Protochlorophyllide Oxidoreductase Research Articles

Loss-of-function of PGL10 impairs photosynthesis and tolerance to high-temperature stress in rice.

High temperature (HT) affects the production of chlorophyll (Chl) pigment and inhibits cellular processes that impair photosynthesis, and growth and development in plants. However, the molecular mechanisms underlying heat stress in rice are not fully understood yet. In this study, we identified two mutants varying in leaf color from the ethylmethanesulfonate mutant library of indica rice cv. Zhongjiazao-17, which showed pale-green leaf color and variegated leaf phenotype under HT conditions. Mut-map revealed that both mutants were allelic, and their phenotype was controlled by a single recessive gene PALE GREEN LEAF 10 (PGL10) that encodes NADPH:protochlorophyllide oxidoreductase B, which is required for the reduction of protochlorophyllide into chlorophyllide in light-dependent tetrapyrrole biosynthetic pathway-based Chl synthesis. Overexpression-based complementation and CRISPR/Cas9-based knockout analyses confirmed the results of Mut-map. Moreover, qRT-PCR-based expression analysis of PGL10 showed that it expresses in almost all plant parts with the lowest expression in root, followed by seed, third leaf, and then other green tissues in both mutants, pgl10a and pgl10b. Its protein localizes in chloroplasts, and the first 17 amino acids from N-terminus are responsible for signals in chloroplasts. Moreover, transcriptome analysis performed under HT conditions revealed that the genes involved in the Chl biosynthesis and degradation, photosynthesis, and reactive oxygen species detoxification were differentially expressed in mutants compared to WT. Thus, these results indicate that PGL10 is required for maintaining chloroplast function and plays an important role in rice adaptation to HT stress conditions by controlling photosynthetic activity.

Fluorination of a conserved tyrosine in POR offers new clues for proton transfer.

Reduction of the 17,18-double bond in the D-ring during chlorophyll biosynthesis is catalyzed by the rare, naturally occurring photoenzyme protochlorophyllide oxidoreductase (POR). A conserved tyrosine residue has been suggested to donate a proton to C18 of the substrate in the past decades. Taylor and colleagues scrutinized the model with a powerful tool that utilized a modified genetic code to introduce fluorinated tyrosine analogues into POR. The presented results show that the suggested catalytically critical tyrosine is unlikely to participate in the reaction chemistry but is required for substrate binding, and instead, a cysteine residue preceding the lid helix is proposed to have the role of proton donor.

The light and hypoxia induced gene ZmPORB1 determines tocopherol content in the maize kernel.

Tocopherol is an important lipid-soluble antioxidant beneficial for both human health and plant growth. Here, we fine mapped a major QTL-qVE1 affecting γ-tocopherol content in maize kernel, positionally cloned and confirmed the underlying gene ZmPORB1 (por1), as a protochlorophyllide oxidoreductase. A 13.7 kb insertion reduced the tocopherol and chlorophyll content, and the photosynthetic activity by repressing ZmPORB1 expression in embryos of NIL-K22, but did not affect the levels of the tocopherol precursors HGA (homogentisic acid) and PMP (phytyl monophosphate). Furthermore, ZmPORB1 is inducible by low oxygen and light, thereby involved in the hypoxia response in developing embryos. Concurrent with natural hypoxia in embryos, the redox state has been changed with NO increasing and H2O2 decreasing, which lowered γ-tocopherol content via scavenging reactive nitrogen species. In conclusion, we proposed that the lower light-harvesting chlorophyll content weakened embryo photosynthesis, leading to fewer oxygen supplies and consequently diverse hypoxic responses including an elevated γ-tocopherol consumption. Our findings shed light on the mechanism for fine-tuning endogenous oxygen concentration in the maize embryo through a novel feedback pathway involving the light and low oxygen regulation of ZmPORB1 expression and chlorophyll content.

Elucidating substrate binding in the light-dependent protochlorophyllide oxidoreductase

Two protochlorophyllide binding modes to the light-dependent protochlorophyllide oxidoreductase were considered: mode B exhibits more thermodynamically favorable binding.

Regulatory mechanism of a light -dependent protochlorophyllide oxidoreductase in chlorophyll biosynthesis and environmental adaptation

Regulatory mechanism of a light -dependent protochlorophyllide oxidoreductase in chlorophyll biosynthesis and environmental adaptation

Mechanistic implications of the ternary complex structural models for the photoenzyme protochlorophyllide oxidoreductase.

