Effects of Overexpression of MtVP1 on Potato Phenotypes and Sugar Metabolism

doi:10.11983/CBB21074

Abstract

Abstract: Type I H⁺-PPase is involved in gluconeogenesis and sucrose catabolism. Different types of mutants of Arabidopsis thaliana type I H⁺-PPase gene fed with different sugars (sucrose, glucose and fructose) showed various phenotypes. Therefore, it is speculated that type I H⁺-PPase may contain other mechanisms that affect sugar metabolism. In order to further clarify the effect of the enzyme on sugar metabolism, phenotypes of the MtVP1 overexpressed potato Weishu 4 were observed under different culture conditions. The fluctuation of sugar content was monitored, and the transcription profile was analyzed. The results showed that the MtVP1 overexpressed potato grew red stem, purple flower, and more developed trichomes. Furthermore, tubers per plant decreased in number, but became larger in size, and shriveled up faster. However, the transgenic plant had significantly decreased starch, glucose, and fructose contents in tubers, and glucose and fructose contents in buds. At the same time, feeding fructose significantly reduced the anthocyanin, and up-regulated the gene expression of fructose-1,6-diphosphate and fructose-2,6-diphosphate by 3-7 fold in the transgenic potato. The results could shed light on further studies of the physiological function of type I H⁺-PPase from the perspective of sugar metabolism.

Key words: type I membrane-associated H⁺-PPase, fructose-1,6-bisphosphatase, fructose-2,6-bisphosphatase, sugar metabolism, fructose metabolism

Jianwu Wang, Wenjuan Wang, Weiwei Xiang, Huiping Dai, Haiqing Wang, Xiangxiang Qu, Furen Kang. Effects of Overexpression of MtVP1 on Potato Phenotypes and Sugar Metabolism[J]. Chinese Bulletin of Botany, 2022, 57(2): 197-208.

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URL: https://www.chinbullbotany.com/EN/10.11983/CBB21074

https://www.chinbullbotany.com/EN/Y2022/V57/I2/197

Figures/Tables 10

Table 1 Primer sequences for qRT-PCR

Primer name	Sequences (5′-3′)
10788-F	TCTTGTACGCTTGTCTTGAGCACTG
10788-R	CTTCCCTTTCTTCGGAATCTTGATA
19189-F	CACTCCAACTGATGATTGCCTCC
19189-R	CCTGCTGCTAGAAGATTGTTCCCT
20363-F	ATACAGCCCGAATGATGAGTGC
20363-R	GCTGCAAGAAGGTTTGTCCCT
19188-F	ACCACTTAAAGCAACCAGGTCC
19188-R	TCCCGCCATACAACAACATTC
24109-F	GAATGAGCAGACGAAGCACCCT
24109-R	ACGAACTTGCAGCCAAGAACAA
30370-F	TTGGGAAGTACCGCCGACTC
30370-R	TGCAAGGGCAGCTACCTCATT
ef1α-F	ATTGGAAACGGATATGCTCCA
ef1α-R	TCCTTACCTGAACGCCTGTCA

Figure 1 Molecular identification and changes of flower color in MtVP1 overexpressed potato (A) PCR result at DNA level; (B) Substrate hydrolysis activity of type I H+-PPase; (C) Changes of flower color. WT: Wild type potato (negative control); P: The expression vector for transgene (positive control); 1-5 indicate different transgenic potato lines. Data are reported as means±SE of three replicates (student’s t-test). * P<0.05; Bars=1 cm

Figure 2 Changes of tubers in MtVP1 overexpressed potato WT: Wild type. Bars=1 cm

Figure 3 Phenotype of tubers in MtVP1 overexpressed potato (A) Tubers after 5 months storage; (B) Tubers (as seed) at the beginning of the budding stage. WT: Wild type. Bars=1 cm

Figure 4 Contents of different sugars in tuber and bud of MtVP1 overexpressed potato (A) Starch contents of tubers; (B) Glucose contents of bud and tuber; (C) Fructose contents of bud and tuber; (D) Trehalose contents of bud and tuber. Data are reported as means±SE of three replicates (student’s t-test). WT: Wild type. * P<0.05, ** P<0.01

Figure 5 Sugar contents in potato sterile seedlings cultured by normal MS medium (A) and anthocyanin content in shoots by various sugar feeding in MS medium (B) Data are reported as means±SE of three replicates (student’s t-test). WT: Wild type. * P<0.05, ** P<0.01

Table 2 Number of differentially expressed genes in potato determined by transcriptome sequencing

Lines	Differentially expressed genes
Lines	Total	Up	Down
StOE1 vs WT	8943	4397	4546
StOE2 vs WT	14121	7045	7076
StOE3 vs WT	14654	7310	7335
StOE1 vs Favorita	5489	3091	2398
StOE2 vs Favorita	8058	4251	3707
StOE3 vs Favorita	12305	6485	5820

Figure 6 Heat map of pairwise comparison among MtVP1 overexpressed potato, wild type (WT) control and commercial cultivar Favorita

Table 3 log2FoldChange value of fructose-1,6-bisphosphatase and fructose-2,6-bisphosphatase transcripts of potato determined by transcriptome sequencing

Lines	Fructose-1,6-bisphosphatase					Fructose-2,6- bisphosphatase
Lines	10788	19189	20363	19188	24109	30370
StOE1 vs WT	0.39262	1.1159	0.8341	0.91068	1.2916	0.77498
StOE2 vs WT	0.28647	1.0103	0.9656	0.66701	1.0852	0.91597
StOE3 vs WT	0.1806	0.83961	1.0418	0.87353	1.3994	0.80856
StOE1 vs Favorita	NSC	0.64089	NSC	0.81127	0.37061	NSC
StOE2 vs Favorita	NSC	0.55679	0.44266	0.58928	0.18557	0.35543
StOE3 vs Favorita	NSC	0.40857	0.54143	0.8185	0.52253	0.27085

Figure 7 Expression profiles of fructose-2,6-bisphosphatase gene (A) and fructose-1,6-bisphosphatase genes (B-F) transcripts in potato Data are reported as means±SE of three replicates (student’s t-test). * P<0.05

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