Chin Bull Bot ›› 2019, Vol. 54 ›› Issue (1): 46-57.doi: 10.11983/CBB18045

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Heterologous Overexpression of Rice OsSAPP3 Gene Promotes Leaf Senescence in Transgenic Arabidopsis

Cui Shengnan1,2,Zhang Yihan1,Xu Fan1,*()   

  1. 1 Key Laboratory of Northeast Rice Biology and Breeding, Ministry of Agriculture, Key Laboratory of Northern Japonica Rice Genetics and Breedings, Ministry of Education, Rice Research Institute, Shenyang Agriculture University, Shenyang 110866, China
    2 Fushun Sifang Senior High School, Fushun 113122, China
  • Received:2018-02-12 Accepted:2018-05-21 Online:2019-07-31 Published:2019-01-01
  • Contact: Xu Fan E-mail:celiafanfan@163.com

Abstract:

Protein phosphatase-catalyzed reversible phosphorylation of proteins is a key aspect of leaf senescence. We screened and cloned a new PP2C gene, OsSAPP3, involved in leaf senescence regulation in rice. The promoter of OsSAPP3 was active in rosette leaves of ProOsSAPP3-GUS transgenic Arabidopsis and its activity increased with leaf age. With heterologous overexpression of OsSAPP3 driven by a CaMV 35S promoter, transgenic Arabidopsis could not grow normally. Therefore, OsSAPP3 heterologous overexpression was induced by the inducible promoter GVG system. Exo- genous inducible heterologous overexpression of OsSAPP3 led to a precocious leaf senescence phenotype in transgenic Arabidopsis including smaller rosette leaves and increased number, premature leaf senescence, and advanced bolting and flowering. Additionally, quantitative RT-PCR showed that exogenous inducible heterologous overexpression of OsSAPP3 upregulated the expression of critical senescence-related transcription factor genes, including SAG12, WRKY6, and NAC2. In summary, OsSAPP3 is a positive factor involved in regulating leaf senescence in rice.

Key words: Arabidopsis, leaf senescence, OsSAPP3, 2C type protein phosphatase

Table 1

Sequences of primers"

Primer name Primer sequence (5'-3') Annotation
CDSOsSAPP3-F CCCGGGATGCTATCTGCTGCGATGGAATACTT PCR
CDSOsSAPP3-R GAACTCTATGTAGGAACTGAGAATGGAGCAAGG PCR
cGVG-SAPP3-F GTCGACATGCTATCTGCTGCGATGGAATAC PCR
cGVG-SAPP3-R ATCGATCTATGTAGGAACTGAGAATGGAGCAA PCR
ProOsSAPP3-F AAGCTTTCATATCCGTTGTTCCGTAGCG PCR
ProOsSAPP3-R TCTAGACCTTCATCTCCGACACCTCCC PCR

Figure 1

GUS histochemical staining of ProOsSAPP3-GUS transgenic Arabidopsis thaliana at different developmental stages (A) From top to bottom, from left to right, the transgenic Arabidopsis thaliana before stain: Stage 0.5, Stage 1.02, Stage 1.04, Stage 1.10, Stage 6.0, Stage 6.9; (B) From top to bottom, from left to right, the transgenic Arabidopsis thaliana was stained in turn, and the seedling age was identical to that before staining; (C) Transgenic Arabidopsis thaliana treated with MOCK solution (hormone solvent); (D) Transgenic Arabidopsis thaliana treated with 10 μmol?L-1 ABA solution; (E) Transgenic Arabidopsis thaliana treated with 10 μmol?L-1 6-BA solution. Bars=5 mm"

Figure 2

Heterologous expression of OsSAPP3 gene lead to abnormal growth and development of transgenic Arabidopsis (A) 35S-OsSAPP3 transgenic Arabidopsis and wild-type Arabidopsis gene expression levels; (B) Wild-type Arabidopsis plant (WT) and 35S-OsSAPP3 transgenic Arabidopsis line (35S-OsSAPP3). The top left panel was the 14-day-old seedling growing in the nutrient soil, the bottom left is the 21-day-old plant growing in the nutrient soil, and the right panel was the plants cultured in nutrient soil for 25 days. Values are means±SD of one representative biological replicate (n=10) out of three, ** P<0.01. Bars=1 cm"

Figure 3

Exogenously induced OsSAPP3 heterologous over expression resulted in the inability of germination of transgenic Arabidopsis (A)-(C) DEX induced 36 h, the germination of transgenic and control seeds; (D)-(F) DEX induced 48 h, the germination of transgenic and control seeds; (G)-(I) DEX induced 72 h, the germination of transgenic and control seeds; (J) The expression of OsSAPP3 induced by exogenous factors at different times; (K) Exogenous induction of WRKY6 expression changes at different times; (L) Gene expression levels of GVG-OsSAPP3 transgenic Arabidopsis homozygous lines. Values are means±SD of one representative biological replicate (n=10) out of three. * P<0.05, ** P<0.01. Bars=1 cm"

"

Number of rosette
leavesa
5% significant
level
1% very significant
level
Rosette leaf
sizeb
5% significant
level
1% very significant
level
CK 9.4 c A 2.84 d C
Line2 10.2 bc A 2.3 ab BC
Line20 11.8 abc A 1.9 bc AB
Line14 12.6 ab A 1.5 cd A

Figure 4

Exogenous-induced OsSAPP3 heterologous over expression promotes premature aging of transgenic Arabidopsis mature seedlings (A) Arabidopsis control (CK) and GVG-OsSAPP3 transgenic Arabidopsis after spray treatment for 5 days; (B) 24-day-old CK and GVG-OsSAPP3 transgenic Arabidopsis plants; (C) The CK and GVG-OsSAPP3 transgenic Arabidopsis bolting process; (D) 34-day-old CK and GVG-OsSAPP3 transgenic Arabidopsis plants; (E) The CK and GVG-OsSAPP3 transgenic Arabidopsis flowering process; (F) CK and GVG-OsSAPP3 transgenic Arabidopsis after spray treatment for 21 days; (G) The chlorophyll contents of the fourth, fifth and sixth leaf of 26-day-old CK and GVG-OsSAPP3 transgenic Arabidopsis. Values are means±SD of one representative biological replicate (n=10) out of three. Bars=1 cm"

Figure 5

Changes of senescence marker gene expression in the fifth and sixth leaves of control (CK) and GVG-OsSAPP3 transgenic Arabidopsis after 30 μmol?L-1 DEX and MOCK solution treatment for 24 hoursValues are means±SD of one representative biological replicate (n=10) out of three. * P<0.05, ** P<0.01."

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