A novel NAP member GhNAP is involved in leaf senescence in Gossypium hirsutum

Abstract

Premature leaf senescence has a negative influence on the yield and quality of cotton, and several genes have been found to regulate leaf senescence. Howeer, many underlying transcription factors are yet to be identified. In this study, a NAP-like transcription factor (GhNAP) was isolated from Gossypium hirsutum. GhNAP has the typical NAC structure and a conserved novel subdomain in its divergent transcription activation region (TAR). GhNAP was demonstrated to be a nuclear protein, and it showed transcriptional activation activity in yeast. Furthermore, the expression of GhNAP was closely associated with leaf senescence. GhNAP could rescue the delayed-senescence phenotype of the atnap null mutant. Overexpression of GhNAP could cause precocious senescence in Arabidopsis. However, down-regulation of GhNAP delayed leaf senescence in cotton, and affected cotton yield and its fibre quality. Moreover, the expression of GhNAP can be induced by abscisic acid (ABA), and the delayed leaf senescence phenotype in GhNAPi plants might be caused by the decreased ABA level and reduced expression level of ABA-responsive genes. All of the results suggested that GhNAP could regulate the leaf senescence via the ABA-mediated pathways and was further related to the yield and quality in cotton.

Keywords: Abscisic acid; GhNAP; Gossypium hirsutum; NAP subfamily; leaf senescence; transcription factor..

Fig. 1.

Structural, phylogenetic, subcellular localization, and transcriptional activation analysis of GhNAP. (A) The NAC domain and TAR of GhNAP. Subdomains are shown by rectangles. (B) Phylogenetic analysis of GhNAP. The phylogenetic tree was constructed using the Bayesian method based on the multiple alignments of 23 NAP protein sequences. The tree is unrooted. The numbers in the clades are posterior probability values. (C) Distribution of 15 putative conserved motifs in the NAP subfamily by the MEME search tool. Different motifs are represented by various boxes. The location of each motif can be estimated using the scale at the bottom. The groups within the NAP subfamily are classified by different brackets according to the phylogenetic relationship. (D) Subcellular localization of GhNAP. The GhNAP–GFP fusion protein and free GFP were transiently expressed in transgenic Nicotiana benthamiana plants expressing RFP–H2B, and the transformed leaves were observed by confocal microscopy. Images in the first column show cells with the GFP signal. Images in the second column show the bright-field view of the same cells. Images in the third column show the same cells with the RFP signal, and the images in the fourth column are the overlays of the bright-field and fluorescent images. Scale bar=20 μm. (E) Transcriptional activation analysis of GhNAP in yeast. The full length and the N- (GhNAP-N) and C-terminal (GhNAP-C) regions of GhNAP were inserted into the pGBKT7 vector. The pGBKT7 plasmid was used as the negative control. The above four constructs were transformed into yeast on SD/–Trp and SD/–Trp/X-α-Gal/AbA media for examination of growth. (This figure is available in colour at JXB online.)

Fig. 2.

Physiological and molecular analysis of GhNAP expression during leaf senescence in cotton. (A–E) Cotton leaves at various developmental stages and GhNAP expression. YL, a young leaf half the size of a fully expanded leaf; NS, a fully expanded, non-senescent leaf; ES, an early senescent leaf, with <50% leaf area yellowing; LS, a late senescent leaf, with >50% leaf area yellowing. Chlorophyll content (B), membrane ion leakage (C), and relative expression of GhNAP (D) and GhCAB (E) at the corresponding stage of (A). Error bars indicate the standard error (n=3). Significant differences between means are represented by different letters. A similar statistical analysis was conducted in the following parts. (F–H) Different parts in the senescing cotton leaf and the relative expression level of GhNAP and GhCAB. B, base; M, middle; T, tip. EF1α was used as the standard control in all qRT-PCR experiments in cotton. (This figure is available in colour at JXB online.)

Fig. 3.

Natural senescence of GhNAP-complemented lines. (A, B) Phenotypes of Col-0 and GhNAP_RE lines under a normal environment after 60 d growth. G1–G5, five groups of detached leaves according to the senescent condition. (C–F) Chlorophyll content (C), membrane ion leakage (D), and relative expression of AtSAG12 (E) and AtCAB (F) in the detached leaves of the five groups. AtActin2 was used as the standard control in all of the qRT-PCR experiments in Arabidopsis. (This figure is available in colour at JXB online.)

Fig. 4.

Natural senescence process of Arabidopsis leaves in Col-0 and GhNAP lines. (A, B) Phenotypes of Col-0 and GhNAP lines under a normal environment after 30 d growth. G1–G3, three groups of detached leaves according to the senescent condition. (C–G) Chlorophyll content (C), membrane ion leakage (D), and relative expression of AtNAP (E), AtSAG12 (F), and AtCAB (G) in the detached leaves of the three groups. (This figure is available in colour at JXB online.)

Fig. 5.

Natural senescence process of Arabidopsis leaves in Col-0, atnap, and GhNAPi lines. (A, B) Phenotypes of Col-0, atnap, and GhNAPi lines under a normal environment after 50 d growth. G1–G5, five groups of detached leaves according to the senescent condition. (C–G) Chlorophyll content (C), membrane ion leakage (D), and relative expression of AtNAP (E), AtSAG12 (F), and AtCAB (G) in the detached leaves of the five groups. (This figure is available in colour at JXB online.)

Fig. 6.

Phenotypes of the GhNAPi and wild-type cotton. (A and C) Wild-type cotton. (B and D) GhNAPi cotton. (A) and (B) were photographed at 60 days after planting (DAP). The circle indicates the non-transgenic seedling leaf with the withered dot after screening with 500mg l^–1 kanamycin. (C) and (D) were photographed at 120 DAP. (This figure is available in colour at JXB online.)

Fig. 7.

Physiological and molecular analysis of cotton leaf in wild-type (WT) and GhNAPi lines. (A–E) Chlorophyll content (A), SPAD value (B), net photosynthetic rate (P_n; C), intercellular CO₂ concentration (C_i; D), and F _v/F _m ratio (E) of cotton leaf from 60 to 150 DAP. (F) Emission spectra image on F _v/F _m of the corresponding leaf at 120 DAP. The bar on the bottom shows the F _v/F _m value. (G, H) Relative expression of GhNAP and GhCAB at 120 DAP. (This figure is available in colour at JXB online.)

Fig. 8.

Relationship between GhNAP and ABA pathways. (A) Distribution of cis-elements in the promoter region of GhNAP. The main cis-elements are indicated as follows: filled circles, ABRE; filled inverted triangles, MYC recognition site; open triangles, MYB recognition site; open diamonds, CAAT. (B) Effects of ABA on GhNAP expression in cotton. (C) Endogenous levels of ABA in WT and GhNAPi lines. (D–G) Expression of ABA-related genes in wild-type and GhNAPi plants. (G) Interaction between GhNAP and the promoter of GhSAG113 by Y1H assay. (This figure is available in colour at JXB online.)