Analysis Of Absorption Redshift Of Light-harvesting Complex Of Photosystem ? From Arabidopsis Thaliana

Photosynthesis provides an energy source for the survival of living things on Earth,and photosynthesis cannot be achieved without the energy balance between the two light systems.The existence of the double-light enhancement effect indicates that the absorption of light energy by photosystem I(PSI)and photosystem II(PSII)has their own unique wavelength range,and the light-harvesting antenna complex I(LHCI)combined with a small amount of red-shifted chlorophyll(red chlorophylls,red Chls),these special extended PSI absorption range to far red light range,improve the utilization of long-wavelength light by plants,and have a great influence on the energy transfer process of PSI complex.The high-resolution structure of the higher plant pea PSI-LHCI complex determines that red Chls exists in the form of chlorophyll pair(Chl a603-Chl a609),located at the interface between LHCI and the core complex,and reveals the amino acid coordination of Chl a603 Chl a609.Studying the effect of the protein environment around red Chls on its energy level is of great significance for elucidating the mechanism of energy transfer and transduction of PSI-LHCI in higher plants.In this paper,the change of the red shift phenomenon by artificially changing the amino acid position of the light-harvesting antenna complex that interacts with the red-shifted chlorophyll pair is of great significance for elucidating the relationship between the structure and function of red Chls.In this study,Arabidopsis thaliana was used as experimental material to investigate the red shift of the chlorophyll absorption spectrum of the whole plant after changing the central magnesium atom coordination amino acids of two chlorophyll molecules Chl a603 and Chl a609 in light-harvesting antenna complex I.The main findings are as follows:1.Screening of LHCI deletion homozygous mutants in Arabidopsis thalianaFirstly,Arabidopsis seeds were planted and homozygous plants were identified.Three kinds of light-harvesting antenna-missing homozygous mutants lhca1,lhca2,lhca3 were successful ly screened.The deletion of LHCA1 in lhca1 led to the expression of three other antenna genes.The decrease of LHCA2 in lhca2 led to a significant increase in the expression of LHCA1 gene;the loss of LHCA3 in lhca3 resulted in a significant increase in the expression of LHCA2 gene.Analysis of the 77 K fluorescence spectrum showed that the emission spectrum of the plant lacking an antenna complex at 440 nm was different from that of the wild type,and there was a certain degree of blue shift.The blue shift of the mutant lhca1 was not obvious,and the blue shift of the mutant lhca2 and lhca3 was basically the same,and a blue shift was observed compared with WT.2.Construction of base-editing mutants of amino acid loci associated with LHCI redshift in Arabidopsis thalianaBy editing the amino acid sites of the red-shifted chlorophyll of the four Arabidopsis lightharvesting antenna complexes for the interaction of a603 and a609,10 base editing vectors were constructed,5 of which were for the chlorophyll a603 magnesium atom.The amino acid site was edited by the vector,and the five were vectors for editing the amino acid site of the chlorophyll a609 magnesium atom.A total of 187 base-editing positive seedlings were obtained from 10 editing vectors.Due to the low editing efficiency of base editing,the sequence analysis of amino acid editing sites showed that the obtained positive seedlings did not find transgenic plants for effective editing.3.Site-directed mutagenesis of Arabidopsis thaliana LHCI red shift-related amino acid siteThe protein stability after targeted mutation of the coordinating amino acid was analyzed by site prediction,and 4-5 candidate mutant amino acids were selected per antenna according to the analysis results.For the LHCA1 gene,four over-expressed vectors with directed mutations were constructed.Among them,3,3,7 and 10 transgenic plants were obtained for H90 G,H90W,H90 R and H90 Y respectively.Five directed mutations were constructed for LHCA2 gene.Expression vector,in which H97 L,H97G,H97 R,H97I,H97 Y obtained 5,3,4,3 and 2 transgenic plants respectively;4 over-expressed overexpression vectors were constructed for LHCA3 gene,among which N103 G,N103C 10 strains,12 strains,11 strains,and 10 transgenic plants were obtained from N103 H and N103 M,respectively.All 83 transgenic plants identified by PCR and Western Blot.The results of 77 K fluorescence spectroscopy indicated that the mutant H90 G and H90 R had no effect on the red shift when the amino acid coordinated with red-shifted chlorophyll in LHCA1 was changed.The mutant H90 Y was blue-shifted;the LHCA2 was changed with redshifted chlorophyll.The amino acid position of the amino acid was blue-shifted compared with the wild type,and there was a red shift phenomenon compared with the mutant;the amino acid coordinated with the red-shifted chlorophyll in LHCA3 was changed to be blue-shifted compared with the wild type.The experimental results show that changing the amino acid coordinated with the red-shifted chlorophyll a603 central magnesium atom will affect the absorption wavelength of red-shifted chlorophyll to varying degrees.