Functional characterization of GhNAC2 promoter conferring hormone- and stress-induced expression: a potential tool to improve growth and stress tolerance in cotton

Abstract

The GhNAC2 transcription factor identified from G. herbaceum improves root growth and drought tolerance through transcriptional reprogramming of phytohormone signaling. The promoter of such a versatile gene could serve as an important genetic engineering tool for biotechnological application. In this study, we identified and characterized the promoter of GhNAC2 to understand its regulatory mechanism. GhNAC2 transcription factor increased in root tissues in response to GA, ethylene, auxin, ABA, mannitol, and NaCl. In silico analysis revealed an overrepresentation of cis-regulatory elements associated with hormone signaling, stress responses and root-, pollen-, and seed-specific promoter activity. To validate their role in GhNAC2 function/regulation, an 870-bp upstream regulatory sequence was fused with the GUS reporter gene (uidA) and expressed in Arabidopsis and cotton hairy roots for in planta characterization. Histochemical GUS staining indicated localized expression in root tips, root elongation zone, root primordia, and reproductive tissues under optimal growth conditions. Mannitol, NaCl, auxin, GA, and ABA, induced the promoter-driven GUS expression in all tissues while ethylene suppressed the promoter activity. The results show that the 870 nt fragment of the GhNAC2 promoter drives root-preferential expression and responds to phytohormonal and stress signals. In corroboration with promoter regulation, GA and ethylene pathways differentially regulated root growth in GhNAC2-expressing Arabidopsis. The findings suggest that differential promoter activity governs the expression of GhNAC2 in root growth and stress-related functions independently through specific promoter elements. This multifarious promoter can be utilized to develop yield and climate resilience in cotton by expanding the options to control gene regulation.

Supplementary information: The online version contains supplementary material available at 10.1007/s12298-024-01411-2.

Keywords: Cotton; Gene regulation; GhNAC2; Hormone signaling; Inducible promoter; Root.

Fig. 1

Expression and in-silico promoter analysis of GhNAC2 transcription factor. a GhNAC2 was significantly induced by the exogenous treatment of GA₃, ACC, ABA, IAA, mannitol, and NaCl. All data are means ± SE (n = 9). Student’s t- test with a threshold p value < 0.05 was used to compare the treated samples with the mock controls (a = p value < 0.001, b = p value < 0.01, and c = p value < 0.05). b Schematic diagram representing the core and distal promoter elements, 5’-UTR, exon/intron arrangement, and 3’-UTR. c Histogram of the over-represented cis-acting elements in the distal promoter region involved in the stress, hormonal, light, and tissue-specific gene regulation. d Histogram representing the TF binding sites occurring in the promoter. (UTR untranslated region, TSS transcription start site)

Fig. 2

Tissue-specific GUS expression driven by GhNAC2 promoter in transgenic Arabidopsis. GUS histo-chemical staining was performed in different tissues of transgenic Arabidopsis lines expressing the Pro_GhNAC2::GUS construct. Tissues expressing the empty pBI101 vector served as control (a: 72 h-old root, f: 15-day-old seedling, k: one-month-old leaf, p: flower, and u: silique). Prominent GUS expression was observed in the root tips and elongation zones of b 36 h-old, c 48 h-old, and d, e 72 h-old seedlings. In the 15-day-old seedlings, g preferential GUS expression was localized in the root tissues: h, i elongation zone and primary root tip, j lateral root tip and lateral root primordia. In the one-month-old soil-grown plants, GUS expression was visible in l leaf veins, m trichomes, n root hairs, along with o the lateral roots. In the two-month-old plants, prominent GUS expression was observed in the q, r stigma and anthers, s pollen grains, t apical buds, v, w immature silique valves and seeds, and x, y mature silique valves. The images represent the results obtained for three independent T₃ generation lines. (RT root tip, EZ elongation zone, DZ differentiation zone, LRP lateral root primordia, LRT lateral root tip, RH root hair, AB apical bud, IV immature silique valve, IS immature seed, MV mature silique valve, and MS mature seed)

Fig. 3

Tissue-specific GUS expression driven by GhNAC2 promoter in cotton hairy roots. GUS localization was visualized in the intact (top) and transverse sections (bottom). a Empty pBI101 and b pBI121 vectors served as the negative and positive control, respectively. GhNAC2 promoter-driven GUS expression was observed in the c elongation zone, d root tip, and e root primordia, except f differentiation zone of cotton hairy roots. The images represent the results obtained for 10–12 independent hairy roots expressing each construct. (EZ elongation zone, DZ differentiation zone, RT root tip, RP root primordia, EC epidermal cell, CC cortical cell, PC pericycle cell)

Fig. 4

Phytohormonal regulation of GhNAC2 promoter in cotton hairy roots. Promoter-driven GUS expression was examined in response to a, f, k Mock treatment, b GA₃, c ACC (an ethylene precursor), d ABA, e IAA, their respective inhibitors: g PAC (GA biosynthesis inhibitor), h AgNO₃ (ethylene signal inhibitor), i TIBA (auxin transport inhibitor), and j Fluridon (ABA biosynthesis inhibitor), and their combinations: l IAA and PAC, m (IAA and ACC, n IAA and AgNO₃, and o ACC and TIBA. Strong GUS expression is indicated by arrows. The images represent the results obtained for 10–12 independent hairy roots for each treatment

Fig. 5

Abiotic stress-induced regulation of GhNAC2 promoter in Arabidopsis (top) and cotton hairy roots (bottom). GUS histochemical staining was performed to study the promoter activity upon treatment with osmotic and salt stress. a Negative control (empty pBI101), b Positive control (empty pBI121), c Mock (no stress), d osmotic stress (mannitol), and e salt stress (NaCl) treatment. The images represent the results obtained for three independent T₃ generation Arabidopsis lines and 10–12 independent cotton hairy roots for each treatment

Fig. 6

Quantitative measurement of the GUS enzyme activity. GUS fluorometric assay was performed in the transgenic cotton hairy root tips to estimate the promoter-driven GUS expression upon a phytohormone and b stress treatment. The GUS activity was determined in terms of the pmol of 4-MU (4-methylumbelliferone) produced per mg of crude protein per min. All data are means ± SE (n = 9)

Fig. 7

GhNAC2-promoted root growth is controlled by phytohormone signaling. a GA₃ differentially increased the root growth in WT and transgenic Arabidopsis lines (L1, L4, L8, and L15) expressing the GhNAC2 gene. Whereas in the presence of PAC, the root growth was decreased. b ACC and AgNO₃ differentially suppressed and promoted root growth in WT and transgenic lines, respectively. c The transgenic lines showed less ABA sensitivity as compared to WT, whereas fluridon decreased the root growth of both WT and transgenic lines. d In the presence of IAA, no significant root stimulation was observed in either WT or transgenic lines. In addition, TIBA suppressed the root growth in both WT and transgenic lines. All data are means ± SE (n = 9). Different letters indicate significant differences between treatments and genotypes based on the Duncan’s multiple range test (a = p value < 0.001, b = p value < 0.01, and c = p value < 0.05)

Fig. 8

Phytohormonal signaling of GhNAC2 transcription factor. Ethylene, ABA, GA, and auxin are the upstream hormonal regulators determining GhNAC2 promoter activity. Further, the GhNAC2 protein regulates the downstream genes involved in the biosynthesis and/or signaling of ethylene, ABA, and GA. This suggested that the actions of GhNAC2 are governed by the ethylene/ABA/GA pathway through a feedback loop