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Literature Sharing | PcNAC25, a NAC transcription factor of Pugionium cornutum(L.) Gaertn conferring enhanced drought and salt stress tolerances in Arabidopsis
Release time:
2025-03-03
Pugionium cornutum (L.) Gaertn (P. cornutum) has strong tolerance to drought, salt and disease, but the tolerance mechanisms for such stresses in P. cornutum are largely unknown. In this study, we identified the PcNAC25 transcription factor gene in P. cornutum. Its open reading frame was revealed to comprise 891 bp, encoding a protein consisting of 297 amino acids, with an isoelectric point of 6.61. Phylogenetic analysis showed that PcNAC25 was most closely related to ANAC019. The expression of PcNAC25 was induced by dehydration, mannitol, heat, cold, salt stresses and abscisic acid (ABA), salicylic acid (SA), and methyl jasmonate (JA) treatments. A subcellular localization analysis confirmed that PcNAC25 was localized in the nucleus. The overexpressing PcNAC25 lines in Arabidopsis had longer roots than wild-type (WT) lines under drought and salt stress. The overexpression of PcNAC25 improved drought and salt tolerance in transgenic Arabidopsis. Under drought and salt stress, PcNAC25 transgenic lines exhibited higher the CAT, POD and SOD activities and scavenging ability of hydroxyl radical than WT, more proline accumulation than WT and less MDA and H2O2 content and superoxide anion production rate than WT. PcNAC25 transgenic lines also exhibited greater reduced water loss rate of detached leaves than WT. Meanwhile, DAB and NBT staining showed that the accumulation of hydrogen peroxide and superoxide anion in PcNAC25 transgenic lines were also less than WT. In addition, overexpressing PcNAC25 enhanced the expression of drought response genes (DREB2A, SOD4, RD29A, NCED3, POD3, P5CS1, PYR1 and SAG13) and salt response genes NHX, SLAH1, SOS1 and NPF6.3. The mentioned above results indicated that PcNAC25 is a positive regulator that activates ROS-scavenging enzymes and enhances root formation in Arabidopsis, which provided a basis for further research on the molecular mechanism of PCNAC25-mediated regulation of drought and salt stress, and also provided gene resources of drought and salt tolerance.
In this study, 48 NAC genes with complete ORF were identified in the transcriptome data of P. cornutum under drought stress. A phylogenetic tree was constructed with 48 NAC amino acid sequences of P. cornutum and NAC family members from other plant species. The result revealed that PcNAC25 was closely related to ANAC019. It is supposed the function of PcNAC25 may have the same function as ANAC019(Fig. 1).
The full-length gene was cloned and the 1271 bp cDNA clone contained an 891 bp open reading frame (ORF), encoding a putative NAC transcription factor of 297 amino acid residues.
Fig. 1. Phylogenetic tree analysis of NAC genes in P. cornutum
To further understand the characteristics of PcNAC25, MEME online tool is used to predict PcNAC25 potential conservative motifs. A total of 10 motifs were identified (Fig. 2), ranging in length from 15 to 50 amino acids. PcNAC25 contains conserved motifs 1–5. The results of conserved motifs were consistent with phylogenetic relationships because members with similar conserved motifs were grouped in the same cluster, indicating that they may have similar functions.
Fig. 2. Conserved motif of PcNAC25 in P. cornutum.
To understand the response of PcNAC25 to different abiotic stresses and hormones, qPCR analysis was performed in P. cornutum. Under control condition (H2O treatment), the expression levels of PcNAC25 did not show any significant change within 24 h (Data not shown). However, the expression level of PcNAC25 showed different changes under different stress treatments. Under dehydration and 250 mmol/L mannitol treatment, the relative expression levels of PcNAC25 increased and peaked to 115-fold at 12 h and 154-fold at 6 h, respectively (Fig. 4A,B). Under 200 mmol/L NaCl treatment, the relative expression of PcNAC25 increased and peaked to 74-fold at 6 h (Fig. 4C) under 42 °C and 4 °C stresses, the relative expression levels of PcNAC25 increased and peaked to 4.98-fold at 3 h and 6.4-fold at 6 h, respectively (Fig. 4D,E). Furthermore, we examined the patterns of PcNAC25 in response to various phytohormones. PcNAC25 expression levels were significantly induced to about 6 to 9-folds by ABA, SA and MeJA (Fig. 4F-H). The expression patterns and maximum expression levels differed among them.
