Cytosolic and plastid forms of 5-enolpyruvylshikimate-3-phosphate synthase in Euglena gracilis are differentially expressed during light-induced chloroplast development

The enzyme 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase (EC 2.5.1.19), the target of the herbicide glyphosate [N-(phosphonomethyl)glycine], exists in two molecular forms in Euglena gracilis. One form has previously been characterized as a monofunctional 59 kDa protein. The other form constitutes a single domain of the multifunctional 165 kDa arom protein. The two enzyme forms are inversely regulated at the protein and mRNA levels during light-induced chloroplast development, as demonstrated by the determination of their enzyme activities after non-denaturing polyacrylamide gel electrophoresis and Northern hybridization analysis with a Saccharomyces cerevisiae ARO1 gene probe. The arom protein and its mRNA predominate in dark-grown cells, and the levels of both decline upon illumination. In contrast, the monofunctional EPSP synthase and its mRNA are induced by light, the increase in mRNA abundance preceding accumulation of the protein. The two enzymes are localized in different subcellular compartments, as demonstrated by comparing total protein patterns with those of isolated organelles. Glyphosate-adapted wild-type cells and glyphosate-tolerant cells of a plastid-free mutant of E. gracilis, W₁₀BSmL, were used for organelle isolation and protein extraction, as these cell lines overproduce EPSP synthase and the arom protein, respectively. Evidence was obtained for the cytosolic localization of the arom protein and the plastid compartmentalization of the monofunctional EPSP synthase. These conclusions are further supported by the observation that EPSP synthase precursor, produced by in vitro translation of the hybrid-selected mRNA, was efficiently taken up and processed to mature size by isolated chloroplasts from photoautotrophic wild-type E. gracilis cells, while the in vitro-synthesized arom protein was not sequestered by isolated Euglena plastids.Dedicated to Prof. Dr. A. Trebst on the occasion of his 65th birthday     

Current views on chloroplast protein import and hypotheses on the origin of the transport mechanism

Most chloroplastic proteins are synthesized as precursors in the cytosol prior to their transport into chloroplasts. These precursors are generally synthesized in a form that is larger than the mature form found inside chloroplasts. The extra amino acids, called transit peptides, are present at the amino terminus. The transit peptide is necessary and sufficient to recognize the chloroplast and induce movement of the attached protein across the envelope membranes. In this review, we discuss the primary and secondary structure of transit peptides, describe what is known about the import process, and present some hypotheses on the evolutionary origin of the import mechanism.Abbreviations DHFR dihydrofolate reductase - EPSP synthase 5-enolpyrovylshikimate-3-phosphate synthase; hsp heat-shock protein - LHCP II light-harvesting chlorophylla/b binding protein - OEE 16, 23, and 33 the 16-, 23-, and 33-kDa proteins of the oxygen-evolving complex - pr precursor - rubisco ribulose-1,5-bisphosphate carboxylase/oxygenase - SS rubisco small subunit     

SULTR3;1 is a chloroplast‐localized sulfate transporter in Arabidopsis thaliana

Plants play a prominent role as sulfur reducers in the global sulfur cycle. Sulfate, the major form of inorganic sulfur utilized by plants, is absorbed and transported by specific sulfate transporters into plastids, especially chloroplasts, where it is reduced and assimilated into cysteine before entering other metabolic processes. How sulfate is transported into the chloroplast, however, remains unresolved; no plastid‐localized sulfate transporters have been previously identified in higher plants. Here we report that SULTR3;1 is localized in the chloroplast, which was demonstrated by SULTR3;1‐GFP localization, Western blot analysis, protein import as well as comparative analysis of sulfate uptake by chloroplasts between knockout mutants, complemented transgenic plants, and the wild type. Loss of SULTR3;1 significantly decreases the sulfate uptake of the chloroplast. Complementation of the sultr3;1 mutant phenotypes by expression of a 35S‐SULTR3;1 construct further confirms that SULTR3;1 is one of the transporters responsible for sulfate transport into chloroplasts.     