The photoenzyme protochlorophyllide oxidoreductase (POR) is an important enzyme for understanding biological H-transfer mechanisms. It uses light to catalyse the reduction of protochlorophyllide to chlorophyllide, a key step in chlorophyll biosynthesis. Although a wealth of spectroscopic data have provided crucial mechanistic insight, a structural rationale for POR photocatalysis has proved challenging and remains hotly debated. Recent structural models of the ternary enzyme-substrate complex, derived from crystal and electron microscopy data, show differences in the orientation of the protochlorophyllide substrate and the architecture of the POR active site, with significant implications for the catalytic mechanism. Here, we use a combination of computational and experimental approaches to investigate the compatibility of each structural model with the hypothesised reaction mechanisms and propose an alternative structural model for the cyanobacterial POR ternary complex. We show that a strictly conserved tyrosine, previously proposed to act as the proton donor in POR photocatalysis, is unlikely to be involved in this step of the reaction but is crucial for Pchlide binding. Instead, an active site cysteine is important for both hydride and proton transfer reactions in POR and is proposed to act as the proton donor, either directly or through a water-mediated network. Moreover, a conserved glutamine is important for Pchlide binding and ensuring efficient photochemistry by tuning its electronic properties, likely by interacting with the central Mg atom of the substrate. This optimal 'binding pose' for the POR ternary enzyme-substrate complex illustrates how light energy can be harnessed to facilitate enzyme catalysis by this unique enzyme.

A novel tetratricopeptide-repeat protein, TTP1, forms complexes with glutamyl-tRNA reductase and protochlorophyllide oxidoreductase during tetrapyrrole biosynthesis.

The biosynthesis of the tetrapyrrole end-products chlorophyll and heme depends on a multifaceted control mechanism that acts primarily at the post-translational level upon the rate-limiting step of 5-aminolevulinic acid synthesis and upon light-dependent protochlorophyllide oxidoreductase (POR). These regulatory processes require auxiliary factors that modulate the activity, stability, complex formation, and subplastidal localization of the relevant proteins. Together, they ensure optimal metabolic flow during the day and at night. As an Arabidopsis homolog of the POR-interacting tetratricopeptide-repeat protein (Pitt) first reported in Synechocystis, we characterize tetrapyrrole biosynthesis-regulating tetratricopeptide-repeat protein1 (TTP1). TTP1 is a plastid-localized, membrane-bound factor that interacts with POR, the Mg protoporphyrin monomethylester cyclase CHL27, glutamyl-tRNA reductase (GluTR), GluTR-binding protein, and FLUORESCENCE IN BLUE LIGHT. Lack of TTP1 leads to accumulation of GluTR, enhanced 5-aminolevulinic acid synthesis and lower levels of POR. Knockout mutants show enhanced sensitivity to reactive oxygen species and a slower greening of etiolated seedlings. Based on our studies, the interaction of TTP1 with GluTR and POR does not directly inhibit their enzymatic activity and contribute to the control of 5-aminolevulinic acid synthesis. Instead, we propose that TTP1 sequesters a fraction of these proteins on the thylakoid membrane, and contributes to their stability.

Far-red light mediates light energy capture and distribution in soybeans (Glycine max L.) under the shade

Shade avoidance syndrome (SAS) is an important measure of how plants respond to shade. Shade conditions are characterized by low light density and low red-to-far-red light (R/FR) ratio. However, low light intensity and low R/FR ratio play considerable and diverse roles in SAS. In the present study, soybean plants were grown in the presence and absence of additional FR at two light levels to investigate the different effects on soybean plants. Compared with normal light intensity, net photosynthetic rate significantly decreased in low light, but increased in low R/FR ratio. Chlorophyll a fluorescence transient curves showed that the absorption of light flux increased under low light intensity and low R/FR ratio, whereas the number of photo reaction centers decreased under low light intensity. In addition, the content of porphyrins and chlorophyll metabolites (magnesium protoporphyrin IX, protochlorophyllide, and chlorophyll a and b) was significantly enhanced by low light intensity and the relative gene expression levels of protoporphyrin IX magnesium chelatase, chlorophyll a oxygenase and protochlorophyllide oxidoreductases involved in the porphyrin pathway were significantly increased. The inhibitory effects of low light on the photosynthetic rate and biomass of soybean plants were alleviated by a low R/FR ratio. A low R/FR ratio optimized light capture and achieved efficient light energy utilization under low light intensity by fine-tuning the chlorophyll level and the relative gene expression level of critical chlorophyll biosynthesis-related enzymes.

