Background: Auxins play an important role in plant growth and development; the auxins responsive gene; auxin/indole-3-acetic acid (Aux/IAA), small auxin-up RNAs (SAUR) and Gretchen Hagen3 (GH3) control their mechanisms. The GH3 genes function in homeostasis by the catalytic activities in auxin conjugation and bounding free indole-3-acetic acid (IAA) to amino acids.
Results: In our study, we identified the GH3 genes in three cotton species; Gossypium hirsutum, Gossypium arboreum and Gossypium raimondii, analyzed their chromosomal distribution, phylogenetic relationships, cis-regulatory element function and performed virus induced gene silencing of the novel Gh_A08G1120 (GH3.5) gene. The phylogenetic tree showed four clusters of genes with clade 1, 3 and 4 having mainly members of the GH3 of the cotton species while clade 2 was mainly members belonging to Arabidopsis. There were no paralogous genes, and few orthologous genes were observed between Gossypium and other species. All the GO terms were detected, but only 14 genes were found to have described GO terms in upland cotton, more biological functions were detected, as compared to the other functions. The GH3.17 subfamily harbored the highest number of the cis-regulatory elements, most having promoters towards dehydration-responsiveness. The RNA expression analysis revealed that 10 and 8 genes in drought and salinity stress conditions respectively were upregulated in G. hirsutum. All the genes that were upregulated in plants under salt stress conditions were also upregulated in drought stress; moreover, Gh_A08G1120 (GH3.5) exhibited a significant upregulation across the two stress factors. Functional characterization of Gh_A08G1120 (GH3.5) through virus-induced gene silencing (VIGS) revealed that the VIGS plants ability to tolerate drought and salt stresses was significantly reduced compared to the wild types. The chlorophyll content, relative leaf water content (RLWC), and superoxide dismutase (SOD) concentration level were reduced significantly while malondialdehyde concentration and ion leakage as a measure of cell membrane stability (CMS) increased in VIGS plants under drought and salt stress conditions.
Conclusion: This study revealed the significance of the GH3 genes in enabling the plant's adaptation to drought and salt stress conditions as evidenced by the VIGS results and RT-qPCR analysis.
Keywords: Cis-regulatory elements; Gretchen Hagen3; Orthologous genes; Virus induced gene silencing.