Import of a precursor protein into chloroplasts is inhibited by the herbicide glyphosate

Import of the precursor to 5-enolpyruvylshikimate-3-phosphate synthase (pEPSPS) into chloroplasts is inhibited by the herbicide glyphosate. Inhibition of import is maximal at glyphosate concentrations of ≥10 μm and occurs only when pEPSPS is present as a ternary complex of enzyme–shikimate-3-phosphate–glyphosate. Glyphosate alone had no effect on the import of pEPSPS since it is not known to interact with the enzyme in the absence of shikimate-3-phosphate. Experiments with wild-type and glyphosate-resistant mutant forms of pEPSPS show that inhibition of import is directly proportional to the binding constants for glyphosate. Inhibition of import is thus a direct consequence of glyphosate binding to the enzyme–shikimate-3-phosphate complex. The potential for non-specific effects of glyphosate on the chloroplast transport mechanism has been discounted by showing that import of another chloroplast-designated protein was unaffected by high concentrations of glyphosate and shikimate-3-phosphate. The mechanism of import inhibition by glyphosate is consistent with a precursor unfolding/refolding model.     

Cytosolic and plastid forms of 5-enolpyruvylshikimate-3-phosphate synthase in Euglena gracilis are differentially expressed during light-induced chloroplast development

The enzyme 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase (EC 2.5.1.19), the target of the herbicide glyphosate [N-(phosphonomethyl)glycine], exists in two molecular forms in Euglena gracilis. One form has previously been characterized as a monofunctional 59 kDa protein. The other form constitutes a single domain of the multifunctional 165 kDa arom protein. The two enzyme forms are inversely regulated at the protein and mRNA levels during light-induced chloroplast development, as demonstrated by the determination of their enzyme activities after non-denaturing polyacrylamide gel electrophoresis and Northern hybridization analysis with a Saccharomyces cerevisiae ARO1 gene probe. The arom protein and its mRNA predominate in dark-grown cells, and the levels of both decline upon illumination. In contrast, the monofunctional EPSP synthase and its mRNA are induced by light, the increase in mRNA abundance preceding accumulation of the protein. The two enzymes are localized in different subcellular compartments, as demonstrated by comparing total protein patterns with those of isolated organelles. Glyphosate-adapted wild-type cells and glyphosate-tolerant cells of a plastid-free mutant of E. gracilis, W₁₀BSmL, were used for organelle isolation and protein extraction, as these cell lines overproduce EPSP synthase and the arom protein, respectively. Evidence was obtained for the cytosolic localization of the arom protein and the plastid compartmentalization of the monofunctional EPSP synthase. These conclusions are further supported by the observation that EPSP synthase precursor, produced by in vitro translation of the hybrid-selected mRNA, was efficiently taken up and processed to mature size by isolated chloroplasts from photoautotrophic wild-type E. gracilis cells, while the in vitro-synthesized arom protein was not sequestered by isolated Euglena plastids.     

Nucleotide binding and dimerization at the chloroplast pre‐protein import receptor,atToc33, are not essential in vivo but do increase import efficiency

The atToc33 protein is one of several pre‐protein import receptors in the outer envelope of Arabidopsis chloroplasts. It is a GTPase with motifs characteristic of such proteins, and its loss in the plastid protein import 1 (ppi1) mutant interferes with the import of photosynthesis‐related pre‐proteins, causing a chlorotic phenotype in mutant plants. To assess the significance of GTPase cycling by atToc33, we generated several atToc33 point mutants with predicted effects on GTP binding (K49R, S50N and S50N/S51N), GTP hydrolysis (G45R, G45V, Q68A and N101A), both binding and hydrolysis (G45R/K49N/S50R), and dimerization or the functional interaction between dimeric partners (R125A, R130A and R130K). First, a selection of these mutants was assessed in vitro, or in yeast, to confirm that the mutations have the desired effects: in relation to nucleotide binding and dimerization, the mutants behaved as expected. Then, activities of selected mutants were tested in vivo, by assessing for complementation of ppi1 in transgenic plants. Remarkably, all tested mutants mediated high levels of complementation: complemented plants were similar to the wild type in growth rate, chlorophyll accumulation, photosynthetic performance, and chloroplast ultrastructure. Protein import into mutant chloroplasts was also complemented to >50% of the wild‐type level. Overall, the data indicate that neither nucleotide binding nor dimerization at atToc33 is essential for chloroplast import (in plants that continue to express the other TOC receptors in native form), although both processes do increase import efficiency. Absence of atToc33 GTPase activity might somehow be compensated for by that of the Toc159 receptors. However, overexpression of atToc33 (or its close relative, atToc34) in Toc159‐deficient plants did not mediate complementation, indicating that the receptors do not share functional redundancy in the conventional sense.     