Low-Temperature-Mediated Promoter Methylation Relates to the Expression of TaPOR2D, Affecting the Level of Chlorophyll Accumulation in Albino Wheat (Triticum aestivum L.).

Chlorophyll is an indispensable photoreceptor in plant photosynthesis. Its anabolic imbalance is detrimental to individual growth and development. As an essential epigenetic modification, DNA methylation can induce phenotypic variations, such as leaf color transformation, by regulating gene expression. Albino line XN1376B is a natural mutation of winter wheat cultivar XN1376; however, the regulatory mechanism of its albinism is still unclear. In this study, we found that low temperatures induced albinism in XN1376B. The number of chloroplasts decreased as the phenomenon of bleaching intensified and the fence tissue and sponge tissue slowly dissolved. We identified six distinct TaPOR (protochlorophyllide oxidoreductase) genes in the wheat genome, and TaPOR2D was deemed to be related to the phenomenon of albinism based on the expression in different color leaves (green leaves, white leaves and returned green leaves) and the analysis of promoters' cis-acting elements. TaPOR2D was localized to chloroplasts. TaPOR2D overexpression (TaPOR2D-OE) enhanced the chlorophyll significantly in Arabidopsis, especially at two weeks; the amount of chlorophyll was 6.46 mg/L higher than in WT. The methylation rate of the TaPOR2D promoter in low-temperature albino leaves is as high as 93%, whereas there was no methylation in green leaves. Correspondingly, three DNA methyltransferase genes (TaMET1, TaDRM and TaCMT) were up-regulated in white leaves. Our study clarified that the expression of TaPOR2D is associated with its promoter methylation at a low temperature; it affects the level of chlorophyll accumulation, which probably causes the abnormal development of plant chloroplasts in albino wheat XN1376B. The results provide a theoretical basis for in-depth analysis of the regulation of development of plant chloroplasts and color variation in wheat XN1376B leaves.

Identification of key genes responsible for green and white colored spathes in Anthurium andraeanum (Hort.).

Modern anthuriums, Anthurium andraeanum (Hort.) are among the most popular flowering plants and widely used for interior decoration. Their popularity is largely attributed to the exotic spathes with different colors. Previous studies have reported color development in red spathe cultivars, but limited information is available on key genes regulating white and green colored spathes. This study analyzed anthocyanin, chlorophyll, and carotenoid contents as well as transcript differences in spathes of eight cultivars that differed in spathe colors ranging from red to white and green. Results showed that increased expression of a transcription factor AaMYB2 was associated with elevated levels of anthocyanin in spathes, but decreased expression of AaMYB2 and increased expression of AaLAR (leucoanthocyanidin reductase) and AaANR (anthocyanidin reductase) were accompanied with the accumulation of colorless proanthocyanidin, thus the white spathe. As to the green colored spathe, chlorophyll content in the green spathe cultivar was substantially higher than the other cultivars. Correspondingly, transcripts of chlorophyll biosynthesis-related genes AaHemB (porphobilinogen synthase) and AaPor (protochlorophyllide oxidoreductase) were highly upregulated but almost undetectable in white and red spathes. The increased expression of AaHemB and AaPor was correlated with the expression of transcription factor AaMYB124. Subsequently, qRT-PCR analysis confirmed their expression levels in nine additional cultivars with red, white, and green spathes. A working model for the formation of white and green spathes was proposed. White colored spathes are likely due to the decreased expression of AaMYB2 which results in increased expression of AaLAR and AaANR, and the green spathes are attributed to AaMYB124 enhanced expression of AaHemB and AaPor. Further research is warranted to test this working model.

Theoretical and Experimental Studies on Plant Light-Dependent Protochlorophyllide Oxidoreductase as a Novel Target for Searching Potential Herbicides.

