CsCHLI plays an important role in chlorophyll biosynthesis of tea plant ( Camellia sinensis )

Chlorophyll biosynthesis is a crucial biological process in plants, and chlorophyll content is one of the most important traits in the yield and quality of tea. Magnesium chelatase is a conserved enzyme complex responsible for the chlorophyll biosynthesis, which composed of the subnuit of CHLI, CHLD and CHLH. In this study, there were positive correlation between the expression of CsCHLI , chlorophyll content and chloroplast structure. The CsCHLI gene structure and functional domain indicated that, its cDNA length was 1275 bp, encodes 424 amino acids, consisted of cTP, AAA+ and AAA lid domain. Meanwhile, the subcellular localization demonstrated that CsCHLI localized in chloroplasts. In addition, protein-protein interaction analysis by yeast two-hybrid (Y2H) and bimolecular fluorescence complementation (BiFC) assays indicated that CsCHLI could interact with CsCHLI to form homodimer, or perhaps interact with CsCHLD and CsCHLH to form heterodimer. Moreover, Arabidopsis transformation displayed that overexpression of CsCHLI could restore the etiolation phenotype of the atchli1 mutant. These findings provide the mechanistic function of CsCHLI and its implications in chlorophyll biosynthesis in tea plant.


Introduction
Tea plant (Camellia sinensis (L) O. Kuntze) is an improtant leaf economic crop broadly cultivated worldwide [1] .Chlorophyll content as a useful agronomic trait, plays a crucial role in tea plant cultivating and breeding [2,3] .Different varieties of tea plants in leaf color are often perceived as more visually appealing, and have a higher price due to their delicate appearance, high content of theanine, as well as umami flavor [4] .In recent years, more and more new tea varieties with albino or etiolation phenotypes were cultivated, including 'Baiye 1 (Anji Baicha)', 'Baicha 1', 'Huangjinya', and 'Zhonghuang 1', etc [5,6] .In general, albino or etiolation buds were known to possess high levels of amino acids, which are essential in enhancing the natural flavor and improving the quality of tea [7,8] .At present, cultivated albino or etiolation tea plants can be categorized to temperature-sensitive, light-sensitive and environmentalinsensitive according to their response to the environment [9,10] .'Baiye 1' is a cultivar of temperature-sensitive albino tea, which displays yellow shoots at low temperatures and transition to green shoots upon exposure to warm temperatures [11] .Previous studies demonstrated that chlorophyll deficiency in albino or etiolation tea plants may be closely related to differential gene expression in the pathways of both chlorophyll biosynthesis and degradation [12] .
Chlorophyll is a crucial pigment that plays the central role in light-harvesting during photosynthesis, and facilitates the transfer of electrons to the photoreaction center [13] .Typically, the chlorophyll biosynthesis involves in three primary stages: (i) the production of 5-aminolevulinic acid (ALA), (ii) the formation of protoporphyrin IX from eight molecules of ALA, and (iii) the synthesis of chlorophyll through the Mg-protoporphyrin pathway [15,16] .The first step in chlorophyll biosynthesis is directed by the Mg-branch of the tetrapyrrole pathway, which involves the ATP-dependent insertion of Mg 2+ into protoporphyrin IX, performed by the magnesium chelatase enzyme complex.The magnesium chelatase enzyme complex composed of three subunits: CHLI, CHLD, and CHLH [17,18] .The CHLI subunit is primarily responsible for the hydrolysis of ATP during enzymatic reactions within the chlorophyll biosynthetic pathway [19] .It is equipped with specific structural motifs required for ATP binding, such as Walker A and B (W-A and W-B) motifs, sensors 1 and 2 (S-1 and S-2) motifs, presensor I and II (PS-I and II) inserts, as well as an arginine finger (ARG-finger) motif.PsCHLI1 has been identified by Wu [20] as the main CHLI subunit, have a crucial role in sustaining Mg 2+ -chelatase activity as well as enhancing the pea's photosynthetic efficiency.Interestingly, the CHLD subunit exhibits an AAA+ module at its N-terminus, which shares significant structural similarity to CHLI.Research has revealed that comparison of Rhodobacter capsulatus subunit CHLI and the AAA+-like module of subunit CHLD (residues 1-238 aa) shows 25% identity for the N-terminal AAA+ core [21] .The CHLD subunit possesses an elongated linker region abundant in acidic residues, as well as a C-terminal integrin I domain (also referred to as von Willebrand factor A or VWFA).In addition, CHLH is a multifunctional protein local-I n P r e s s ized in the plastid that acts as a crucial gene related to leaf pigmentation by regulating chlorophyll biosynthesis and chloroplast ultrastructure.Regarding Mg 2+ -chelatase, the ATPase function is carried out by the AAA-type CHLI subunit, while the binding of protoporphyrin IX (Proto IX) and metal insertion take place on CHLH [22] .
A number of mutations of Mg 2+ -chelatase subunit have been reported in chlorophyll-deficient mutants in Arabidopsis, rice and maize [23−25] .However, the interaction mechanism between subunit of the Mg 2+ -chelatase and the function of Mg 2+chelatase in chlorophyll biosynthesis and metabolism in albino tea plants has not been investigated.In this study, the relationship between gene expression of CsCHLI, CsCHLD and CsCHLH of Mg 2+ chelating enzyme and the chlorophyll content in tea leaves were characterized.Then, the CsCHLI gene was cloned, the subcellular localization and the mechanism in chlorophyll synthesis were analyzed by yeast two-hybrid (Y2H) and bimolecular fluorescence complementation (BiFC).Moreover, transgenic Arabidopsis was used to validate the role of CsCHLI in chlorophyll.This study provides in-depth insights into the interaction mechanism of Mg 2+ chelating enzyme, as well as the functional of CsCHLI in chlorophyll biosynthesis of tea plant.These results can supply improtant theoretical for the albino mechanism, meanwhile serve the valuable industrial support for the tea breeding.

