A Novel Senescence-Specific Gene (ZmSAG39) Negatively Regulates Darkness and Drought Responses in Maize

Abstract

The papain-like cysteine proteases (PLCPs) is a subfamily of cysteine proteases that plays an important role in leaf senescence, and some of its members are involved in the regulation of plant growth and development under stress. In this study, we cloned a new gene, ZmSAG39, from maize. Expression profile analysis showed that ZmSAG39 was induced by darkness and drought treatments. In addition, the ZmSAG39 overexpression in maize accelerated the senescence of maize leaves under darkness and drought treatments. However, the knockout of ZmSAG39 in maize enhanced the resistance of maize to darkness and drought stresses and reduced the degree of senescence of maize leaves. Under drought stress, compared with WT plants, the knockout lines had a higher seed germination rate, seedling survival rate and chlorophyll content, and lower reactive oxygen species (ROS) level and malondialdehyde (MDA) content. In addition, quantitative real-time PCR (qRT-PCR) analysis showed that ZmSAG39 negatively regulated some stress-related genes but positively regulated senescence-related genes under darkness and drought stress conditions. To summarize, these results indicate that ZmSAG39 is a senescence-related gene and plays a negative role in response to darkness and drought stresses. This study laid a theoretical foundation for the innovation of maize germplasm resources with high quality, high yield and strong stress resistance.

Keywords: ZmSAG39; darkness stress; drought stress; leaf senescence; maize.

Figure 1

Protein sequence analysis and phylogenic tree analysis of ZmSAG39. (A) Phylogenetic analysis of Papain-like cysteine proteases (PLCP)-encoding enzymes from At (Arabidopsis thaliana), Os (Oryza sativa) and Zm (Zea mays). A neighbor-joining tree was generated using MEGAX with 1000 bootstrap replicates. Different background colors showed different subgroups. (B) Multiple sequence alignment of predicted protein sequences belonging to a subgroup of 10 closely related PLCPs. C1A (papain) domain is highlighted by lines. ERFNIN motif is highlighted by continuous arrows. The catalytic triad Cys, His and Asn and also the Glu active site residue are indicated by the arrows.

Figure 1

Protein sequence analysis and phylogenic tree analysis of ZmSAG39. (A) Phylogenetic analysis of Papain-like cysteine proteases (PLCP)-encoding enzymes from At (Arabidopsis thaliana), Os (Oryza sativa) and Zm (Zea mays). A neighbor-joining tree was generated using MEGAX with 1000 bootstrap replicates. Different background colors showed different subgroups. (B) Multiple sequence alignment of predicted protein sequences belonging to a subgroup of 10 closely related PLCPs. C1A (papain) domain is highlighted by lines. ERFNIN motif is highlighted by continuous arrows. The catalytic triad Cys, His and Asn and also the Glu active site residue are indicated by the arrows.

Figure 2

Response of ZmSAG39 to abiotic stress. (A,B) Expression patterns of ZmSAG39 in maize leaves under dark and drought stress conditions. Data are shown as mean ± SD from three independent experiments.

Figure 3

Dark stress tolerance test in the wild-type (WT) and transgenic plants. (A) Leaf senescence phenotype of WT and transgenic lines after dark treatment. (B–D) Total chlorophyll content, ion leakage rate and malondialdehyde (MDA) content of different genotypes. Using Student’s t-test, asterisks indicate statistically significant differences (* p < 0.05; ** p < 0.01). Data are shown as mean ± SD from three independent experiments.

Figure 4

Drought stress tolerance test in the WT and transgenic plants. (A) Seeds of WT and transgenic lines germinated on filter paper soaked in different solutions of PEG6000: water without PEG6000; 8% PEG8000; 12% PEG6000; (B) Leaf senescence phenotype of WT and transgenic lines after drought treatment. Scale bar = 1 cm. (C,D) Statistics of germination rate and root length for different genotypes. (E) Survival rate of WT and transgenic plants after re-watering for 3 days. (F–H) After treatment with drought for 14 days, the total chlorophyll content, ion leakage rate and MDA content of isolated leaves of different genotypes. Using Student’s t-test, asterisks indicate statistically significant differences (* p < 0.05; ** p < 0.01). Data are shown as mean ± SD from three independent experiments.

Figure 5

Expression levels of related genes in WT and transgenic plants under normal condition and dark treatment. Three-leaf stage maize leaves were treated in dark for 5 days. The expression levels of related genes during dark stress were analyzed by qRT-PCR. (A–I) Expression analysis of stress-related genes, Chlorophyll-related genes and senescence-related genes. The expression level was normalized to that of maize ZmACTIN1. Using Student’s t-test, asterisks indicate statistically significant differences (* p < 0.05; ** p < 0.01). Data are shown as mean ± SD from three independent experiments.

Figure 6

Expression levels of related genes in WT and transgenic plants under normal condition and drought treatment. Three-leaf stage maize seedlings were withheld in water for 7 days. The expression levels of related genes during drought stress were analyzed by qRT-PCR. (A–I) Expression analysis of stress-related genes, Chlorophyll-related genes and senescence-related genes. The expression level was normalized to that of maize ZmACTIN1. Using Student’s t-test, asterisks indicate statistically significant differences (* p < 0.05; ** p < 0.01). Data are shown as mean ± SD from three independent experiments.

Figure 7

ZmSAG39 can promote the accumulation of reactive oxygen species (ROS). (A) Diaminobenzidine (DAB) staining. Maize leaves were stained with DAB after drought treatment. (B) Nitro-blue tetrazolium chloride (NBT) staining. Maize leaves were stained with NBT after drought treatment. (C,D) Accumulation of H₂O₂ and O₂⁻ in leaves of different lines. (E–H) Analysis of superoxide dismutase (SOD), catalase (CAT), peroxidase (POD) and ascorbic acid peroxidase (APX) activity. Using Student’s t-test, asterisks indicate statistically significant differences (* p < 0.05; ** p < 0.01). Data are shown as mean ± SD from three independent experiments.

Figure 8

Subcellular localization and yeast two-hybrid test of ZmSAG39 protein. (A) Subcellular localization analysis of ZmSAG39 proteins in tobacco cells. The scale bar represent 50 μm. (B) Interaction testing between pGBKT7-ZmSAG39 and pGADT7-GmYKT62. Positive control: pGADT7-T +pGBKT7-53; negative control: pGADT7-T+pGBKT7-Lam.