NAC transcription factor GbNTL9 modifies the accumulation and organization of cellulose microfibrils to enhance cotton fiber strength.

Introduction: Fiber strength is a critical determinant of fiber quality, with stronger fibers being highly preferred in the cotton textile industry. However, the genetic basis and the specific regulatory mechanism underlying the formation of cotton fiber strength remain largely unknown.

Objectives: To explore fiber strength-related genes, QTL mapping, map-based cloning, and gene function verification were conducted in a backcross inbred line BS41 derived from interspecific hybridization between upland cotton and sea-island cotton.

Methods: Upland cotton Emian22 (E22) and an interspecific backcross inbred line (BIL) BS41 were used as parents to construct secondary segregation populations for BSA and QTL mapping of fiber strength. The candidate gene GbNTL9 was identified through map-based cloning and expression analysis. The function of NTL9 was determined through transgenic experiments and cytological observations. The regulatory mechanisms of NTL9 were explored using RNA-seq, RT-qPCR, yeast two-hybrid, bimolecular fluorescence complementation, and yeast one-hybrid.

Results: A major QTL for fiber strength, qFS-A11-1, was mapped to a 14.6-kb genomic region using segregating populations from E22 × BS41. GbNTL9, which encodes a NAC transcription factor, was identified as the candidate gene. Overexpression of both upland cotton genotype NTL9 and sea-island genotype NTL9 in upland cotton enhanced fiber strength by facilitating the dense accumulation and orderly organization of cellulose microfibrils within the cell wall. Transcriptomic analysis revealed that NTL9 inhibited the expression of genes involved in secondary wall synthesis, such as CESA4, CESA7, and CESA8, thereby delaying cell wall cellulose deposition and altering the microfibril deposition pattern. NTL9 interacted with MYB6 and functioned as a downstream gene in the ethylene signaling pathway. Additionally, an effective gene marker NTL9-24 was developed to distinguish haplotypes from G. barbadense and G. hirsutum for fiber quality breeding program.

Conclusion: Our findings demonstrate that GbNTL9 positively regulates fiber strength through altering the microfibril deposition pattern, and provide a new insight into the molecular mechanism underlying fiber strength.