Overexpression of REDUCED WALL ACETYLATION C increases xylan acetylation and biomass recalcitrance in Populus.

Plant lignocellulosic biomass, i.e. secondary cell walls of plants, is a vital alternative source for bioenergy. However, the acetylation of xylan in secondary cell walls impedes the conversion of biomass to biofuels. Previous studies have shown that REDUCED WALL ACETYLATION (RWA) proteins are directly involved in the acetylation of xylan but the regulatory mechanism of RWAs is not fully understood. In this study, we demonstrate that overexpression of a Populus trichocarpa PtRWA-C gene increases the level of xylan acetylation and increases the lignin content and S/G ratio, ultimately yielding poplar woody biomass with reduced saccharification efficiency. Furthermore, through gene coexpression network and expression quantitative trait loci (eQTL) analysis, we found that PtRWA-C was regulated not only by the secondary cell wall hierarchical regulatory network but also by an AP2 family transcription factor HARDY (HRD). Specifically, HRD activates PtRWA-C expression by directly binding to the PtRWA-C promoter, which is also the cis-eQTL for PtRWA-C. Taken together, our findings provide insights into the functional roles of PtRWA-C in xylan acetylation and consequently saccharification and shed light on synthetic biology approaches to manipulate this gene and alter cell wall properties. These findings have substantial implications for genetic engineering of woody species, which could be used as a sustainable source of biofuels, valuable biochemicals, and biomaterials.

Chemo-Enzymatic Synthesis of Long-Chain Oligosaccharides for Studying Xylan-Modifying Enzymes.

Plant research is hampered in several aspects by a lack of pure oligosaccharide samples that closely represent structural features of cell wall glycans. An alternative to purely chemical synthesis to access these oligosaccharides is chemo-enzymatic synthesis using glycosynthases. These enzymes enable the ligation of oligosaccharide donors, when activated for example as α-glycosyl fluorides, with suitable acceptor oligosaccharides. Herein, the synthesis of xylan oligosaccharides up to dodecasaccharides is reported, with glycosynthase-mediated coupling reactions as key steps. The xylo-oligosaccharide donors were protected at the non-reducing end with a 4-O-tetrahydropyranyl (THP) group to prevent polymerization. Installation of an unnatural 3-O-methylether substituent at the reducing end xylose of the oligosaccharides ensured good water solubility. Biochemical assays demonstrated enzymatic activity for the xylan acetyltransferase XOAT1 from Arabidopsis thaliana, xylan arabinofuranosyl-transferase XAT3 enzymes from rice and switchgrass, and the xylan glucuronosyltransferase GUX3 from Arabidopsis thaliana. In case of the glucuronosyltransferase GUX3, MALDI-MS/MS analysis of the reaction product suggested that a single glucuronosyl substituent was installed primarily at the central xylose residues of the dodecasaccharide acceptor, demonstrating the value of long-chain acceptors for assaying biosynthetic glycosyltransferases.

Network-driven intracellular cAMP coordinates circadian rhythm in the suprachiasmatic nucleus.

The mammalian central circadian clock, located in the suprachiasmatic nucleus (SCN), coordinates the timing of physiology and behavior to local time cues. In the SCN, second messengers, such as cAMP and Ca2+, are suggested to be involved in the input and/or output of the molecular circadian clock. However, the functional roles of second messengers and their dynamics in the SCN remain largely unclear. In the present study, we visualized the spatiotemporal patterns of circadian rhythms of second messengers and neurotransmitter release in the SCN. Here, we show that neuronal activity regulates the rhythmic release of vasoactive intestinal peptides from the SCN, which drives the circadian rhythms of intracellular cAMP in the SCN. Furthermore, optical manipulation of intracellular cAMP levels in the SCN shifts molecular and behavioral circadian rhythms. Together, our study demonstrates that intracellular cAMP is a key molecule in the organization of the SCN circadian neuronal network.

Real time monitoring of cold Ca2+ dependent transcription and its modulation by NCX inhibitors.

Real-time monitoring of cellular temperature responses is an important technique in thermal biology and drug development. Recent study identified that Na+/Ca2+ exchanger (NCX)-dependent Ca2+ influx transduces cold signals to circadian clock in mammalian cultured cells. The finding raised an idea that cellular responses to the cold signals can be analyzed by monitoring of clock gene expression. We found that Per1 and Per2 were up-regulated after culture at 27 °C compared to 37 °C in Rat-1 fibroblasts. In order to monitor cold-Ca2+-dependent transcription in living cells, we developed a luciferase-based real-time reporting system by using Per1 promoter, Per2 promoter, Ca2+/cAMP-response elements (CRE) or NFAT-binding elements. We found that benzyloxyphenyl NCX inhibitor KB-R7943 and SN-6, but not SEA-0400 or YM-244769 inhibited the cold induction of Per2. Our study established a real-time monitoring system for cold Ca2+ signaling which can be applied to evaluation of drugs.