Herbicide resistance is a prevalent problem that has posed a foremost challenge to crop production worldwide. Light-dependent enzyme NADPH: protochlorophyllide oxidoreductase (LPOR) in plants is a metabolic target that could satisfy this unmet demand. Herein, for the first time, we embarked on proposing a new mode of action of herbicides by performing structure-based virtual screening targeting multiple LPOR binding sites, with the determination of further bioactivity on the lead series. The feasibility of exploiting high selectivity and safety herbicides targeting LPOR was discussed from the perspective of the origin and phylogeny. Besides, we revealed the structural rearrangement and the selection key for NADPH cofactor binding to LPOR. Based on these, multitarget virtual screening was performed and the result identified compounds 2 affording micromolar inhibition, in which the IC50 reached 4.74 μM. Transcriptome analysis revealed that compound 2 induced more genes related to chlorophyll synthesis in Arabidopsis thaliana, especially the LPOR genes. Additionally, we clarified that these compounds binding to the site enhanced the overall stability and local rigidity of the complex systems from molecular dynamics simulation. This study delivers a guideline on how to assess activity-determining features of inhibitors to LPOR and how to translate this knowledge into the design of novel and effective inhibitors against malignant weed that act by targeting LPOR.

Plastid Genome Evolution of Two Colony-Forming Benthic Ochrosphaera neapolitana Strains (Coccolithales, Haptophyta)

Coccolithophores are well-known haptophytes that produce small calcium carbonate coccoliths, which in turn contribute to carbon sequestration in the marine environment. Despite their important ecological role, only two of eleven haptophyte plastid genomes are from coccolithophores, and those two belong to the order Isochrysidales. Here, we report the plastid genomes of two strains of Ochrosphaera neapolitana (Coccolithales) from Spain (CCAC 3688 B) and the USA (A15,280). The newly constructed plastid genomes are the largest in size (116,906 bp and 113,686 bp, respectively) among all the available haptophyte plastid genomes, primarily due to the increased intergenic regions. These two plastid genomes possess a conventional quadripartite structure with a long single copy and short single copy separated by two inverted ribosomal repeats. These two plastid genomes share 110 core genes, six rRNAs, and 29 tRNAs, but CCAC 3688 B has an additional CDS (ycf55) and one tRNA (trnL-UAG). Two large insertions at the intergenic regions (2 kb insertion between ycf35 and ycf45; 0.5 kb insertion in the middle of trnM and trnY) were detected in the strain CCAC 3688 B. We found the genes of light-independent protochlorophyllide oxidoreductase (chlB, chlN, and chlL), which convert protochlorophyllide to chlorophyllide during chlorophyll biosynthesis, in the plastid genomes of O. neapolitana as well as in other benthic Isochrysidales and Coccolithales species, putatively suggesting an evolutionary adaptation to benthic habitats.

Chloroplast SRP43 and SRP54 independently promote thermostability and membrane binding of light-dependent protochlorophyllide oxidoreductases.

Protochlorophyllide oxidoreductase (POR), which converts protochlorophyllide into chlorophyllide, is the only light-dependent enzyme in chlorophyll biosynthesis. While its catalytic reaction and importance for chloroplast development are well understood, little is known about the post-translational control of PORs. Here, we show that cpSRP43 and cpSRP54, two components of the chloroplast signal recognition particle pathway, play distinct roles in optimizing the function of PORB, the predominant POR isoform in Arabidopsis. The chaperone cpSRP43 stabilizes the enzyme and provides appropriate amounts of PORB during leaf greening and heat shock, whereas cpSRP54 enhances its binding to the thylakoid membrane, thereby ensuring adequate levels of metabolic flux in late chlorophyll biosynthesis. Furthermore, cpSRP43 and the DnaJ-like protein CHAPERONE-LIKE PROTEIN of POR1 concurrently act to stabilize PORB. Overall, these findings enhance our understanding of the coordinating role of cpSPR43 and cpSRP54 in the post-translational control of chlorophyll synthesis and assembly of photosynthetic chlorophyll-binding proteins.

Expression of RsPORB Is Associated with Radish Root Color.

Radish (Raphanus sativus) plants exhibit varied root colors due to the accumulation of chlorophylls and anthocyanins compounds that are beneficial for both human health and visual quality. The mechanisms of chlorophyll biosynthesis have been extensively studied in foliar tissues but remain largely unknown in other tissues. In this study, we examined the role of NADPH:protochlorophyllide oxidoreductases (PORs), which are key enzymes in chlorophyll biosynthesis, in radish roots. The transcript level of RsPORB was abundantly expressed in green roots and positively correlated with chlorophyll content in radish roots. Sequences of the RsPORB coding region were identical between white (948) and green (847) radish breeding lines. Additionally, virus-induced gene silencing assay with RsPORB exhibited reduced chlorophyll contents, verifying that RsPORB is a functional enzyme for chlorophyll biosynthesis. Sequence comparison of RsPORB promoters from white and green radishes showed several insertions and deletions (InDels) and single-nucleotide polymorphisms. Promoter activation assays using radish root protoplasts verified that InDels of the RsPORB promoter contribute to its expression level. These results suggested that RsPORB is one of the key genes underlying chlorophyll biosynthesis and green coloration in non-foliar tissues, such as roots.

