**1. Introduction**

Cotton is a valued economic crop worldwide. The long growth cycle of cotton coupled with its large genome size have rendered many available traditional methods complicated and labor-intensive in analyzing its gene function [1]. *Gossypium hirsutum*, commonly referred as upland cotton, is the most popular cotton germplasm due to its high yield. About 90% of all cotton cultivars being produced globally are derived from upland cotton. Due to climate change, crops are exposed to various abiotic stresses affecting plant growth, development, yield components, and productivity [2]. Among them, drought and salinity are the harshest environmental adversities, causing dramatic losses in cotton production [3]. Drought stress induces extensive crop loss, and predictions have revealed that it will intensify in the future [3]. It is estimated that no less than 6% of landmass globally is affected by salinity [4]. Sodium chloride is the primary salt responsible for soil

**Citation:** Kilwake, J.W.; Umer, M.J.; Wei, Y.; Mehari, T.G.; Magwanga, R.O.; Xu, Y.; Hou, Y.; Wang, Y.; Shiraku, M.L.; Kirungu, J.N.; et al. Genome-Wide Characterization of the SAMS Gene Family in Cotton Unveils the Putative Role of *GhSAMS2* in Enhancing Abiotic Stress Tolerance. *Agronomy* **2023**, *13*, 612. https://doi.org/ 10.3390/agronomy13020612

Academic Editor: Tristan Edward Coram

Received: 28 April 2022 Revised: 24 May 2022 Accepted: 25 May 2022 Published: 20 February 2023

**Copyright:** © 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

salinity, and its continued accumulation poses a severe threat to farmers worldwide as agriculture productivity dwindles due to considerable defects in plant growth [5,6]. The presence of sodium chloride in high concentrations usually induces deficiency diseases (the unavailability of crucial nutrients for plants' healthy growth) and disrupts cellular ionic balance [7].

Plants have developed complex and dynamic mechanisms to adapt to these stressful environments, including various morphological, physiological, and molecular changes [8]. The common strategies employed by plants to tolerate drought and salt stresses are the reinforcement and maintenance of biological membranes' structure and properties and the escalated synthesis of antioxidant enzymes [3,9]. Many gene families, such as S-adenosyl-L-methionine synthase (SAMS), are involved in the dynamic complex regulatory networks of plants' stress responses to modulate continued development and enhance stress tolerance [10]. The SAMS genes contain a methionine binding site and an ATP binding motif in their N-terminal and C-terminal domain, respectively [10]. They catalyze the combination of methionine and ATP to produce SAM (S-Adenosyl-L-methionine), a critical molecule involved in essential biological processes in eukaryotic cells [11]. SAM provides methyl groups for DNA, RNA, lipids, and proteins methylation and participates in transsulfuration reactions and the biosynthesis of polyamine, nicotianamine, and lignin [11–14]. Moreover, SAM is the precursor for synthesizing ethylene and polyamines (PAs), which are essential for plant growth, development, and responses to environmental stresses [15–18].

Regarding the importance of SAMS, studies have focused on SAMS' function in regulating plants' stress response. The overexpression of the potato *SbSAMS* improved drought and salt stress tolerance in transgenic *Arabidopsis* plants [2]. In rice, the knockdown of *OsSAMS1*, 2, and 3 altered the histones and DNA methylation, leading to late flowering [19]. The overexpression of the Sugar Beet M14 *SAMS2* in transgenic *Arabidopsis* enhanced its tolerance to oxidative stress and salt [14]. The targeted reduction of PAs biosynthesis induced a decrease in pollen viability and plant length and promoted sensitivity to abiotic stress in rice [20]. The overexpression of *Medicago sativa subsp. falcata SAMS1* induced oxidation and polyamine synthesis in transgenic tobacco plants, improving their tolerance to chilling and freezing stress [21]. The overexpression of the cucumber *CsSAMS1* and its interacting protein *CsCDPK6* promoted ethylene and PAs biosynthesis, leading to the enhancement of salt stress tolerance in transgenic tobacco [22]. The SAMS gene family has been well studied in diverse monocotyledonous and dicotyledonous plants such as rice, sugar beet M14, *Arabidopsis*, barley, tomato, soybean, sunflower, sorghum, *Medicago truncatula*, eggplant, *Triticum urartu* [11], and *Chorispora bungeana* [23]. However, in upland cotton, no study has focused on SAMS genes and their potential to enhance stress tolerance.

Moreover, it was recently found that *GhCBL10* plays a central role in upland cotton's tolerance to salt stress [24]. Therefore, it is of particular interest to identify the *GhSAMS* with strong co-expression interaction with *GhCBL10* for the targeted improvement of cotton plants' tolerance to multiple abiotic stresses.

In the present study, SAMS genes were identified in upland cotton, and their structure, chromosomal distribution, subcellular localization, phylogeny, cis-acting elements, and conserved motifs were revealed through comprehensive bioinformatic analyses. We performed yeast two-hybrid experiments and detected the *GhSAMS* that exhibited the strongest co-expression relationship with *GhCBL10*. Furthermore, we explored the expression patterns of *GhSAMS* genes in response to salt and drought treatments, and the most promising *GhSAMS* for enhancing plant tolerance to multiple abiotic stresses was identified and functionally validated via transgenic experiments. Our data represent important resources for deciphering *GhSAMSs* in plant functions and insights into the complex molecular regulatory networks of abiotic stress response in cotton.

#### **2. Materials and Methods**

#### *2.1. Protein Identification and Physiochemical Analysis of SAMS Genes in Gossypium hirsutum*

SAMS proteins were retrieved from three Pfam domain accessions in the NAU assembly: PF00438 (1), PF02772 (2), and PF02773 (3). The three accession domains carry 16, 17, and 16 genes, respectively, though the gene names are similar. Pfam Scan was specifically used to query the genes (https://www.ebi.ac.uk/Tools/pfa/pfamscan/; accessed on 5 May 2020), and SMART search provided the identity of SAMS genes present in *Gossypium hirsutum* (http://smart.emblheidelberg.de/smart/; accessed on 20 May 2020). *Gh-SAMS* genes' identity was further confirmed via the official website of the Cotton genomic database (https://cottonfgd.org/; accessed on 29 May 2020), using PF02772. The physical and chemical properties of *GhSAMS* proteins (excluding the scaffolded gene), including the instability index, protein length, isoelectric point (pI), grand average of hydropathy (GRAVY), and molecular weight (MW), were predicted by ExPASy ProtParam software [25].
