Molecular traits and functional analysis of Rapid Alkalinization Factors (RALFs) in four Gossypium species

Rapid Alkalinization Factors (RALFs) are plant-secreted, cysteine-rich polypeptides which are known to play essential roles in plant developmental processes and in several defense mechanisms. So far, RALF polypeptides have not been investigated in the Gossypium genus. In this study, 42, 38, 104 and 120 RALFs were identified from diploid G. arboreum and G. raimondi and tetraploid G. hirsutum and G. barbadense, respectively. These were further divided into four groups. Protein characteristics, sequence alignment, gene structure, conserved motifs, chromosomal location and cis-element identification were comprehensively analyzed. Whole genome duplication (WGD) /segmental duplication may be the reason why the number of RALF genes doubled in tetraploid Gossypium species. Expression patterns analysis showed that GhRALFs had different transcript accumulation patterns in the tested tissues and were differentially expressed in response to various abiotic stresses. Furthermore, GhRALF41-3 over-expressing (OE) plants showed reduction in root length and developed later with short stems and small rosettes than that of the wild type. The GhRALF14-8 and GhRALF27-8 OE plants, especially the latter, showed increase in seed abortion. Both transgenic Arabidopsis and VIGS cotton demonstrate that three GhRALFs are negative regulators in response to salt stress. Our systematic analyses provided insights into the characterization of RALF genes in Gossypium, which forms genetic basis for further exploration in their potential applications in cotton production.