Ultrastructural localisation by protein A-gold immunocytochemistry of 5-enolpyruvylshikimic acid 3-phosphate synthase in a plant cell culture which overproduces the enzyme

Recently we have shown that cultured cells of the higher plant Corydalis sempervirens Pers., adapted to growth in the presence of high concentrations of the herbicide glyphosate, a potent specific inhibitor of the shikimate pathway enzyme 5-enolpyruvylshikimic acid 3-phosphate (EPSP) synthase (EC 2.5.1.19, 3-phosphoshikimate 1-carboxyvinyltransferase) oversynthesize the EPSP synthase protein (Smart et al., 1985, J. Biol. Chem. 260, 16338–16346). We now report that the EPSP synthase protein can be detected in cells of the adapted as well as of the non-adapted strain by the use of protein A-colloidal gold immunocytochemistry. The overproduced EPSP synthase in the glyphosate-adapted cells is located exclusively in the plastid and we find no evidence for the existence of extra-plastidic EPSP synthase in either strain.Abbreviations EPSP 5-enolpyruvylshikimic acid 3-phosphate     

Subcellular localization of the common shikimate-pathway enzymes in Pisum sativum L.

5-Enolpyruvylshikimate 3-phosphate (EPSP) synthase (3-phosphoshikimate 1-carboxyvinyltransferase; EC 2.5.1.19), 3-dehydroquinate dehydratase (EC 4.2.1.10) and shikimate: NADP⁺ oxidoreductase (EC 1.1.1.25) were present in intact chloroplasts and root plastids isolated from pea seedling extracts by sucrose and modified-silica density gradient centrifugation. In young (approx. 10-d-old) seedling shoots the enzymes were predominantly chloroplastic; high-performance anion-exchange chromatography resolved minor isoenzymic activities not observed in density-gradientpurified chloroplasts. The initial enzyme of the shikimate pathway, 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase (EC 4.1.2.15) was also associated with intact density-gradient-purified chloroplasts. 3-Dehydroquinate synthase (EC 4.6.1.3) and shikimate kinase (EC 2.7.1.71) were detected together with the other pathway enzymes in stromal preparations from washed chloroplasts. Plastidic EPSP synthase was inhibited by micromolar concentrations of the herbicide glyphosate.Abbreviations DAHP 3-deoxy-d-arabino-heptulosonate 7-phosphate - DEAE diethylaminoethyl - DHQase 3-dehydroquinate dehydratase - DTT dithiothreitol - EPSP 5-enolpyruvylshikimate 3-phosphate - SORase shikimate:NADP⁺ oxidoreductase     

Chloroplast transport of a ribulose bisphosphate carboxylase small subunit-5-enolpyruvyl 3-phosphoshikimate synthase chimeric protein requires part of the mature small subunit in addition to the transit peptide

Ribulose bisphosphate carboxylase small subunit protein is synthesized in the cytoplasm as a precursor and transported into the chloroplast where the amino-terminal portion, the transit peptide, is removed proteolytically. To obtain chloroplast delivery of the 43-kDa 5-enolpyruvyl 3-phosphoshikimate (EPSP) synthase of Salmonella typhimurium, we constructed fusion proteins between the bacterial EPSP synthase and the ribulose bisphosphate carboxylase small subunit. A fusion protein consisting of the transit peptide fused to the EPSP synthase was not transported in vitro or in vivo into chloroplasts. A second fusion protein consisting of the transit peptide and 24 amino acids of the mature small subunit fused to the EPSP synthase was transported both in vitro and in vivo into chloroplasts. It was processed into two polypeptides of 46 and 47 kDa, respectively. This heterogeneity in processing was not caused by the presence of the aroA start codon, since its removal resulted in the same pattern. Substituting 24 different amino acids for the 24 amino acids of the mature small subunit resulted in a fusion protein that was not transported into the chloroplast. It was concluded that a portion of the mature small subunit was needed for efficient chloroplast delivery.     

