Scientia Agricultura Sinica ›› 2021, Vol. 54 ›› Issue (24): 5266-5276.doi: 10.3864/j.issn.0578-1752.2021.24.009

• HORTICULTURE • Previous Articles     Next Articles

The Role and Mechanism of Tomato SlNAC29 Transcription Factor in Regulating Plant Senescence

WANG Ping(),ZHENG ChenFei,WANG Jiao,HU ZhangJian,SHAO ShuJun,SHI Kai()   

  1. College of Agriculture and Biotechnology, Zhejiang University, Hangzhou 310058
  • Received:2021-02-06 Accepted:2021-05-03 Online:2021-12-16 Published:2021-12-28
  • Contact: Kai SHI E-mail:11916061@zju.edu.cn;kaishi@zju.edu.cn

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Abstract:

【Background】 Tomato（Solanum lycopersicum）is an important horticultural crop with continuous flower bud differentiation and fruiting. Premature senescence seriously limits tomato plants growth period, crop yield and fruit quality. NAC (NAM, ATAF1/2 and CUC2) transcription factor family regulates leaf senescence process in Arabidopsis, rice and other plants. Nevertheless, the roles of tomato NAC transcription factor in the regulation of leaf senescence have not been well understood. SlNAP2 (NAC-like, activated by apetala3/pistillata) is known to be involved in the regulation of tomato leaf senescence. 【Objective】 SlNAC29 transcription factor is the homologous gene of SlNAP2 in tomato, while its function remains largely unclear. In this study, the role of SlNAC29 and its underlying mechanism in leaf senescence was investigated, which can provide some scientific basis for tomato senescence regulation and germplasm innovation. 【Method】Condine Red (CR) was used as the wild-type background in this study. qRT-PCR was used to analyze the relative expression of SlNAC29 in tomato leaves at different senescence stages. Slnac29 homozygous mutant lines and the OE: SlNAC29 stable overexpression lines were generated through CRISPR/Cas9 gene editing and over-expression approaches, respectively. Using these lines, plant growth phenotypes, chlorophyll content, leaf photosynthesis, transcription of senescence- and chlorophyll degradation- related genes were analyzed under both natural and dark-induced senescence conditions. The clustering heat map was used to analyze the relative expression of 29 genes, including senescence-, chlorophyll degradation- and ABA biosynthesis/signaling-associated genes. Based on gene expression profiles, four of them were selected to electrophoretic mobility shift analysis (EMSA) to identify the SlNAC29-target gene during senescence process. 【Result】The relative expression of SlNAC29 was significantly up-regulated in early senescent and senescent leaves, as compared with young and mature leaves. Under natural growth condition, the Slnac29 mutant lines showed no differences with the wild-type in terms of plant growth phenotypes and photosynthetic rate. By contrast, the height of OE: SlNAC29 plant was shorter than wild-type plants, OE: SlNAC29 plants also showed lower chlorophyll content and photosynthetic rate, which were only 25% and 50% of the wild-type control, respectively. Under dark-induced senescence condition, the leaves of wild-type plants turned yellow and the chlorophyll content decreased significantly. The senescent phenotypes were alleviated in Slnac29 mutant lines, which not only have significant higher chlorophyll content, but also showed higher transcript level of senescence-associated genes (SAGs) and chlorophyll degradation-related genes. On the contrary, the dark-induced senescence effect was aggravated in OE: SlNAC29. Cluster analysis showed that several genes, especially SAGs and chlorophyll degradation-related genes SlSAG12, SlAGT1, SlSGR1 and SlNYC1, were significantly up-regulated in OE: SlNAC29 plants. The EMSA analysis showed that SlNAC29 could directly bind to the promoter of SlAGT1 (Glyoxylate aminotransferase), a member of SAGs. Moreover, the relative expression of SlAGT1 in OE: SlNAC29 was significantly higher than that of wild-type and Slnac29 plants. 【Conclusion】 SlNAC29 transcription factor is involved in the regulation of leaf senescence in tomato plants, which promotes the senescence process under dark conditions. SlNAC29 may directly bind to the promoter region of the senescence-related gene SlAGT1 to regulate its transcriptional expression.

