Heterologous Overexpression of Autophagy-related Gene OsATG8b from Rice Confers Tolerance to Nitrogen/Carbon Starvation and Increases Yield in Arabidopsis

doi:10.11983/CBB18064

Abstract

Abstract: Nitrogen is an essential element for plant growth and development and plays an important role in plant yield and quality. Autophagy is a conserved degradation-recycle pathway of cellular components in eukaryotes that plays an important role in nitrogen remobilization during plant growth and grain formation. We identified an autophagy core gene OsATG8b in rice and obtained 2 independent 35S-OsATG8b transgenic Arabidopsis homozygous lines. The expression of OsATG8b responded to nitrogen starvation in rice. Overexpression of OsATG8b promoted the growth and development of transgenic Arabidopsis, with rosette leaves larger than wild-type leaves. In addition, the yield increased significantly, by 15.16%. In addition, overexpression of OsATG8b could significantly enhance autophagic activity in leaves of transgenic Arabidopsis under nitrogen deficiency and effectively alleviate the growth inhibition of transgenic Arabidopsis caused by nitrogen and carbon stress. OsATG8b may be a good candidate gene for increasing nitrogen use efficiency and yield.

Key words: autophagy, OsATG8b, nitrogen remobilization, yield

Xiaoxi Zhen,Haoran Liu,Xin Li,Fan Xu,Wenzhong Zhang. Heterologous Overexpression of Autophagy-related Gene OsATG8b from Rice Confers Tolerance to Nitrogen/Carbon Starvation and Increases Yield in Arabidopsis[J]. Chinese Bulletin of Botany, 2019, 54(1): 23-36.

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URL: https://www.chinbullbotany.com/EN/10.11983/CBB18064

https://www.chinbullbotany.com/EN/Y2019/V54/I1/23

Figures/Tables 9

Table 1 The information of primers

Primer name	Sequence (5′-3′)	Function
cOsATG8b-F	CCATTCAAGTGGATGGCCAAGAGCTCGTTCAAGC	Gene cloning
cOsATG8b-R	GGTGACCTAGAGCAGCCCAAAGGTGTTCTCG	Gene cloning
cpOsATG8b-F	AAGCTTAAAATTAAATAAGACGAACAGTCAAACG	Gene cloning
cpOsATG8b-R	CCATGGCGCTCCTTCCTGCACACAAT	Gene cloning
rtOsATG8b-F	GCTGATCTTACCGTTGGGCA	Real-time RT-PCR
rtOsATG8b-R	ATCAGAGCAGCTGTTGGTGG	Real-time RT-PCR
rtAtAMT1-F	GCCTCTGCTGACTACTCCAACTT	Real-time RT-PCR
rtAtAMT1-R	GACCAGAACCAGTGAGAGACGA	Real-time RT-PCR
rtAtNR1-F	AGGATGGGCTAGTAAGCATAAGG	Real-time RT-PCR
rtAtNR1-R	GCAAACTGAATCATAGGCGGTG	Real-time RT-PCR
rtAtGS2-F	CACCAAACCTTACTCTCTGACA	Real-time RT-PCR
rtAtGS2-R	CACTATCTTCACCAGGTGCTTG	Real-time RT-PCR
rtAtGDH1-F	GCTTTAGCAGCAACAAACAGAA	Real-time RT-PCR
rtAtGDH1-R	TGAGCCAATGCGTTCACTTC	Real-time RT-PCR
rtACTIN1-F	ACCATTGGTGCTGAGCGTTT	Real-time RT-PCR
rtACTIN1-R	CGCAGCTTCCATTCCTATGAA	Real-time RT-PCR
rtTIP41-F	GTATGAAGATGAACTGGCTGACAAT	Real-time RT-PCR
rtTIP41-R	ATCAACTCTCAGCCAAAATCGCAAG	Real-time RT-PCR

Figure 1 Identification of OsATG8b as a nitrogen deficiency inducible/responsive gene in leaves and roots of rice seedlings (A) The rice seedlings cultured with N-sufficient (NS) solution for 14 days and transferred to the same NS solution, low N (NL) solution and the N-deficient (ND) solution, the expression of OsATG8b gene in leaves after 1 day and 3 days treatment; (B) The expression of OsATG8b gene in roots after 1 day and 3 days treatment. Values are means±SD, n=16, three biological replicates were performed. ** indicate significant differences in NS solution compared with NL and ND solution (P<0.01) (Student’s t-test).