The potato granule bound starch synthase chloroplast transit peptide directs recombinant proteins to plastids

The chloroplast targeting transit sequence from potato granule bound starch synthase (gbss) was used to direct the accumulation of recombinant proteins to the plastid stroma. The potato gbss transit sequence was fused to the N-terminus of the green fluorescent protein (GFP) and the Catharanthus roseus strictosidine synthase (Str1) enzyme. Fluorescence microscopy confirmed that the recombinant gbss-GFP fusion protein was exclusively targeted to the plastid stroma in tobacco suspension cells, demonstrating that the transit sequence was functional in vivo. The Str1 fusion protein accumulated to high levels in plastids isolated from transgenic plants. We conclude that the potato gbss transit sequence is functional and directs import of recombinant proteins into the chloroplast stroma.     

Identification of the first glyphosate transporter by genomic adaptation

The soil bacterium Bacillus subtilis can get into contact with growth-inhibiting substances, which may be of anthropogenic origin. Glyphosate is such a substance serving as a nonselective herbicide. Glyphosate specifically inhibits the 5-enolpyruvyl-shikimate-3-phosphate (EPSP) synthase, which generates an essential precursor for de novo synthesis of aromatic amino acids in plants, fungi, bacteria and archaea. Inhibition of the EPSP synthase by glyphosate results in depletion of the cellular levels of aromatic amino acids unless the environment provides them. Here, we have assessed the potential of B. subtilis to adapt to glyphosate at the genome level. In contrast to Escherichia coli, which evolves glyphosate resistance by elevating the production and decreasing the glyphosate sensitivity of the EPSP synthase, B. subtilis primarily inactivates the gltT gene encoding the high-affinity glutamate/aspartate symporter GltT. Further adaptation of the gltT mutants to glyphosate led to the inactivation of the gltP gene encoding the glutamate transporter GltP. Metabolome analyses confirmed that GltT is the major entryway of glyphosate into B. subtilis. GltP, the GltT homologue of E. coli also transports glyphosate into B. subtilis. Finally, we found that GltT is involved in uptake of the herbicide glufosinate, which inhibits the glutamine synthetase.     

The rice RING E3 ligase,OsCTR1, inhibits trafficking to the chloroplasts of OsCP12 and OsRP1, and its overexpression confers drought tolerance in Arabidopsis

Plant growth under low water availability adversely affects many key processes with morphological, physiological, biochemical and molecular consequences. Here, we found that a rice gene, OsCTR1, encoding the RING Ub E3 ligase plays an important role in drought tolerance. OsCTR1 was highly expressed in response to dehydration treatment and defense‐related phytohormones, and its encoded protein was localized in both the chloroplasts and the cytosol. Intriguingly, the OsCTR1 protein was found predominantly targeted to the cytosol when rice protoplasts transfected with OsCTR1 were treated with abscisic acid (ABA). Several interacting partners were identified, which were mainly targeted to the chloroplasts, and interactions with OsCTR1 were confirmed by using biomolecular fluorescence complementation (BiFC). Interestingly, two chloroplast‐localized proteins (OsCP12 and OsRP1) interacted with OsCTR1 in the cytosol, and ubiquitination by OsCTR1 led to protein degradation via the Ub 26S proteasome. Heterogeneous overexpression of OsCTR1 in Arabidopsis exhibited hypersensitive phenotypes with respect to ABA‐responsive seed germination, seedling growth and stomatal closure. The ABA‐sensitive transgenic plants also showed improvement in their tolerance against severe water deficits. Taken together, our findings lend support to the hypothesis that the molecular functions of OsCTR1 are related to tolerance to water‐deficit stress via ABA‐dependent regulation and related systems.     

Chloroplast protein import inhibition by a soluble factor from wheat germ lysate

Protein import into chloroplasts occurs post-translationally in vitro. The precursor proteins are generally synthesised in a reticulocyte lysate- or wheat germ lysate-derived system and imported out of this system into chloroplast. These complex soluble protein mixtures are likely to contain factors, which influence somehow the import competence and import efficiency. Here we describe a heat-stable soluble proteinaceaous factor, which inhibits protein import into chloroplasts in vitro. The inhibitor interacts directly with the precursor protein and renders it import incompetent. This mode of action is supported by two observations: firstly, binding of the precursor to the chloroplast surface is diminished in the presence of the inhibitor. Secondly, when chloroplasts were loaded with precursor proteins under conditions, which allow only binding but not import the inhibitor was unable to abolish the subsequent translocation step.     

