G65V Substitution in Actin Disturbs Polymerization Leading to Inhibited Cell Elongation in Cotton

Abstract

The importance of the actin cytoskeleton for proper cell development has been well established in a variety of organisms. Actin protein sequences are highly conserved, and each amino acid residue may be essential for its function. In this study, we report the isolation and characterization of GhLi 1 from an upland cotton mutant Ligon lintless-1 (Li1), which harbors the G65V substitution in its encoded actin protein. Li1 mutants exhibit pleiotropic malformed phenotypes, including dwarf plants, distorted organs, and extremely shortened fibers. Cytological analysis showed that the actin cytoskeleton was disorganized and the abundance of F-actin was decreased in the Li1 cells. Vesicles were aggregated into patches, and excessive cellulose synthase complexes were inserted into the plasma membrane during the secondary cell wall biosynthesis stage, which dramatically affected the morphology of the Li1 cells. Molecular model prediction suggested that the G65V substitution may affect the three-bodied G-actin interaction during F-actin assembly. Biochemical assays demonstrated that the recombinant GhLi1 protein disturbs actin dynamics by inhibiting the nucleation and elongation processes. Therefore, our findings demonstrate that the G65V substitution in actin had dominant-negative effects on cell elongation, by disturbing actin polymerization and actin cytoskeleton-based biological processes such as intracellular transportation.

A Modified Actin (Gly65Val Substitution) Expressed in Cotton Disrupts Polymerization of Actin Filaments Leading to the Phenotype of Ligon Lintless-1 (Li1) Mutant

“…Actin is one of the most conserved proteins in eukaryotes. Structural predictions suggested that the G65V substitution would affect the polymerization capability of GhACT17DM, as confirmed by previous in vitro assays [ 12 ]. Importantly, our live-cell observations revealed that Li 1 cells showed defective dynamic behavior of the actin cytoskeleton, compared with that of the wild-type control ( Figure 4 ).…”

“…Thyssen et al also identified Gh_D04G0865 as the Li 1 candidate, labeled by a SNP marker, CFB11927 (23,783,515 bp in G. hirsutum Chr D04), using sequencing-mapping and VIGS [ 11 ]. The experiment carried out by Thyssen et al used a 354-bp fragment from the conservative domain of actin C-terminal sharing high similarity among actin members that would not discriminate the actin families in VIGS, which resulted in a Li 1 -like phenotype but in a different mechanism [ 12 ]. Benefiting from the whole genome sequencing of upland cotton [ 25 ], we now know that 37 actin genes with complete NBDs sharing high identity occur in the TM-1 genome, including Gh_D04G0865 ( Figure S3 , labeled by black square).…”

“…Benefiting from the whole genome sequencing of upland cotton [ 25 ], we now know that 37 actin genes with complete NBDs sharing high identity occur in the TM-1 genome, including Gh_D04G0865 ( Figure S3 , labeled by black square). Therefore, Sun et al used a 3′UTR of Gh_D04G0865 for VIGS to show that it caused little change in wild-type seedlings and partially recovered the curled leaves to be normal in Li 1 seedlings after specifically inhibiting Gh_D04G0865 expression [ 12 ]. Sun et al also discovered that the G65V substitution in Gh_D04G0865 disturbed filament polymerization in vitro but failed to generate gene-overexpression cotton.…”

“…Li 1 not only has seeds with very short lint fibers, but also displays other abnormal phenotypes such as twisted stems, curled leaves, and short plants, which have been confirmed to be caused by a dominant gene and result in a low level of survival in the homozygote [ 2 , 3 ]. The Li 1 locus has been mapped to Chr.22 (D) [ 4 , 5 , 6 ], and the candidates for the Li 1 gene vary among different studies [ 4 , 7 , 8 , 9 ] or lack supportive evidence of their roles in causing the mutation [ 10 , 11 , 12 ].…”

“…Accordingly, the architecture of the actin cytoskeleton is severely deformed and the microtubule organization is moderately altered, while vesicle trafficking is dramatically disrupted [ 24 ]. Using sequencing-mapping and virus-induced gene silencing (VIGS), Thyssen et al [ 11 ] and Sun et al [ 12 ] recently indicated that the actin molecule encoded by Gh_D04G0865 could be the best candidate for producing the Li 1 mutant. Thyssen et al [ 11 ] proposed “Poison subunit” model based on structural modeling of actin monomer and polymer, whereas Sun et al [ 12 ] experimentally proved that model by in vitro polymerization of defective actin unit.…”