Analysis of FLA Gene Family in Four Poaceae Species and Functional Characterization of SiFLA6 in Saline–Alkali Tolerance

Fasciclin-like arabinogalactan proteins (FLAs) are ubiquitous in the plant kingdom and play crucial roles throughout the entire life cycle of plant growth and development. To gain valuable genetic insights and to systematically dissect the biological functions of FLAs, we performed a comprehensive genome-wide analysis of the FLA gene family in four Poaceae species, including foxtail millet, green foxtail, switchgrass, and sorghum, along with functional characterization of key candidate genes. A total of 121 FLA family members were identified across the four species. Phylogenetic tree analysis classified these FLAs into four distinct subfamilies, within which the genes exhibited highly conserved gene structures and conserved motifs. The promoters of most FLA genes contained abundant cis-acting elements associated with stress and hormone responses. Furthermore, the saline–alkali tolerance-associated candidate gene SiFLA6 was cloned, and T3-generation homozygous overexpression lines (OE-1 and OE-2) were generated via genetic transformation. Subcellular localization testing using the OE-1 line confirmed that SiFLA6 is located in the endoplasmic reticulum. Phenotypic assays showed that root growth of the OE-1 and OE-2 lines was less inhibited under saline–alkali stress than that of wild-type Arabidopsis thaliana (WT). Consistent with the phenotypic changes, the OE-1 line exhibited significantly higher shoot fresh and dry biomass, chlorophyll content, and activities of SOD, CAT, and POD, as well as higher K+/Na+ and Ca2+/Na+ ratios compared with WT plants. In contrast, the accumulation of H2O2 and MDA in the overexpression line was markedly reduced. Collectively, these results indicate that SiFLA6 overexpression improves plant saline–alkali tolerance.