A plastid envelope location of Arabidopsis ent-kaurene oxidase links the plastid and endoplasmic reticulum steps of the gibberellin biosynthesis pathway

We have used fusions of gibberellin biosynthesis enzymes to green fluorescent protein (GFP) to determine the subcellular localization of the early steps of the pathway. Gibberellin biosynthesis from geranylgeranyl diphosphate is catalysed by enzymes of the terpene cyclase, cytochrome P450 mono-oxygenase and 2-oxoglutarate-dependent dioxygenase classes. We show that the N-terminal pre-sequences of the Arabidopsis thaliana terpene cyclases copalyl diphosphate synthase (AtCPS1) and ent-kaurene synthase (AtKS1) direct GFP to chloroplasts in transient assays following microprojectile bombardment of tobacco leaves. The AtKS1-GFP fusion is also imported by isolated pea chloroplasts. The N-terminal portion of the cytochrome P450 protein ent-kaurene oxidase (AtKO1) directs GFP to chloroplasts in tobacco leaf transient assays. Chloroplast import assays with 35S-labelled AtKO1 protein show that it is targeted to the outer face of the chloroplast envelope. The leader sequences of the two ent-kaurenoic acid oxidases (AtKAO1 and AtKAO2) from Arabidopsis direct GFP to the endoplasmic reticulum. These data suggest that the AtKO1 protein links the plastid- and endoplasmic reticulum-located steps of the gibberellin biosynthesis pathway by association with the outer envelope of the plastid.     

Cloning of an Arabidopsis thaliana gene encoding 5-enolpyruvylshikimate-3-phosphate synthase: sequence analysis and manipulation to obtain glyphosate-tolerant plants

Summary 5-enolpyruvylshikimate-3-phosphate synthase (EPSPs), the target of the herbicide glyphosate, catalyzes an essential step in the shikimate pathway common to aromatic amino acid biosynthesis. We have cloned an EPSP synthase gene from Arabidopsis thaliana by hybridization with a petunia cDNA probe. The Arabidopsis gene is highly homologous to the petunia gene within the mature enzyme but is only 23% homologous in the chloroplast transit peptide portion. The Arabidopsis gene contains seven introns in exactly the same positions as those in the petunia gene. The introns are, however, significantly smaller in the Arabidopsis gene. This reduction accounts for the significantly smaller size of the gene as compared to the petunia gene. We have fused the gene to the cauliflower mosaic virus 35 S promoter and reintroduced the chimeric gene into Arabidopsis. The resultant overproduction of EPSPs leads to glyphosate tolerance in transformed callus and plants.     

Purification and Properties of 5-Enolpyruvylshikimate-3-Phosphate Synthase from Dark-Grown Seedlings of Sorghum bicolor

5-Enolpyruvylshikimate-3-phosphate (EPSP) synthase (3-phospho-shikimate 1-carboxyvinyltransferase; EC 2.5.1.19) was purified 1300-fold from etiolated shoots of Sorghum bicolor (L.) Moench. Native polyacrylamide gel electrophoresis revealed three barely separated protein bands staining positive for EPSP synthase activity. The native molecular weight was determined to be 51,000. Enzyme activity was found to be sensitive to metal ions and salts. Apparent K_m values of 7 and 8 micromolar were determined for the substrates shikimate-3-phosphate and phosphoenolpyruvate (PEP), respectively. The herbicide glyphosate was found to inhibit the enzyme competitively with respect to PEP (K_i = 0.16 micromolar). Characterization studies support the conclusion of a high degree of similarity between EPSP synthase from S. bicolor, a monocot, and the enzyme from dicots. A similarity to bacterial EPSP synthase is also discussed. Three EPSP synthase isozymes (I, II, III) were elucidated in crude homogenates of S. bicolor shoots by high performance liquid chromatography. The major isozymes, II and III, were separated and partially characterized. No significant differences in pH activity profiles and glyphosate sensitivity were found. This report of isozymes of EPSP synthase from S. bicolor is consistent with other reports for shikimate pathway enzymes, including EPSP synthase.     