Rational enzyme design for controlled functionalization of acetylated xylan for cell-free polymer biosynthesis.

Xylan O-acetyltransferase 1 (XOAT1) is involved in O-acetylating the backbone of hemicellulose xylan. Recent structural analysis of XOAT1 showed two unequal lobes forming a cleft that is predicted to accommodate and position xylan acceptors into proximity with the catalytic triad. Here, we used docking and molecular dynamics simulations to investigate the optimal orientation of xylan in the binding cleft of XOAT1 and identify putative key residues (Gln445 and Arg444 on Minor lobe & Asn312, Met311 and Asp403 on Major lobe) involved in substrate interactions. Site-directed mutagenesis coupled with biochemical analyses revealed the major lobe of XOAT1 is important for xylan binding. Mutation of single key residues yielded XOAT1 variants with various enzymatic efficiencies that are applicable to one-pot synthesis of xylan polymers with different degrees of O-acetylation. Taken together, our results demonstrate the effectiveness of computational modeling in guiding enzyme engineering aimed at modulating xylan and redesigning plant cell walls.

Na+/Ca2+ exchanger mediates cold Ca2+ signaling conserved for temperature-compensated circadian rhythms.

Circadian rhythms are based on biochemical oscillations generated by clock genes/proteins, which independently evolved in animals, fungi, plants, and cyanobacteria. Temperature compensation of the oscillation speed is a common feature of the circadian clocks, but the evolutionary-conserved mechanism has been unclear. Here, we show that Na+/Ca2+ exchanger (NCX) mediates cold-responsive Ca2+ signaling important for the temperature-compensated oscillation in mammalian cells. In response to temperature decrease, NCX elevates intracellular Ca2+, which activates Ca2+/calmodulin-dependent protein kinase II and accelerates transcriptional oscillations of clock genes. The cold-responsive Ca2+ signaling is conserved among mice, Drosophila, and Arabidopsis The mammalian cellular rhythms and Drosophila behavioral rhythms were severely attenuated by NCX inhibition, indicating essential roles of NCX in both temperature compensation and autonomous oscillation. NCX also contributes to the temperature-compensated transcriptional rhythms in cyanobacterial clock. Our results suggest that NCX-mediated Ca2+ signaling is a common mechanism underlying temperature-compensated circadian rhythms both in eukaryotes and prokaryotes.

AtFUT4 and AtFUT6 Are Arabinofuranose-Specific Fucosyltransferases.

The bulk of plant biomass is comprised of plant cell walls, which are complex polymeric networks, composed of diverse polysaccharides, proteins, polyphenolics, and hydroxyproline-rich glycoproteins (HRGPs). Glycosyltransferases (GTs) work together to synthesize the saccharide components of the plant cell wall. The Arabidopsis thaliana fucosyltransferases (FUTs), AtFUT4, and AtFUT6, are members of the plant-specific GT family 37 (GT37). AtFUT4 and AtFUT6 transfer fucose (Fuc) onto arabinose (Ara) residues of arabinogalactan (AG) proteins (AGPs) and have been postulated to be non-redundant AGP-specific FUTs. AtFUT4 and AtFUT6 were recombinantly expressed in mammalian HEK293 cells and purified for biochemical analysis. We report an updated understanding on the specificities of AtFUT4 and AtFUT6 that are involved in the synthesis of wall localized AGPs. Our findings suggest that they are selective enzymes that can utilize various arabinogalactan (AG)-like and non-AG-like oligosaccharide acceptors, and only require a free, terminal arabinofuranose. We also report with GUS promoter-reporter gene studies that AtFUT4 and AtFUT6 gene expression is sub-localized in different parts of developing A. thaliana roots.

Heterologous expression of plant glycosyltransferases for biochemistry and structural biology.

Much of the carbon captured by photosynthesis is converted into the polysaccharides that constitute plant cell walls. These complex macrostructures are composed of cellulose, hemicellulose, and pectins, together with small amounts of structural proteins, minerals, and in many cases lignin. Wall components assemble and interact with one another to produce dynamic structures with many capabilities, including providing mechanical support to plant structures and determining plant cell shape and size. Despite their abundance, major gaps in our knowledge of the synthesis of the building blocks of these polymers remain, largely due to ineffective methods for expression and purification of active synthetic enzymes for in vitro biochemical analyses. The hemicellulosic polysaccharide, xyloglucan, comprises up to 25% of the dry weight of primary cell walls in plants. Most of the knowledge about the glycosyltransferases (GTs) involved in the xyloglucan biosynthetic pathway has been derived from the identification and carbohydrate analysis of knockout mutants, lending little information on how the catalytic biosynthesis of xyloglucan occurs in planta. In this chapter we describe methods for the heterologous expression of plant GTs using the HEK293 expression platform. As a demonstration of the utility of this platform, nine xyloglucan-relevant GTs from three different CAZy families were evaluated, and methods for expression, purification, and construct optimization are described for biochemical and structural characterization.