Key words: tomato, SlNAC29, transcription regulation, senescence, SlAGT1

Table 1

Primers"

引物名称
Primer name		 基因编号
Accession number		 正向引物
Forward primer (5′-3′)		 反向引物
Reverse primer (5′-3′)		 用途
Amplification
E-SlNAC29 Solyc05g007770 TTggcgcgccATGGTTGGAAAAATTAGCTC CGGggtaccCTGAGATTGAAATGTTGG 基因扩增
Gene amplification
CRISPR-Slnac29 Solyc05g007770 GATTgTTCGATCCCTGGGTATTACC AAACGGTAATACCCAGGGATCGAAc
SlNAC29-GFP Solyc05g007770 CgagctcATGGTTGGAAAAATTAGCTC TCCcccgggtCTGAGATTGAAATGTTGGATTC
RT- SlNAC29 Solyc05g007770 CCAAATAGAGCAGCTGTGTCA TTGGTGGTTTGCCTTTGTAA qRT-PCR
RT- SlAGT1 Solyc10g076250 TGCCATAAACCCTGACCCAT CTATCCCACCAACACCCTGT
RT- SlSAG12 Solyc02g076910 GTGGCTAATCAACCGGTGTC CACTTGCAGCACCATATCCC
RT- SlSGR1 Solyc08g080090 TTCTCAGTTGCAAGGTTGGT CCTTGAGAACCACAGGGAGT
RT- SlNYC1 Solyc07g024000 CAGGCAGCTTCAATCATCCC TAGGTCGGTTAGGACCATGC
RT- SlACTIN Solyc03g078400 TGTCCCTATTTACGAGGGTTATGC CAGTTAAATCACGACCAGCAAGAT
SlAGT1 Solyc10g076250 AATTGAAATTACGTAAATGGAACGTAACCAAAATTATATC GATATAATTTTGGTTACGTTCCATTTACGTAATTTCAATT EMSA
SlSAG12 Solyc02g076910 GTTTTGAAGATGCACACACGAAAATACATGTACAATTGAT ATCAATTGTACATGTATTTTCGTGTGTGCATCTTCAAAAC
SlSGR1 Solyc08g080090 CAATCAATTTATACACGCCTTAACTTATTTTTAAAAATGC GCATTTTTAAAAATAAGTTAAGGCGTGTATAAATTGATTG
SlNYC1 Solyc07g024000 CTAGCATTTTAATATATGAATACACGTGAACAAAATTTCC GGAAATTTTGTTCACGTGTATTCATATATTAAAATGCTAG

Fig. 1

SlNAC29 relative expression in different senescence stage leaves A: Protein sequence alignment of SlNAC29 and SlNAP2. *: Amino acids identical in two proteins are highlighted; B: Relative expression of SlNAC29 in wild-type leaves at different senescence stages by qRT-PCR. ★: Significantly different at P<0.05 level (n=3); YL: Young leaves, ML: Mature leaves, ES: Early senescent leaves, SL: Senescent leaves; C: Subcellular localization of SlNAC29. Scale bar=50 μm"

Fig. 2

Plant construction and growth phenotypes of mutated- and overexpressed-SlNAC29 plants A: Schematic illustration of the sgRNA from CRISPR/Cas 9 editing Slnac29 mutant lines; B: Detection of the SlNAC29-HA fusion protein in two independent homozygous SlNAC29-overexpressed lines by western blot; C: Phenotype of mutated- and overexpressed- SlNAC29 in 5 weeks; D: Chlorophyll content of mutated- and overexpressed-SlNAC29 plants; E: Photosynthetic rate of mutant and overexpressed SlNAC29. The chlorophyll content and photosynthetic rate were measured from the second and third mature leaves in 5 weeks-old plants. Each error bar represents the mean ± SD (n=3). Different letters indicate significant differences at P<0.05, respectively. The same as below"

Fig. 3

Effects of SlNAC29 mutation and overexpression on dark induced leaf senescence A, B: Dark-induced leaf senescence phenotype and chlorophyll content. C: Relative expression of senescence related genes in leaves under control and dark-induced senescence"

Table 2

SlNAC29 core binding site of the target gene promoter"

目标基因
Target gene		 登录号
Accession number		 靶标位点个数
NAC core binding site number
SlAGT1 Solyc10g076250 2
SlSAG12 Solyc02g076910 3
SlSGR1 Solyc08g080090 3
SlNYC1 Solyc07g024000 5

Fig. 4

Gene identification of SlNAC29 binding and transcription activating A: Heatmap showing the fold change of 29 senescence-associated genes, chlorophyll degradation and ABA-associated genes. Red, upregulated; blue, downregulated (as presented by the color bar); B: Electrophoretic mobility shift assay detection of SlNAC29-His binding to SlSAG12, SlAGT1, SlSGR1 and SlNYC1; C: SlAGT1 gene expression in SlNAC29 mutation and overexpression materials"

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