Multiomics Comparison And Physiological Characteristics Of Different Colour Shoots Of Camellia Sinensis Var. Huangjinya

Huangjinya is a photosensitive chlorotic tea variety.The color of shoots is regulated by light intensity.The new shoots are green under weak natural light.And when the light intensity is up to 200-270μmol photons·m^-2·s^-1,the leaves begin to be chlorotic.With the increase of light intensity,the degree of chlorisis gradually deepens,and physiological stress would occur under strong natural light conditions.The color changes of new shoots in Huangjinya along with the natural light intensity provides a good experimental material for the study of photoregulation of chlorophyll metabolism and photosynthesis.It is helpful to explore the specific pathways and modes of light regulation of chlorophyll metabolism and photosynthesis,and to elucidate its light response mechanism.In order to eliminate the adverse physiological and metabolic disturbances caused by excessive light,three different natural light intensities were used to treat Huangjinya in the early stage of this study,and new shoot materials with different colors were obtained.By measuring the photosynthetic physiological indexes（photosynthesis,chlorophyll fluorescence parameters,etc.）,stress physiological indexes（activity of antioxidant enzyme,content of active oxygen,etc.）and the structure of chloroplast membrane system,the chlorotic materials and control materials suitable for exploring the chlorotic mechanism of Huangjinya were reasonably determined.Subsequently,a comparative study of metabolomics and proteomics were carried out on the two materials,and the expression levels of key enzymes in chlorophyll,carotenoid metabolic pathway,photosynthetic electron transfer chain component proteins and key enzymes genes in dark reaction process were analyzed.And the molecular mechanism of leaf color response to light intensity of Huangjinya was explored from multiple dimensions.At the same time,a gene of CsHIPP26.1 protein was screened and cloned for the first time from the shoots of Huangjinya,which responded positively to the changes of light intensity and was highly expressed,and its function was explored.The main results were as follows:（1）The Huangjinya were treated with different shading degrees under natural light to obtain three kinds of color leaves in turn:tender green(180μmol photons·m^-2·s^-1,numbered Y+S),yellow(720μmol photons·m^-2·s^-1,numbered Y)and bright yellow(1620μmol photons·m^-2·s^-1,numbered Hs).The analysis of physiological characteristics showed that the content of O₂^-,H₂O₂,Fv/Fm and the structure of chloroplast membrane system of chlorotic leaves（Y）were similar to those of tender green leaves（Y+S）.There was no significant difference between Y and Y+S,and they were in the physiological state without adversity stress,but there were significant differences in photosynthetic physiological characteristics between Y and Y+S.Y and Hs had no significant difference inΦPSⅡ,qP,characteristics of photosynthetic response curve,light saturation point and light compensation point.They had similar photosynthetic physiological characteristics of chlorotic leaves,but Hs was in adversity physiological state.It could be concluded that Y not only has the chlorotic physiological characteristics of Hs,but also is in a stress-free physiological state similar to Y+S.Y could well reflect the genetic and physiological characteristics of Huangjinya.Y+S,as a control material with normal leaf color,can be used together with Y as an appropriate material for studying the molecular mechanism of light response of Huangjinya.（2）The low chlorophyll content in chlorotic leaves was due to the blocked chlorophyll synthesis and enhanced degradation of intermediate products.There were two main hindrances to chlorophyll synthesis in chlorotic leaves.The first hindrance point was from the synthesis of coproporphyrinogen III（COPP III）to protoporphyrin IX（PPIX）,of which the synthesis site transited from chloroplast matrix to thylakoid membrane.It was speculated that the abnormality of thylakoid membrane structure was the main reason for the obstruction of synthesis at this stage.The second hindered point was located in the Pchlide to chlorophyllide b,while the significant decrease expression of POR and CAO was the reason for its blocked synthesis.In addition,the gene and protein expression of ACD1 in chlorotic leaves increased significantly,which further led to the decreased synthesis of chlorophyll a and b.The xanthophyll cycle of chlorotic leaves was significantly enhanced,and the content of intermediate metabolites in flavonoid synthesis pathway was significantly increased.It was speculated that the chlorotic leaves under strong light of‘Huangjinya’was due to the color presentation of carotenoids and flavonoids after chlorophyll content was significantly reduced.（3）The decrease of electron transfer efficiency enhanced the sensitivity of chlorotic leaves to light.The proteins contents of D1（PsbA）,D2（PsbD）and Cytb559（PsbE）complexes and PSbO and PSbP in photoreaction center of chlorotic leaves were significantly decreased,which reduced the generation and transfer efficiency of donor side electrons.And the decrease of plastid quinone pool（PQ and PQH₂）and the significant decrease of PC protein further inhibited the photosynthetic electron transfer efficiency between PSI and PSI in chlorotic leaves,resulting in the decrease of photosynthetic efficiency.On one hand,the decrease of electron transfer efficiency enhanced the sensitivity of chlorotic leaves to light.But the dark reaction process of photosynthesis was not affected.（4）The function of HIPP26.1,a protein highly expressed in response to light intensity in Huangjinya,was investigated.It was found that the gene and protein expression of HIPP26.1increased with the increase of light intensity.Moreover,apple callus by overexpressing CsHIPP26.1 could significantly improve the tolerance to strong light stress.A CsPIF4 protein interacting with CsHIPP26.1 was found by yeast two-hybrid system.And it was found that CsPIF4 could interact with CsHIPP26.1 in nucleus by BiFC and pull-down verification.