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

doi:10.11983/CBB18045

Abstract

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

Shengnan Cui,Yihan Zhang,Fan Xu. Heterologous Overexpression of Rice OsSAPP3 Gene Promotes Leaf Senescence in Transgenic Arabidopsis[J]. Chinese Bulletin of Botany, 2019, 54(1): 46-57.

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URL: https://www.chinbullbotany.com/EN/10.11983/CBB18045

https://www.chinbullbotany.com/EN/Y2019/V54/I1/46

Figures/Tables 7

Table 1 Sequences of primers

Primer name	Primer sequence (5'-3')	Annotation
CDS_OsSAPP3-F	CCCGGGATGCTATCTGCTGCGATGGAATACTT	PCR
CDS_OsSAPP3-R	GAACTCTATGTAGGAACTGAGAATGGAGCAAGG	PCR
cGVG-SAPP3-F	GTCGACATGCTATCTGCTGCGATGGAATAC	PCR
cGVG-SAPP3-R	ATCGATCTATGTAGGAACTGAGAATGGAGCAA	PCR
Pro_OsSAPP3-F	AAGCTTTCATATCCGTTGTTCCGTAGCG	PCR
Pro_OsSAPP3-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 leaves^a	5% significant level	1% very significant level	Rosette leaf size^b	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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