Fig. 4. Expression patterns of PcNAC25 under different treatments in P. cornutum (the one-way ANOVA analysis)
The subcellular localization of PcNAC25 in tobacco leaf cells was examined by transient transformation experiment. Under confocal microscopy, 35 S::GFP fluorescence signals in empty vectors were localized in the cell membrane, cytoplasm, and nucleus, while 35 S::PcNAC25::GFP fusion protein was transiently expressed and localized in the nucleus, indicating that PcNAC25 localizes in the nucleus, where it can function as a TF (Fig. 5).
Fig. 5. Subcellular localization analysis of PcNAC25
Transgenic Arabidopsis plants overexpressing PcNAC25 genes driven by the UBQ promoter were generated (Fig. 6A). Eight transgenic lines were obtained and three independent homozygous lines (PcNAC25-1, 25−2 and 25−6) were investigated further (Fig. 6B,C).
Fig. 6. Construction of vector and acquisition of overexpressing PcNAC25 of Arabidopsis
The effect of PcNAC25 over-expression was examined on drought tolerance by morphological analysis. To determine the germination rate of PcNAC25 transgenic seeds under drought stress, the 1/2 MS medium containing mannitol was used to simulate the drought stress. Overexpressing seeds and root length did not show any significant difference from wild-type (WT) seeds on control medium (Fig. 7). On the medium containing 250 mmol/L mannitol, the overexpressing PcNAC25 seeds showed higher germination speed, bigger cotyledon size and the root length longer than the WT seeds (Fig. 7).
Fig. 7. Determination of seed germination rate and root length of PcNAC25 overexpression lines in control and 250 mmol/L mannitol treatment
3-weekold seedlings were used to drought tolerance test. Under control condition, PcNAC25 overexpressing (OE) lines showed no difference in either vegetative or reproductive growth from WT line. However, after 12 days of dehydration and mannitol treatment, WT line displayed severely wilting (all rosette leaves were severely curled and some were dead), while most OE lines still had green and fully expanded rosette leaves. 3 days after rewatering, only the OE lines survived (Fig. 8A).
Fig. 8. Phenotypes and physiological determination of PcNAC25 transgenic Arabidopsis under dehydration and 200 mmol/L mannitol treatment.
Given that overexpression of PcNAC25 gene can improve Arabidopsis drought tolerance. Therefore, we explored whether the phenotypic changes in the transgenic plants correlated with changes in the expression profiles of drought-responsive genes. A large number of genes have been identified involved in Arabidopsis stress tolerance. 9 genes were selected for expression determination (ED29A, P5CS1, DREB2A, NCED3, SOD4, POD3, SAG13, CAT2, PYR1). qPCR results showed that the expression levels of these 9 genes were low and there was no significant difference between OE lines and the WT in the control treatment. However, under drought conditions, the transcript levels of these genes were increased. Notably, five genes (ED29A, DREB2A, SOD4, POD3 and CAT2) showed significantly higher expression level in OE lines than the WT (Fig. 10). These results indicated that overexpressing PcNAC25 may induce the expression of these genes and improve the drought resistance of Arabidopsis by some metabolic pathways.
Fig. 10. Expression analysis of drought-related genes in overexpressing PcNAC25 Arabidopsis.
Performance of overexpressing PcNAC25 plants under salt stress was investigated. On the 1/2 MS medium containing 75 mM NaCl, the seeds of the three OE lines exhibited significantly higher germination rate and longer root length than those of the WT (Fig. 11).
Fig. 11. Determination of seed germination rate and root length of overexpressing PcNAC25 in control and 75 mmol/L NaCl treatment
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