GhWRKY40 Interacts with an Asparaginase GhAPD6 Involved in Fiber Development in Upland Cotton (Gossypium hirsutum L.)

Abstract

Fiber quality improvement is a primary goal in cotton breeding. Identification of fiber quality-related genes and understanding the underlying molecular mechanisms are essential prerequisites. Previously, studies determined that silencing the gene GhWRKY40 resulted in longer cotton fibers; however, both the underlying mechanisms and whether this transcription factor is additionally involved in the regulation of cotton fiber strength/fineness are unknown. In the current study, we verified that GhWRKY40 influences the fiber strength, fiber fineness, and fiber surface structure by using virus-induced gene silencing (VIGS). Potential proteins that may interact with the nucleus-localized GhWRKY40 were screened in a yeast two-hybrid (Y2H) nuclear-system cDNA library constructed from fibers at 0, 10, and 25 days post-anthesis (DPA) in two near-isogenic lines differing in fiber length and strength. An aspartyl protease/asparaginase-related protein, GhAP_D6, was identified and confirmed by Y2H and split-luciferase complementation assays. The expression of GhAP_D6 was approximately 30-fold higher in the GhWRKY40-VIGS lines at 10 DPA and aspartyl protease activity was significantly upregulated in the GhWRKY40-VIGS lines at 10-20 DPA. This study suggested that GhWRKY40 may interact with GhAP_D6 to regulate fiber development in cotton. The results provide a theoretical reference for the selection and breeding of high-quality cotton fibers assisted by molecular technology.

Keywords: GhWRKY40; Gossypium hirsutum; aspartyl protease; fiber quality; split-luciferase complementation assay; yeast two-hybrid assay.

Figure 1

Variation in fiber quality indicators and changes in the fiber surface and internal structure in GhWRKY40-VIGS lines. (A,B): (A) Fineness of a single fiber (linear density, Lin.Den), strength of the fiber (Tenac), and force required to break a fiber (F_max). One hundred fibers per replicate were measured, with three replicates analyzed. ** p < 0.01. (B): Electron micrographs of the changes in fiber surface and internal structure when GhWRKY40 was silenced.

Figure 2

GhWRKY40 was functional in the nucleus. RFP: nucleoplasmic marker, GFP: green fluorescence field, BF: brightfield, and Merge: superimposed field.

Figure 3

Construction of the cotton fiber nuclear-system Y2H library and the GhWRKY40-interacting protein screening. (A): Seven lanes from left to right are markers 1–6: A1-0 DPA, A1-10 DPA, A1-25 DPA, A29-0 DPA, A29-10 DPA, and A29-25 DPA. (B): Identification of the capacity of the secondary library of the nuclear system. (C): Identification of the size and recombination rate of the random insertion fragment of the nuclear-system library. (D–F): Primary screening and rescreening of GhWRKY40 interactors on the TDO and QDO/AbA/X-α-Gal media.

Figure 4

Screening of GhWRKY40 interactors in the Y2H and split-luciferase complementation assays. (A): Screening of GhWRKY40 interactors in the Y2H assays; BD was GhWRKY40 (abbreviated as WRKY40), and AD from top to bottom was located on spots 22, 25, 5, 10, 45, and 46. (B): Screening of GhWRKY40 interactors in the split-luciferase complementation assays.

Figure 5

Gene structure and transcript level of GhAP_D6 in A1, A29, and GhWRKY40-VIGS lines. (A): Gene structure of GhAP_D6 and its transcript level in A1 and A29. (B): Transcript level of GhWRKY40 was significantly downregulated at 10 DPA in A29. (C): Transcript level of GhAP_D6 was significantly upregulated when GhWRKY40 was downregulated at 10 DPA in A29. * 0.01 < p ≤ 0.05, ** p ≤ 0.01.

Figure 6

Aspartyl protease activity included the following: (A) indoleacetic acid oxidase (IAAO) activity; (B) peroxidase (POD) activity; and (C) activity when GhWRKY40 was downregulated compared with that of the negative control. ** p ≤ 0.05.