FC2 stabilizes POR and suppresses ALA formation in the tetrapyrrole biosynthesis pathway.

During photoperiodic growth, the light-dependent nature of chlorophyll synthesis in angiosperms necessitates robust control of the production of 5-aminolevulinic acid (ALA), the rate-limiting step in the initial stage of tetrapyrrole biosynthesis (TBS). We are interested in dissecting the post-translational control of this process, which suppresses ALA synthesis for chlorophyll synthesis in dark-grown plants. Using biochemical approaches for analysis of Arabidopsis wild-type (WT) and mutant lines as well as complementation lines, we show that the heme-synthesizing ferrochelatase 2 (FC2) interacts with protochlorophyllide oxidoreductase and the regulator FLU which both promote the feedback-controlled suppression of ALA synthesis by inactivation of glutamyl-tRNA reductase, thus preventing excessive accumulation of potentially deleterious tetrapyrrole intermediates. Thereby, FC2 stabilizes POR by physical interaction. When the interaction between FC2 and POR is perturbed, suppression of ALA synthesis is attenuated and photoreactive protochlorophyllide accumulates. FC2 is anchored in the thylakoid membrane via its membrane-spanning CAB (chlorophyll-a-binding) domain. FC2 is one of the two isoforms of ferrochelatase catalyzing the last step of heme synthesis. Although FC2 belongs to the heme-synthesizing branch of TBS, its interaction with POR potentiates the effects of the GluTR-inactivation complex on the chlorophyll-synthesizing branch and ensures reciprocal control of chlorophyll and heme synthesis.

Auxin inhibits chlorophyll accumulation through ARF7-IAA14-mediated repression of chlorophyll biosynthesis genes in Arabidopsis.

Auxin is a well-known important phytohormone in plant that plays vital roles in almost every development process throughout plant lifecycle. However, the effect of auxin on the metabolism of chlorophyll, one of the most important pigments involved in the photosynthesis, was intertwined and the underlying mechanism remained to be explored. Here, we found the auxin-defective yuc2 yuc6 double mutant displayed dark-green leaf color with higher chlorophyll content than wildtype, suggesting a negative regulatory role of auxin in chlorophyll biosynthesis. The chloroplast number and structure in mesophyll cells were altered and the photosynthetic efficiency was improved in yuc2 yuc6. In addition, the chlorophyll level was significantly improved during seedling de-etiolation in yuc2 yuc6 mutant, and decreased dramatically under IAA treatment, confirming the inhibitory role of auxin in chlorophyll biosynthesis. The analyses of gene expression in mature leaves and de-etiolation seedlings suggested that auxin suppressed the expression of many chlorophyll biosynthesis genes, especially PROTOCHLOROPHYLLIDE OXIDOREDUCTASE A (PORA) and GENOMES UNCOUPLED 5 (GUN5). Yeast-one-hybrid and luciferase assays demonstrated that the AUXIN RESPONSE FACTOR 2 (ARF2) and ARF7 bind to the promoter of PORA and GUN5 to suppress their expression with the help of INDOLE-3-ACETIC ACID14 (IAA14). Collectively, our research explicitly unraveled the direct inhibitory role of auxin in chlorophyll biosynthesis, and provided new insight into the interplay between auxin signaling and chlorophyll metabolism.

Regulation of chlorophyll biosynthesis by light-dependent acetylation of NADPH:protochlorophyll oxidoreductase A in Arabidopsis

Chlorophylls are the major pigments that harvest light energy during photosynthesis in plants. Although reactions in chlorophyll biogenesis have been largely known, little attention has been paid to the post-translational regulation mechanism of this process. In this study, we found that four lysine sites (K128/340/350/390) of NADPH:protochlorophyllide oxidoreductase A (PORA), which catalyzes the only light-triggered step in chlorophyll biosynthesis, were acetylated after dark-grown seedlings transferred to light via acetylomics analysis. Etiolated seedlings with K390 mutation of PORA had a lower greening rate and decreased PORA acetylation after illumination. Importantly, K390 of PORA was found extremely conserved in plants and cyanobacteria via bioinformatics analysis. We further demonstrated that the acetylation level of PORA was increased by exposing the dark-grown seedlings to the histone deacetylase (HDAC) inhibitor TSA. Thus, the HDACs probably regulate the acetylation of PORA, thereby controlling this non-histone substrate to catalyze the reduction of Pchlide to produce chlorophyllide, which provides a novel regulatory mechanism by which the plant actively tunes chlorophyll biosynthesis during the conversion from skotomorphogenesis to photomorphogenesis.