Table 2 Bolting and flowering times of the wild-type and 35S-OsATG8b transgenic Arabidopsis

	WT	L-13	L-14
Bloting time (d)	36.56±1.58	30.78±2.07**	31.39±1.91**
Flowering time (d)	42.67±1.75	35.94±1.98**	36.61±1.94**

Table 3 Yield related characteristics of the wild-type and 35S-OsATG8b transgenic Arabidopsis

	Total number of siliques	Yield per plant (mg)	Thousand grain weight (mg)
WT	35.74±3.86	85.34±7.89	14.87±0.23
L-13	46.26±3.13**	100.13±6.02**	16.36±0.21**
L-14	48.22±3.62**	99.77±5.76**	17.54±0.41**

Figure 2 Over-expression of OsATG8b promotes growth and development of transgenic Arabidopsis 8-day-old seedlings were transferred to vermiculite-nutritional soil (1:3, v/v). (A) Expression level of OsATG8b in 14-day-old seedlings of 35S-OsATG8b transgenic lines and wild type (WT); (B) Panels from top to bottom show phenotypic observations of transgenic lines and WT of Arabidopsis at 10, 17 and 24 days after transfer to soil, respectively; (C) Phenotype of transgenic lines and WT at 42 days after transfer to soil; (D) The maximum rosette radius of 35S-OsATG8b transgenic lines and WT at different seedling age; (E) The plant height; (F) The total chlorophyll content; (G) The soluble protein content. Days: Days after germination. Values are means±SD, n=24, * P<0.05, ** P<0.01 (Student’s t-test), three biological replicates were performed. Bars=1 cm

Figure 3 Overexpression of OsATG8b enhances tolerance to N deficiency in transgenic Arabidopsis (A) 7-day-old seedlings of 35S-OsATG8b transgenic lines and wild type (WT) were transferred to 1/2MS medium for horizontal culture with sufficient (NS) or deficient (ND) N for 9 days. (B)-(D) The fresh weight, chlorophyll content and soluble protein content in rosette leaves of WT and 35S-OsATG8b transgenic Arabidopsis under NS or ND for 9 days, respectively; (E) The phenotype of 7-day-old seedlings of 35S-OsATG8b transgenic lines and WT were transferred to vertical plates with NS or ND for 9 days; (F)-(H) The primary root length, the shoot weight and the root weight of WT and transgenic Arabidopsis lines under NS or ND for 9 days, respectively; (I), (J) 10-day-old seedlings of ProOsATG8b-GUS transgenic Arabidopsis were transferred to ND and 10 μmol·L-1 SA for 24 h, respectively. Mock represented that the seedlings without treated. Values are means±SD, n=16, * and ** indicate significant (P<0.05) and extremely significant (P<0.01) differences between transgenic lines and WT (Student’s t-test), three biological replicates were performed. Bars=5 mm

Figure 4 Overexpression of OsATG8b in Arabidopsis enhanced tolerance to carbon starvation induced by dark treatment (A) 7-day-old seedlings of 35S-OsATG8b transgenic lines and wild type (WT) were transferred to darkness for 10 days (The left is before treatment, and the right is after treatment); (B) The chlorophyll content determination. Values are means±SD, n=10, * indicate significant difference between transgenic lines and WT (P<0.05) (Student’s t-test), three biological replicates were performed. Bars=5 mm

Figure 5 Overexpression of OsATG8b in Arabidopsis enhanced the autophagic activity under N deficient condition 7-day-old seedlings of transgenic line (L-14) and wild type (WT) were transferred to in N-deficient (ND) liquid medium with 1 μmol·L-1 ConA for 12 h, MDC-stained autophagosomes in leaves were observed by confocal microscopy. Bars=10 μm

Figure 6 Overexpression of OsATG8b in Arabidopsis changes the expression of genes in nitrogen metabolic 7-day-old seedlings of transgenic lines and wild type (WT) were transferred to 1/2MS medium with sufficient (NS) or deficient (ND) nitrogen for 14 days. (A) The expression of genes related to nitrogen metabolic in rosette leaves of 35S-OsATG8b transgenic lines and WT; (B) The expression of genes related to nitrogen metabolic in roots of 35S-OsATG8b transgenic lines and WT. Values are means±SD, n=10, * and ** indicate significant (P<0.05) and extremely significant (P<0.01) differences between transgenic lines and WT (Student’s t-test), respectively, three biological replicates were performed.

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