The NAC-type transcription factor CaNAC46 regulates the salt and drought tolerance of transgenic Arabidopsis thaliana

Abstract

Background: The NAC (NAM, ATAF1/ATAF2, and CUC2) transcription factors belong to a large family of plant-specific transcription factors in monocot and dicot species. These transcription factors regulate the expression of stress tolerance-related genes that protect plants from various abiotic stresses, including drought, salinity, and low temperatures.

Results: In this study, we identified the CaNAC46 transcription factor gene in Capsicum annuum. Its open reading frame was revealed to comprise 921 bp, encoding a protein consisting of 306 amino acids, with an isoelectric point of 6.96. A phylogenetic analysis indicated that CaNAC46 belongs to the ATAF subfamily. The expression of CaNAC46 was induced by heat, cold, high salt, drought, abscisic acid, salicylic acid, and methyl jasmonate treatments. Thus, CaNAC46 may be important for the resistance of dry pepper to abiotic stresses. A subcellular localization analysis confirmed that CaNAC46 is localized in the nucleus. The overexpression of CaNAC46 improved the tolerance of transgenic Arabidopsis thaliana plants to drought and salt stresses. The CaNAC46-overexpressing lines had longer roots and more lateral roots than wild-type lines under prolonged drought and high salt stress conditions. Additionally, CaNAC46 affected the accumulation of reactive oxygen species (ROS). Moreover, CaNAC46 promoted the expression of SOD, POD, RD29B, RD20, LDB18, ABI, IAA4, and P5CS. The malondialdehyde contents were higher in TRV2-CaNAC46 lines than in wild-type plants in response to drought and salt stresses. Furthermore, the expression levels of stress-responsive genes, such as ABA2, P5CS, DREB, RD22, CAT, and POD, were down-regulated in TRV2-CaNAC46 plants.

Conclusions: Under saline and drought conditions, CaNAC46 is a positive regulator that activates ROS-scavenging enzymes and enhances root formation. The results of our study indicate CaNAC46 is a transcriptional regulator responsible for salinity and drought tolerance and suggest the abiotic stress-related gene regulatory mechanisms controlling this NAC transcription factor are conserved between A. thaliana and pepper.

Keywords: NAC transcription factor; Pepper; Stress tolerance; Transgenic plants.

Fig. 1

Alignment and phylogenetic analysis of CaNAC46 transcription factor. A Alignment of protein sequences of CaNAC46 and ATAF subfamily from other plants. B Phylogenetic tree of CaNAC46 and NAC members from other plant species. At/A: Arabidopsis thaliana, Ca: Capsicum annuum Os/O: Oryza sativa, Sl: Solanum lycopersicum. The protein sequences used to construct the tree were ATAF1 (At1g01720), ATAF2 (AT5G08790), ANAC036 (At2g17040), ANAC098 (At5g53950), OsNAC005 (LOC_Os11g08210), OsNAC006 (LOC_Os01g66120), ONAC072 (LOC_Os09g32260), ONAC022 (LOC_Os03g04070), SlNAC053 (Solyc06g060230), SlNAC048 (Solyc05g055470), SlNAC067 (Solyc07g053590), SlNAC091 (Solyc00g008000), StNAC53 (CAC42087.1), CaNAC52 (Solyc05g002477), CaNAC71 (Capana07g002159), CaNAC101 (Capana12g002456), CaNAC20 (Capana02g000302), CaNAC46 (Capana05g000569). The phylogenetic tree was constructed based on peptide sequences using the Neighbor-Joining method. CaNAC46 is marked by solid box

Fig. 2

Subcellular Localization and Stress-Responsive Expression. A Nuclear localization of CaNAC46 in tobacco cells. B Expression patterns of CaNAC46 in response to stress treatments. Superscript letters indicate significant differences. Three independent biological experiments were performed (P < 0.05)

Fig. 3

Over-expression of CaNAC46 in Arabidopsis improves salt and drought stress tolerance. A The seedlings of transgenic and WT plants were cultured with containing 1/2 MS, 1/2 MS with 200 mM NaCl and 1/2 MS with 10% PEG6000 agar medium for 7 days. B Performance of 3-week-old WT, CaNAC46-OE1 and OE1 plants grown under drought stress for 3 weeks and were re-watered for 3 days (upper); 3-week-old WT, CaNAC46-OE1 and OE1 plants were watered 200 mM NaCl for 3 weeks (below). C The number and length of roots were counted at 1-week-old seedling stage. Three independent biological experiments were performed. Superscript letters indicate significant differences (P < 0.05)

Fig. 4

The physiological and stress-responsive gene expression analysis under stress treatments. A The activities of SOD, POD, and CAT were measured in the WT and CaNAC46-overexpressing plants under normal and stress conditions. B The expression levels of stress-responsive genes were in examined by qRT-PCR analyses the WT and CaNAC46-overexpressing plants before and after the abiotic stress treatments. Three independent biological experiments were performed. Superscript letters indicate significant differences (P < 0.05)

Fig. 5

Suppression of CaNAC46 leads to salt and drought sensitivity in pepper. The role of CaNAC46 for salt and drought sensitivity was assessed by VIGS. A The TRV2-CaNAC46 and WT plants were subjected to drought stress by dehydration and salinity stress by watering with a 200 mM NaCl solution for 3 weeks. B DAB staining was in TRV2-CaNAC46 and WT leaves than in WT leaves after for 3 weeks of the salt and drought treatments. C The MDA content of TRV2-CaNAC46 and WT plants was measured under drought and salinity stresses. D The expression levels of stress-responsive genes were in examined by qRT-PCR analyses the WT and TRV2-CaNAC46 plants before and after the abiotic stress treatments. Three independent biological experiments were performed. Superscript letters indicate significant differences (P < 0.05)