Influence of Light of Different Spectral Compositions on Growth Parameters, Photosynthetic Pigment Contents and Gene Expression in Scots Pine Plantlets.

The photoreceptors of red light (phytochromes) and blue light (cryptochromes) impact plant growth and metabolism. However, their action has been barely studied, especially in coniferous plants. Therefore, the influence of blue (maximum 450 nm), red (maximum 660 nm), white light (maxima 450 nm + 575 nm), far-red light (maximum 730 nm), white fluorescent light and dark on seed germination, growth, chlorophyll and carotenoid contents, as well as the transcript levels of genes involved in reception, photosynthesis, light and hormonal signaling of Scots pine plantlets, was investigated. The highest values of dry weight, root length and photosynthetic pigment contents were characteristic of 9-day-old plantlets grown under red light, whereas in the dark plantlet length, seed vigor, seed germination, dry weight and pigment contents were decreased. Under blue and white lights, the main studied morphological parameters were decreased or close to red light. The cotyledons were undeveloped under dark conditions, likely due to the reduced content of photosynthetic pigments, which agrees with the low transcript levels of genes encoding protochlorophyllide oxidoreductase (PORA) and phytoene synthase (PSY). The transcript levels of a number of genes involved in phytohormone biosynthesis and signaling, such as GA3ox, RRa, KAO and JazA, were enhanced under red light, unlike under dark conditions. We suggest that the observed phenomena of red light are the most important for the germination of the plantlets and may be based on earlier and enhanced expression of auxin, cytokinin, gibberellin and jasmonate signaling genes activated by corresponding photoreceptors. The obtained results may help to improve reforestation technology; however, this problem needs further study.

Gossypium hirsutum gene of unknown function Gohir.A03G0737001 encodes a potential Chaperone-like Protein of protochlorophyllide oxidoreductase (CPP1).

A gene of unknown function identified in Gossypium hirsutum , Gohir.A03G0737001.1, was studied using sequence and bioinformatic tools. The encoded protein (referred to here as GhCPP1-A0A1U8HKT6) was predicted to function as a Chaperone-like protein of protochlorophyllide oxidoreductase (CPP1), which is involved with initiation of photochemical reactions of chlorophyll biosynthesis. Sequence analysis indicates it is embedded in the chloroplast envelope membrane through four transmembrane regions and contains a J-like domain that is structurally similar to the J domain of DnaJ/Hsp40 "holdase" chaperone proteins.

Establishment of a Virus-Induced Gene-Silencing (VIGS) System in Tea Plant and Its Use in the Functional Analysis of CsTCS1.

Tea (Camellia sinensis [L.] O. Kuntze) is an important global economic crop and is considered to enhance health. However, the functions of many genes in tea plants are unknown. Virus-induced gene silencing (VIGS) mediated by tobacco rattle virus (TRV) is an effective tool for the analysis of gene functions, although this method has rarely been reported in tea plants. In this study, we established an effective VIGS-mediated gene knockout technology to understand the functional identification of large-scale genomic sequences in tea plants. The results showed that the VIGS system was verified by detecting the virus and using a real-time quantitative reverse transcription PCR (qRT-PCR) analysis. The reporter gene CsPOR1 (protochlorophyllide oxidoreductase) was silenced using the vacuum infiltration method, and typical photobleaching and albino symptoms were observed in newly sprouted leaves at the whole plant level of tea after infection for 12 d and 25 d. After optimization, the VIGS system was successfully used to silence the tea plant CsTCS1 (caffeine synthase) gene. The results showed that the relative caffeine content was reduced 6.26-fold compared with the control, and the level of expression of CsPOR1 decreased by approximately 3.12-fold in plants in which CsPOR1 was silenced. These results demonstrate that VIGS can be quickly and efficiently used to analyze the function of genes in tea plants. The successful establishment of VIGS could eliminate the need for tissue culture by providing an effective method to study gene function in tea plants and accelerate the process of functional genome research in tea.