually used to determine the cellular damage within the organism's tissues [3]. Oxidative stress in living organisms is dictated by the level of MDA and ROS contents accumulated at a particular time [14]. ROS production is often promoted by reducing the usage of absorption light energy caused by Calvin cycle enzyme inhibition under abiotic stress conditions. [67]. The *GhSAMS2* knockdown in VIGS plants incapacitated the scavenging ability of excess ROS, resulting in acute oxidative stress and high H2O2 and MDA accumulation. The VIGS-*GhSAMS2* plants showed deficiency in terms of CAT and POD activities compared to the control plants, supporting the deterioration of enzymatic oxidation defense systems [68]. These results demonstrate that *GhSAMS2* (Gh\_A08G1067) is a promising gene for enhancing upland cotton and other crops' tolerance to drought and salt stress through molecular breeding. Moreover, they confirm the successful gene knockdown and effectiveness of the tobacco virus rattle vector [3,32]. Further functional characterization of identified *GhSAMSs* via subsequent knockout and overexpression coupled with transcriptomic and metabolomic analyses is required to understand cotton plant stress response mechanisms better.