Chloroplast ultrastructure observation
Chloroplast ultrastructure observations were performed as described [26] .The freshly harvested second leaf was cut into 1 × 1 mm 2 pieces and subsequently immersed in a 2.5% glutaraldehyde solution (v/v), at 4 °C for 12 h to effectively fix the samples.Dehydration and infiltration were conducted using a gradient mixture of acetone and resin, then the ultrathin sections were observed using an HT-7700 transmission electron microscope (TEM; Hitachi, Japan).

Chlorophyll content determination
The leaves of tea and Arabidopsis were isolated by using 80% (v/v) ice-cold acetone and homogenized with zirconia beads.The extracts were then centrifuged at 10,000 × g for 1 min at 4 °C.The absorbance of the extracts solution were measured using UV-1600 spectrophotometer (Shimadzu, Kyoto, Japan) at 645, and 663 nm, respectively.The content of chlorophyll a and b were calculated as described [26] .Three experimental replica-tions were conducted.

RNA extraction and RT-qPCR analysis
Total RNA was extracted from tea plant leaves using a Tissue RNA Isolation Kit (Beibei Biotechnology Co., Ltd., Zhengzhou, China) following the protocol.RNA quality and integrity were evaluated by electrophoresis on the 1.2% (w/v) agarose gel.Furthermore, cDNA was obtained using a HiScript II 1st Strand cDNA Synthesis Kit (Vazyme Biotechnology Co., Ltd., Nanjing, China).The sequence of CsCHLI (CSS0004540.1)and CsACTIN (CSS0008920.1)were obtained from the Tea Plant Information Archive (TPIA; http://tpia.teaplant.org).The specific primers of RT-qPCR were designed with Primer 7.0 software and listed in Supplemental Table S1.RT-qPCR analysis was conducted with the Bio-Rad Real-time thermal cycler CFX96 with SYBR Premix ExTaq™ Kit (Takara Co. Ltd., Japan).Finally, the 2 −ΔΔCᴛ method was used to determine the relative expression levels.Three biological replicates per sample were used.

