Responses of Choloroplast Specific Protein Profile to Different Stomatal Densities in Nicotiana benthamiana

doi:10.13560/j.cnki.biotech.bull.1985.2022-0506

Abstract

Abstract:

CO₂ enters chloroplast through stomata. How chloroplast is in response to stomatal density in molecular level is unknown yet. In this study, transgenic Nicotiana benthamiana lines, showing either significantly higher（HSD）or lower（LSD）leaf stomatal density, respectively, were generated through regulation of NtEPF 2（Nicotiana benthamiana EPIDERMAL PATTERNING FACTOR 2）expression in vivo. Transgenic lines were characterized with chlorophyll accumulation, photosynthetic parameters and leaf chloroplast differential expression proteins（DEPs）based on iTRAQ（isobaric tags for relative and absolute quantitation）. The response of chloroplast composition to stomatal density variation was expected to be revealed. The results showed that accumulation of DEPs, metabolizing PSI-PSII-LHCII complex formation and accelerating its electron transfer were up-regulated significantly, following down-regulation of DEPs for PSI photoprotection and light damage repair in HSD lines. Therefore, chlorophyll accumulation and net photosynthetic rate were 43% and 67% higher in HSD lines than control. On the other hand, attributing to strong photophosphorylation ATP biosynthesis at the early stage of leaf development, chlorophyll accumulation and photosynthetic parameter in LSD lines were significantly higher than in control. Chlorophyll accumulation went down along with growing leaf age. The result indicated that the increase in stomatal density accelerated PSI-PSII-LHCII complex formation and its electron transfer, and reduced photoprotection. Thus, chlorophyll accumulations in leaf at all ages were maintained at a higher level. Reversely, the decrease in stomatal density resulted in an upside-down mode for chlorophyll accumulations along with leaf ages owing to unbalance between ATP biosynthesis and carbon fixation. The result not only contributes to further understanding how stomata work, also broadens our view to rise carbon fixation through stomata regulation.

Key words: iTRAQ, NtEPF 2, protein, chlorophyll, chloroplast, stomatal density, Nicotiana benthamiana

SA Shi-juan, WU Han-yu, WEN Yuan, CHEN Xue-na, ZHENG Rui, YAO Xin-ling. Responses of Choloroplast Specific Protein Profile to Different Stomatal Densities in Nicotiana benthamiana[J]. Biotechnology Bulletin, 2023, 39(2): 193-202.

Figures/Tables 8

Table 1 Primers and sequences used in the study

名称Name		序列Sequence（5'-3'）
EP	EPF	TCCCGCCTTCAGTTTAGC
EP	EPR	CCCTTACGTCAGTGGAGATATC
EP+	EP+F	TCTAGCCAAAGCCTACGTCCAT
EP+	EP+R	AGGGAAACAAGGTCCACAAGCA
EP-	EP-F	AGCCAAAGCCTACGTCCATAT
EP-	EP-R	TTGGATACATCTCCATTCCTAACA

Fig. 1 Assay on stomatal density, chlorophyll accumulation and photosynthesis as well as stomata observation for ntepf-m and ntepf-p lines A: Relative accumulation of StEPF 2 mRNA. B: Assay on leaf stomatal density. C: Assay on stomatal conductance. D: Assay on net photosynthesis rate. E: Assay on chorophyll content. F: Stomatal observation for StEPF 2 overexpression lines. G: Stomatal observation for control lines. H: Stomatal observation for StEPF 2 inhibition expression lines. Data in statistics analysis from 3 repetitions, ** for P<0.05 difference significant level

Table 2 GO enrichment with DEPs sharing >60% similarity with orthologs in Arabidopsis

	生物过程 Biological process	分子功能 Molecular function	细胞组分 Cellular components
ntepf-m	120	69	72
ntepf-p	123	55	98

Fig. 2 Chloroplast common GO IDs in GO enrichment of DEPs identified from the leaves of up- or down-stomatal density

Table 3 GO enrichment with stomatal density rising specific DEPs

GO ID	U/R比值U/R ratio
光系统II反应中心Photosystem II reaction center（GO:0009539）	0.30
光系统II放氧复合物Photosystem II oxygen evolving complex（GO:0009654）	0.33
光系统II Photosystem II（GO:0009523）	0.27
光系统I反应中心Photosystem I reaction center（GO:0009538）	0.36
光系统I Photosystem I（GO:0009522）	0.36
光系统Photosystem（GO:0009521）	0.31
细胞色素b6f复合物Cytochrome b6f complex（GO:0009512）	0.33
叶绿体Chromoplast（GO:0009509）	0.67
叶绿体类囊体膜蛋白复合物Chloroplast thylakoid membrane protein complex（GO:0098807）	0.12
叶绿体基质类囊体 Chloroplast stromal thylakoid（GO:0009533）	0.30

Fig. 3 Accumulation variation of co-expreesion DEPs resulting from the leaves of up- or down-stomatal density A: Expression-lowing 3 DEPs along with rising stomatal density. B: Expression-lowing 12 DEPs along with rising stomatal density. C: Expression-rising 7 DEPs along with increased stomatal density

Fig. 4 Accumulation variation of specific DEPs in response to rising stomatal density A: PSII-PSI complex DEP accumulations up-regulated by high stomatal density. B: Electron transfer DEP accumulations down-regulated by high stomatal density. C: PSII component DEP accumulations down-regulated by high stomatal density

Fig. 5 Accumulation variation of specific DEPs in responses to lowering stomatal density A: DEP accumulations up-regulated by low stomatal density. B: DEP accumulations down-regulated by low stomatal density

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