Mode of action of glyphosate in Candida maltosa

The broad-spectrum herbicide glyphosate inhibits the growth of Candida maltosa and causes the accumulation of shikimic acid and shikimate-3-phosphate. Glyphosate is a potent inhibitor of three enzymes of aromatic amino acid biosynthesis in this yeast. In relation to tyrosine-sensitive 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase and dehydroquinate synthase, the inhibitory effect appears at concentrations in the mM range, but 5-enolpyruvylshikimate 3-phosphate (EPSP) synthase is inhibited by micromolar concentrations of glyphosate. Inhibition of partially purified EPSP synthase reaction by glyphosate is competitive with respect to phosphoenolpyruvate (PEP) with a K _i -value of 12 M. The app. K _m for PEP is about 5-fold higher and was 62 M. Furthermore, the presence of glyphosate leads to derepression of many amino acid biosynthetic enzymes.Abbreviations DAHP 3-deoxy-D-arabino-heptulosonate 7-phosphate - EPSP synthase 5-enolpyruvylshikimate 3-phosphate synthase - PEP phosphoenolpyruvate - S-3-P shikimate-3-phosphate     

Transport of proteins into chloroplasts

The import of cytoplasmically synthesized proteins into chloroplasts involves an interaction between at least two components; the precursor protein, and the import apparatus in the chloroplast envelope membrane. This review summarizes the information available about each of these components. Precursor proteins consist of an amino terminal transit peptide attached to a passenger protein. Transit peptides from various precurosrs are diverse with respect to length and amino acid sequence; analysis of their sequences has not revealed insight into their mode of action. A variety of foreign passenger proteins can be imported into chloroplasts when a transit peptide is present at the amino terminus. However, foreign passenger proteins are not imported as efficiently as natural passenger proteins, and some chimeric precursor proteins are not imported into chloroplasts at all. Therefore, the passenger protein, as well as the transit peptide, influences the import process. Import begins by binding of the precursor to the chloroplast surface. It has been suggested that this binding is mediated by a receptor, but evidence to support this hypothesis remains incomplete and a receptor protein has not yet been characterized. Protein translocation requires energy derived from ATP hydrolysis, although there are conflicting reports as to where hydrolysis occurs and it is unclear how this energy is utilized. The mechanism(s) whereby proteins are translocated across either the two envelope membranes or the thylakoid membrane is not known.Abbreviations EPSP 5-enolpyruvyulshikimate-3-phosphate - LHCP Chlorophyll a/b binding protein of the light-harvesting complex - NPT-II Neomycin phosphotransferase II - PC Plastocyanin - Pr Precursor - Rubisco Ribulose-1,5,-bisphosphate carboxylase/oxygenase - SS Small subunit of Rubisco     

Helper component‐proteinase enhances the activity of 1‐deoxy‐D‐xylulose‐5‐phosphate synthase and promotes the biosynthesis of plastidic isoprenoids in Potato virus Y‐infected tobacco

Virus‐infected plants show strong morphological and physiological alterations. Many physiological processes in chloroplast are affected, including the plastidic isoprenoid biosynthetic pathway [the 2C‐methyl‐D‐erythritol‐4‐phosphate (MEP) pathway]; indeed, isoprenoid contents have been demonstrated to be altered in virus‐infected plants. In this study, we found that the levels of photosynthetic pigments and abscisic acid (ABA) were altered in Potato virus Y (PVY)‐infected tobacco. Using yeast two‐hybrid assays, we demonstrated an interaction between virus protein PVY helper component‐proteinase (HC‐Pro) and tobacco chloroplast protein 1‐deoxy‐D‐xylulose‐5‐phosphate synthase (NtDXS). This interaction was confirmed using bimolecular fluorescence complementation (BiFC) assays and pull‐down assays. The Transket_pyr domain (residues 394–561) of NtDXS was required for interaction with HC‐Pro, while the N‐terminal region of HC‐Pro (residues 1–97) was necessary for interaction with NtDXS. Using in vitro enzyme activity assays, PVY HC‐Pro was found to promote the synthase activity of NtDXS. We observed increases in photosynthetic pigment contents and ABA levels in transgenic plants with HC‐Pro accumulating in the chloroplasts. During virus infection, the enhancement of plastidic isoprenoid biosynthesis was attributed to the enhancement of DXS activity by HC‐Pro. Our study reveals a new role of HC‐Pro in the host plant metabolic system and will contribute to the study of host–virus relationships.