Gene structure and motif analysis
The gene structure of CsCHLI, including base pair coordinates, was visualized using TBtools software by integrating the findings from the Pfam search and GFF3 gene structure information.The conserved motifs were analyzed utilizing NCBI Conserved Domains (https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi).The analysis of conserved domains was conducted on the online MEME software (v.4.9.1)(Multiple Expectation Maximizations for Motif Elicitation; http://memesuite.org/tools/meme).The motifs were carried out in Classic Mode on the online MEME software, with the number parameter set to less than 10 and all other options maintained at default settings.Afterward, the MEME outcomes were downloaded as a MAST file and visualized in TBtools, where the motif positions were shown.

Phylogenetic analysis
To obtain the amino acid sequences of CsCHLI and ten other homologs, BLASTP was performed on the National Center for Biotechnology Information website (NCBI; https://blast.ncbi.nlm.nih.gov).Sequence alignment was accomplished with ClustalW, and the phylogenetic analysis was generated using MEGA-X software by the Neighbor-Joining method with 1000 bootstrap replications.The phylogenetic tree was constructed using Geneious 9.0.

Vectors construction
The sequences of genomic DNA or cDNA of CsCHLI were amplifed from young leaves of 'Shaancha 1'.For subcellular location analysis, the open reading frame (ORF) sequence of CsCHLI without the stop codon was amplified use the primers of 3302Y-CHLI-F and 3302Y-CHLI-R.The target PCR fragment was recovered and combined into the 3302Y vector with enhanced yellow fluorescent protein (EYFP) and 35S promoter to construct the recombinant vectors 35S::CsCHLI-EYFP by the ClonExpress II One Step Cloning Kit (Vazyme Biotech, China).For Arabidopsis transformation, full-length of CsCHLI was amplified use the primers of 1300-CHLI-F and 1300-CHLI-R, then the digested fragment with BamHI was recovered and inserted into 1300, resulting in the recombinant plasmids 35S::CsCHLI-GFP.For the Y2H assay, CsCHLI (CSS0004540.1),CsCHLD (CSS0026907.1),and CsCHLH (CSS0016317.1)CDS were cloned by corresponding specific primers, the fragments were recovered and inserted into pGBKT7 (BD) and pGADT7 (AD) vocters by the ClonExpress II One Step Cloning Kit to constructed the recombinant plasmids BD-CsCHLI, AD-CsCHLI, BD-CsCHLD, AD-CsCHLD and BD-CsCHLH, respectively.For the BiFC assay, CsCHLI, CsCHLD, and CsCHLH were cloned by corresponding specific primers, the target fragments were recovered and inserted into the pNYFP and pCYFP vectors by the ClonExpress II One Step Cloning Kit to constructed the 35S::CsCHLI-YFP C , 35S::CsCHLI-YFP N , 35S::CsCHLD-YFP C , and 35S::CsCHLH-YFP N plasmids, respectively.

Subcellular localization
The 35S promoter-driven fusions of CsCHLI-EYFP was transformed into Agrobacterium tumefaciens (strain GV3101).Transient transformation method was employed to inject the cells into the leaves of six-week-old tobaccos (N.benthamiana) using a needleless syringe.After 48-72 h, the infiltrated leaves were harvested, and EYFP fluorescence was quantified using a laser scanning confocal microscope (Leica, Wetzlar, Germany).

Bimolecular fluorescence complementation assay (BiFC)
BiFC assays were conducted following the previously described [27] .The CDSs of CsCHLI, CsCHLD, and CsCHLH were inserted into pSPYNE-35S and pSPYCE-35S, which contain the C-terminal and N-terminal regions of yellow fluorescent protein (YFP), respectively.The two plasmids were co-transformed into A. tumefaciens strain GV3101 cells, then the six-week-old tobacco leaves were transiently infiltrated.After infiltration, plants were grown at 22 °C with 16 h photoperiod before detection.After 2 d, the fluorescence signal in the injected area of the leaves were observed using a confocal laser scanning microscope (Leica, Wetzlar, Germany).YFP was visualized by exciting with an argon laser at 514 nm.

