The bZIP transcription factor CibZIP43 positively regulates salt and heat tolerance in pecan (Carya illinoinensis)

Abstract

Pecan (Carya illinoinensis) is an economically and ecologically important tree species, but its growth is frequently challenged by salt stress and heat stress. bZIP transcription factors are key regulators of plant responses to abiotic stresses. This study aimed to functionally characterize the pecan transcription factor CibZIP43 and reveal its role in salt and heat stress tolerance. The CibZIP43 gene was overexpressed in Arabidopsis, and the results showed that CibZIP43 overexpression (OE) in Arabidopsis significantly enhanced the tolerance of transgenic plants to salt and heat stress. Physiological and biochemical analyzes showed that CibZIP43 OE lines exhibited better growth and improved physiological indices under salt and heat stress, including increased chlorophyll content, relative water content, and antioxidant enzyme activities, along with reduced membrane lipid peroxidation and proline accumulation. Moreover, CibZIP43 OE enhanced plant adaptation to salt and heat stress by regulating the expression of Na⁺/K⁺ transporter-related and heat stress-related genes. These findings uncover new molecular mechanisms and provide potential genetic resources for breeding for stress-tolerance in pecan varieties.

Keywords: CibZIP43; Pecan; Salt and heat tolerance.

Fig. 1

Expression patterns analysis of CibZIP genes in different tissues

Fig. 2

CibZIP43 binds to G-box. A Yeast one-hybrid assays of CibZIP43 and G-box. B Dual-Luciferase reporter assays. For each leaf, one area was infiltrated. All experiments were performed at least 3 times, and representative results are shown

Fig. 3

GUS staining and activity analysis of proCibZIP43 under salt and heat stress. A CibZIP43 promoter GUS staining. B CibZIP43 promoter GUS activity analysis. And at least 3 independent replication groups were set for each experiment

Fig. 4

Phenotypes of CibZIP43 overexpressing Arabidopsis seedlings under salt and heat stress. A Phenotypes of seed germination under salt and heat stress. B Statistical graph of changes in seed germination rate at different times. C Seed germination under heat treatment. D Root length and root length statistics of seedlings under salt stress. E Phenotypes of seedlings under heat stress. The asterisks represent significant differences calculated by the T-test (*P < 0.05, **P < 0.01)

Fig. 5

Phenotypic analysis of CibZIP43 transgenic seedlings under salt and heat stress. A Phenotypes of transgenic seedlings under salt stress. B Phenotypes of transgenic seedlings under heat stress. C-D Analysis of chlorophyll content of transgenic CibZIP43 under salt and heat stress. E-F Analysis of relative water content in transgenic CibZIP43 under salt and heat stress. The asterisks represent significant differences calculated by the T-test (*P < 0.05, **P < 0.01)

Fig. 6

ROS clearance ability of A. thaliana overexpressed with CibZIP43 under salt and heat stress. A, B DAB staining analysis. C, D NBT staining analysis. E, F CAT activity analysis. G, H POD activity analysis. The asterisks represent significant differences calculated by the T-test (*P < 0.05, **P < 0.01)

Fig. 7

REL, MDA and proline contents of CibZIP43 overexpression in Arabidopsis thaliana. A, B Relative electrolyte leakage of transgenic Arabidopsis under salt and heat stresses. C, D MDA content of CibZIP43 transgenic Arabidopsis. E, F Analysis of proline content in CibZIP43 transgenic Arabidopsis. The asterisks represent significant differences calculated by the T-test (*P < 0.05, **P < 0.01)

Fig. 8

PCA principal component (PCA) analysis. A PCA analysis under salt stress. B PCA analysis under heat stress

Fig. 9

Effect of CibZIP43 transgenic plants on Na + and K + operation under salt stress. A Na⁺ content, K⁺ content and K⁺/Na⁺ ratio. B Expression levels of SOS1, NHX1 and NHX2. The asterisks represent significant differences calculated by the T-test (*P < 0.05, **P < 0.01)

Fig. 10

Expression of stress-related genes in transgenic plants under salt treatment. The asterisks represent significant differences calculated by the T-test (*P < 0.05, **P < 0.01)

Fig. 11

CibZIP43 over-expression regulates thermal response gene expression under heat stress. The asterisks represent significant differences calculated by the T-test (*P < 0.05, **P < 0.01)