Effect Of Shading And Temperature On The Leaf Color Of The Light-sensetive Albino Tea Plant ’Huangjinya’ And Its Potential Mechanism

Cultivar ‘Huangjinya’ is the most widely planted light-sensitive albino tea plant in current China.Its newly sprouted buds and leaves are golden yellow with high amino acid content,can be used to process high quality tea.‘Huangjinya’ has been considered as an excellent germplasm for studying the molecular mechanism of leaf color variation and its interaction with the environment since its albino trait can be stably inherited,but is easily influenced by environmental conditions such as light.This thesis aims to clarify the effects of shading and temperature on the leaf color of the ‘Huangjinya’ and their potential molecular mechanisms through monitoring the content of photosynthetic pigments,observing the ultrastructure of chloroplasts,analyzing gene differential expression,and immunoblotting the PSⅡ / LHCⅡ supercomplexes and their subunits under different shading(0-144h)and temperature(25-5°C)conditions.This study will lay the foundation for revealing the genetic regulation mechanism of shoot albinism and developing efficient cultivation measures of the ’Huangjinya’.The main research results are as follows:(1)The restoration of the structure and function of thylakoid in chloroplasts under low light is an important physical-chemical basis for the transformation of the newly sprouted leaves from yellow to green in ‘Huangjinya’ after being shaded.A large number of cystic voids with blurry structure completely fill up the chloroplasts in leaves of’Huangjinya’ grown under natural light.Significant inhibition of lipid metabolism,less accumulation of photosynthetic pigments,and abnormal chlorophyll fluorescence kinetics curves and photosynthetic parameters have also been observed.Along with the extension of shading time,the structure of chloroplasts and their subcellular organelles gradually recovers,with formation of the flat stroma lamellae and stacked grana lamellae of thylakoids,a significant increase in the content of photosynthetic pigments,as well as normalization of the chlorophyll fluorescence kinetics curve and parameters such as the activity and the maximum photochemical quantum yield of the unit photosystem II(PSⅡ)reaction center.The obstacle of thylakoid membrane assembly may be the critical inducement for the albino shoots of ’Huangjinya’ grown under natural light.(2)Up-regulatory expression of the genes encoding the light harvesting complex II(LHCⅡ)subunits is a key prerequisite for the normalization of thylakoid membrane structure and function in the leaf of ’Huangjinya’ after being shaded.7154 differentially expressed genes(DEGs)are co-expressed during the short-term shading process,and mainly enriched in pathways such as “photosynthesis”,“photosynthesis-antenna proteins”,and “glycerolipid synthesis”.Compared with leaf of ‘Huangjinya’ grown under natural light,the expression levels of the Psb A,Psb D,Psb C,and Psb B,encoding the subunits of the PSⅡ reaction center complex in the “photosynthesis” pathway,and Lhcb1.x,Lhcb2.x,Lhcb3.x,Lhcb4,Lhcb5,and Lhcb6,encoding LHCⅡ subunits in the“photosynthesis-antenna protein” pathway,are all upregulated in leaves along with an extension of shading time.Combined with the changes in expression of the genes related to PSⅡ and LHCⅡ subunits in leaf of ‘Fudingdabaicha’ before and after being shaded,it is clear that the suppressed expression of the LHCⅡ subunits-encoding genes,especially the Lhcb1.x is closely related to the albino shoots of ‘Huangjinya’,and the expression of these genes is primarily regulated by the plastid retrograde signaling pathway GUN1-PTM-ABI4(GENOMES UNCOUPLED 1-PHD type transcription factor with transmembrane domains-ABSCISIC ACID INSENSITIVE 4).(3)The increased concentration of the LHCⅡ subunits promotes the assembly and accumulation of photosynthetic pigment-protein complexes in the thylakoid membranes of ‘Huangjinya’ after be shaded.In leaves of ‘Huangjinya’ grown under natural light,PSⅡ reaction center subunits such as D1,D2,CP43,and CP47 are present at low concentrations,while LHCⅡ subunits including Lhcb1,Lhcb2,Lhcb4,and Lhcb6 can’t be detected by the immunoblotting,and few photosynthetic pigment-protein complexes can be observed in the thylakoid membrane.Along with an extension of shading time,the concentrations of PSⅡ reaction center subunits and LHCⅡ subunits significantly increase in leaf of ‘Huangjinya’,leading to an accumulation of photosynthetic pigmentprotein complexes such as LHCⅡ trimers in the thylakoid membrane.Combining the changes in PSⅡ,LHCⅡ,and photosynthetic pigment-protein complexes in leaves of‘Fudingdabaicha’ before and after being shaded,the albino shoots of ‘Huangjinya’ under natural light is mainly associated with the impaired assembly of photosynthetic pigment-protein complexes in the thylakoid membrane.This assembly impairment is caused by the limitation of the LHCⅡ trimers formation due to the low concentration of subunit Lhcb1,but isn’t related to the synthesis of PSⅡ reaction center and inner antenna subunits.(4)The increased concentration of subunit Lhcb5 may be closely related to the partial green restoration of the albino shoots of ‘Huangjinya’ grown at high temperature.During the late autumn to early winter,the environmental temperature decreases from25°C to 5°C,the leaf color of ‘Huangjinya’ changes from “golden yellow with slight green” to “golden yellow” and finally to “yellowish-white”,with the intensified degradation of thylakoid structure,and significant decrease in the content of photosynthetic pigments and photosynthetic efficiency.It implies that the leaf color of ‘Huangjinya’can be influenced by temperature besides the light.The transcription and translation of the PSⅡ subunit-encoding genes are significantly down-regulated along with a decrease of temperature.The decline of the level of PSⅡ subunits in ‘Huangjinya’ may be related to the severe deficiency of LHCⅡ subunit accumulation.Lhcb1,Lhcb2,Lhcb4,and Lhcb6 can’t be detected by the immunoblotting in leaf of ‘Huangjinya’ grown at different temperatures except that the Lhcb5 can be done at 25°C and 15°C.The protein level of the LHCⅡ subunits changes inconsistently with the expression of their corresponding genes along with temperature decreasing.This suggests that a significant obstacle of translation and/or transportation occurs in the genes encoding the LHCⅡ subunits of the ‘Huangjinya’.