Transgenic Arabidopsis generation
The recombinant vector was introduced into A. tumefaciens strain GV3101 using the electroporation transformation method.Using the floral dip method [28] , 35S::CsCHLI-GFP was transformed into Ler-0 wild type (Ler-0) and atchli1 mutant in Ler-0 wild type background, respectively.Transgenic Arabidopsis plants were selected in the T 3 transgenic plants for phenotypic identification, chlorophyll content measurement, and gene expression analysis [29] .T 1 transgenic plants were identified through the detection of GFP fluorescence as well as PCR amplification.The results were analyzed using DSDecode (http://dsdecode.scgene.com/home/) to decode the sequence and detect mutations.

Statistical analyses
The determination of chlorophyll and molecular analyses were conducted using at least three independent biological replicates.Statistical analyses were performed using Minitab 17.0 statistical software (Minitab Inc., UK).To detect statistically significant differences between mean values, one-way ANOVA was conducted with Fisher's least significant difference (LSD) test at 5%.

The chlorophyll content and expression of CsCHLI gene declined in albino tea leaves
To investigate the function of Mg 2+ -Chelatase genes in the chlorophyll biosynthetic of tea leaves.The ultrastructure, chlorophyll contents, and the expression levels of the Mg 2+ -Chelatase genes in tea leaves were determined.As shown in Fig. 1a, BY1, which was an albino tea plant, exhibited a pale green or blanched color compared to normal green tea.Ultrastructural analysis revealed a significant reduction in the number of chloroplasts in the mesophyll cells, which exhibited defects and lacked stacked thylakoid structures in BY1 leaves (Fig. 1b).In addition, the plastids of albino BY1 mesophyll cells showed a reduced number of osmiophilic granules and were devoid of starch granules.The further supported by measurements of chlorophyll a and b content in mature leaves, which showed significant differences between albino BY1 and green SC1 or LJ43 tea plants ( Fig. 1c).Furthermore, the expression levels of CsCHLI, CsCHLD, and CsCHLH genes involved in Mg 2+ -Chelatase were analyzed by RT-qPCR (Fig. 1d).The relative expression levels of CsCHLI was found to be significantly lower in the BY1 cultivars compared to CsCHLD and CsCHLH, while the expression levels of CsCHLH displayed no significant difference in difference cultivars.These results implied that CsCHLI could play crucial role in the biology of chlorophyll of tea plant.Therefore, the molecular function and mechanism of CsCHLI were conducted in the next research.

Cloning, characterization,and phylogenetic analysis of CsCHLI
The full-length of CsCHLI was successfully cloned from the total RNA of 'Shaancha 1'.According to the sequencing results, CsCHLI was 1275 bp, encoded a polypeptide of approximately 46 kDa, consisted of 424 amino acids (aa), the theoretical pI was 5.42 (Fig. 2a, 2b, Supplemental Table S2).Based on TargetP analysis, CsCHLI contained a chloroplastic transit peptide (CTP) at the N-terminus.
To investigate the functional domain and conserved motif of the CsCHLI proteins, multiple sequence were alignmented.As shown in Fig. 2, CHLI was highly conserved and shared multiple motifs in different species, such as Walker A, Walker B, Sensor I, Arginine fingers (R finger), and Sensor II.Furthermore, the AAA + (ATPases Associated with Diverse Cellular Activities) domain of CsCHLI was similar to that of AAA lid domain (Fig. 2b,  2c).Previous studies concluded that the domain of CsCHLI is responsible for binding the ATP α and β phosphates through the Walker-A motif, as well as coordinating the water molecule required for ATP hydrolysis via the Walker-B and sensor I motifs [30] .Therefore, the CsCHLI might have had the specific or exclusive function in ATP hydrolysis activity.
In order to better understand the evolutionary connections between Mg 2+ -chelatase proteins across various species, a phylogenetic tree was generated using sequence data from cyanobacterium, monocots, and dicots, including 1 cyanobacteria (Ssp.PCC6803), 3 monocotyledonous (O.sativa, S. italica, S. bicolor) and 6 dicotyledonous (A.thaliana, G. max, T. cacao, C. arabica, A. chinensis, N. tabacum) (Fig. 2d).The phylogenetic I n P r e s s analysis showd that, CsCHLI was in the branch of dicotyledons, and has more closely genetic relationship with A. chinensis.This result suggested that CHLI may have evolved before the divergence of dicots and monocots, and CsCHLI was more homology to AcCHLI.

Subcellular localization of CsCHLI
The gene's function strongly depends on their distribution in cells.For examining the localization of CsCHLI protein, the fused vectors expressing CsCHLI-EYFP under the 35S promoter were constructed.As shown in Fig. 3, free EYFP (35S::EYFP) fluorescence was visualized in both cytosol and nucleus.In contrast, the fluorescent signals of CsCHLI-EYFP was overlapped with those of the auto-fluorescence of chlorophyll in chloroplast.These results indicated that, CsCHLI could be located in the chloroplast.

Protein-protein interactions of CsCHLI
To investigate the interacting partners of CsCHLI in regulatory complexes, the Y2H and BiFC assays were conducted .As shown in Fig. 4, all combination yeast cells were normal grown on the SD-TL medium indicated that the combination voters were co-transfored into yeast.In the SD-TLHA medium, yeast cells that co-contained a combination of CsCHLI-AD and CsCHLI-BD or CsCHLD-AD and CsCHLD-BD constructs could grow effectively, implied that CsCHLI could interacted with CsCHLI and CsCHLD.However, the yeast carrying CsCHLH-AD or CsCHLH-BD failed to grow on SD-TLHA medium, probability due to it was more distance of genetic system between yeast and plant.
To further indentify the protein-protein interaction of CsCHLI in plant, the BiFC assays were performed.Following the coexpression of CsCHLI-YFP N and CsCHLI-YFP C , as well as CsCHLH-YFP N and CsCHLD-YFP C , strong YFP fluorescence signals were detected in the leaves of N. benthamiana (Fig. 5).In contrast, the negative control plasmids did not elicit any fluorescent response in the cells (Supplemental Fig. S1).These results demonstrated that the CsCHLI protein could interate with CsCHLD and CsCHLH in plant cells.

Overexpression of CsCHLI functionally complements atchli1 mutation
To further demonstrate the function of CsCHLI in vivo, CsCHLI was overexpressed in Arabidopsis corresponding mutants, atchli1.As shown in Fig. 6, compared to Ler-0, CsCHLI could partially restore the lethal yellowing phenotype of atchli1 in different transgenic lines (Fig. 6a).In addition, 35S::CsCHLI-GFP could located in chloroplast whatever in Ler-0 and atchli1 (Fig. 6c).Moverover, the contents of chlorophyll-a and chlorophyll-b of transgenic lines atchli1-SI were significantly restored (Fig. 6d).These results indicated that CsCHLI could located in chloroplast, and partically restored the reduced chlorophyll content and yellow leaf phenotype.

Discussion
As well known, theanine undergoes hydrolysis to form glutamate, which serves as a substrate for chlorophyll synthesis in tea plant [31] .Owing to their special leaf color and high level of theanine, tea cultivars exhibiting albino or etiolation pheno-

I n P r e s s
Mg 2+ -chelatase is a crucial rate-limiting enzyme in the biosynthesis of chlorophyll, and therefore represents a promising target for improving photosynthetic efficiency.Previous studies have demonstrated that knockdown of the gene of CHLI could lead to various physiological and biochemical changes, such as significant reduction in chlorophyll accumulation and yellow-leaf phenotype in Arabidipsis, pea and strawberry [32−34] .In this study, the functional of Mg 2+ -chelatase I subunits (CsCHLI) in chlorophyll biosynthesis in albino tea plants were investigated.Compared to BY1, SC1 and LJ43, BY1 leaves were exhibited the defect of chloroplast structure, including granular stacks and thylakoids, lower chlorophyll content, and significantly decreased expression level of CsCHLI.These results indicate that the gene expression of CsCHLI may be related to the chlorophyll biosynthesis in albino tea cultivars, which could provide an explanation for the differential accumulation of chlorophyll in the tea cultivars.
Analyzing phylogenetic relationships is extremely important for elucidating genes evolution.CHLI was a highly conserved protein, to date, it have been identified in a wide range of plants and bacterial species such as cyanobacteria, Arabidopsis, rice, soybean, tobacco, tomato, etc., indicating CHLI have an important roles in different species [35−37] .As shown in Fig. 2, CsCHLI had closely phylogenetic relationships with dicotyledonous species, suggesting that CsCHLI may potentially serve similar functions in both chlorophyll biosynthesis and metabolism with other dicotyledonous plant.Wei [1] .reported that C. sinensis and A. chinensis have more closely relationship and diverged from a shared lineage about 80 Mya ago.Meanwhile, the similar result was also found in the multiple sequence alignment and phylogenetic analysis of CHLI, CHLI  were highly conserved during the evolution from cyanobacterium to higher plant and the CHLI of C. sinensis and A. chinensis were clustered into one subgroup.
In diverse plant species, the defect of CHLI may cause to etiolation phenotype.Such as, the defect of CHLI Arabidopsis mutant, atchli1, presented yellow leaf and lead to the deficient formation of the photosynthetic apparatus.The silencing of pea CHLI was characterized by underdeveloped thylakoid membranes, altered chloroplast nucleoid structure, and malformed antenna complexes, which resulted in etiolation phenotype [17] .Many studies shown that, CHLI exhibited the ability to interact with CHLD and CHLH, forming a heterologous complex known as CHLI-D-H.In this study, the analysis of BiFC implied that CsCHLI probably interacted with CsCHLI, CsCHLD, and CsCHLH to form homodimer or heterodimers, however no interaction was detected between CsCHLH and CsCHLI or CsCHLD through Y2H method.Similar results were obtained in the interaction of magnesium chelatase in Soybean [38] .This was probably due to the higher molecular weight of CsCHLH (~152 kDa), it maybe unable to fold correctly, thus may not be worked in Y2H.Additionally, it may also because the interaction between CsCHLH and CsCHLI or CsCHLD probably involved in other plant proteins, tobacco could provided it.Howerer, it was not presented in yeast strain.
Previous studies have demonstrated that CHLI could restore the etiolation phenotype of corresponding mutants.This was confirmed in wheat mutant chli [39] , cucumber mutant c528 [40] , strawberry mutant p240 [34] .In order to demonstrate the function of CsCHLI in vivo, overexpression of CsCHLI were transformed into Ler-0 and atchli mutant (Ler-0 wild type background), respectively.As shown in Fig. 6, 35::CsCHLI-GFP could able to locate in chloroplast, and recover the etiolation phenotype of Arabidopsis mutant atchli1 to a normal green leaf phenotype, meanwhile has not significant affected the chlorophyll content of Ler-0.Therefore, the increased level of CsCHLI expression observed in the atchli mutant may serve as a compensatory feedback mechanism to counteract the lowchlorophyll stress in the tea, induced by the decreased activity of Mg-chelatase.

Conclusion
Taken together, the function and mechanism of CsCHLI in chlorophyll biosynthesis were identified in tea and Arabidopsis.The analysis between phenotype and gene expression indicated that, the gene expression of CsCHLI has positive correspondence with the chlorophyll content and the chloroplast ultrastructure of albinism tea leaves.Phylogenetic tree analysis confirmed that CHLI were highly conserved from algae to higher plants, and the genetic relationship of CHLI was more closely between C. sinensis and A. sinensis.The BiFC analysis showed that CsCHLI has a potential interaction with CsCHLI, CsCHLD and CsCHLH to form protein complex.Furthermore, the overexpression of CsCHLI could restore the chlorophyll-deficiency phenotype of atchli1 Arabidipsis mutant.This study indicated that CsCHLI located in chloroplast, has a potential protein-protein interaction with CsCHLD and CsCHLH to form heterologous complex, and performed an important function in the chlorophyll biosynthesis in plant.Our results provide a theoretical basis for the meachanism of albinism tea leave, and will be beneficial for the albino or etiolation breeding of tea plants.I n P r e s s reviewed and approved the final manuscript.