Molecular Mechanism of Polysaccharide Acetylation by the Arabidopsis Xylan O-acetyltransferase XOAT1.

Xylans are a major component of plant cell walls. O-Acetyl moieties are the dominant backbone substituents of glucuronoxylan in dicots and play a major role in the polymer-polymer interactions that are crucial for wall architecture and normal plant development. Here, we describe the biochemical, structural, and mechanistic characterization of Arabidopsis (Arabidopsis thaliana) xylan O-acetyltransferase 1 (XOAT1), a member of the plant-specific Trichome Birefringence Like (TBL) family. Detailed characterization of XOAT1-catalyzed reactions by real-time NMR confirms that it exclusively catalyzes the 2-O-acetylation of xylan, followed by nonenzymatic acetyl migration to the O-3 position, resulting in products that are monoacetylated at both O-2 and O-3 positions. In addition, we report the crystal structure of the catalytic domain of XOAT1, which adopts a unique conformation that bears some similarities to the α/ß/α topology of members of the GDSL-like lipase/acylhydrolase family. Finally, we use a combination of biochemical analyses, mutagenesis, and molecular simulations to show that XOAT1 catalyzes xylan acetylation through formation of an acyl-enzyme intermediate, Ac-Ser-216, by a double displacement bi-bi mechanism involving a Ser-His-Asp catalytic triad and unconventionally uses an Arg residue in the formation of an oxyanion hole.

Transplanting fecal material from wild-type mice fed black raspberries alters the immune system of recipient mice.

By constantly stimulating intestinal immunity, gut microbes play important regulatory roles, and their possible involvement in human physical and mental disorders beyond intestinal diseases suggests the importance of maintaining homeostasis in the gut microbiota. Both transplantation of fecal microbiota and dietary interventions have been shown to restore microbial homeostasis in recipients. In the current study with wild-type mice, we combined these two approaches to determine if transplanting fecal material from mice fed black raspberries (BRB, 5%) altered recipients' immune system. The donors received a control or 5% BRB diet, and fecal transplantation was performed every other day 15 times into recipients fed control diet. Afterward, we used flow cytometry to analyze populations of CD3+ T, CD4+ T, CD8+ T cells, and NK cells among bone marrow cells, splenocytes, and peripheral blood mononuclear cells (PBMCs) collected from the recipients. We found that BRB-fecal material that contained both fecal microbiota and their metabolites increased NK cell populations among bone marrow cells, splenocytes, and PBMCs, and raised levels of CD8+ T cells in splenocytes. Our findings suggest that fecal transplantation can modulate the immune system and might therefore be valuable for managing a range of physical and mental disorders.

Effects of Dietary Interventions on Gut Microbiota in Humans and the Possible Impacts of Foods on Patients' Responses to Cancer Immunotherapy.

The gut microbiota-the community of microorganisms in the gut-has been implicated in many physical and mental disorders in addition to intestinal diseases. Diets are the most studied and promising factors for altering it. Indeed, certain dietary interventions that increase fiber intake rapidly change levels of certain nutrients that can modify the composition of the microbiota, promoting richness and diversity. Recent intriguing evidence from several human clinical trials suggested that the composition and diversity of patients' gut microbiotas at baseline can influence their responses to cancer immunotherapy. If the factors that influence the gut microbiota were fully understood, it is conceivable that manipulating them could boost therapeutic responses in cancer patients. In this review, we investigate the possibility of using fruits, vegetables, or whole grains to enhance response to cancer therapies in humans, as current evidence suggests that these dietary components can manipulate and enhance diversity of the gut microbiota. Accordingly, dietary interventions with locally available fruits, vegetables, and whole grains might be an affordable and safe approach to enhancing the diversity of the gut microbiota before immunotherapy, in turn improving patients' responses to their treatments.

Black Raspberries Suppress Colorectal Cancer by Enhancing Smad4 Expression in Colonic Epithelium and Natural Killer Cells.

Innate immune cells in the tumor microenvironment have been proposed to control the transition from benign to malignant stages. In many cancers, increased infiltration of natural killer (NK) cells associates with good prognosis. Although the mechanisms that enable NK cells to restrain colorectal cancer (CRC) are unclear, the current study suggests the involvement of Smad4. We found suppressed Smad4 expression in circulating NK cells of untreated metastatic CRC patients. Moreover, NK cell-specific Smad4 deletion promoted colon adenomas in DSS-treated ApcMin/+ mice and adenocarcinomas in AOM/DSS-treated mice. Other studies have shown that Smad4 loss or weak expression in colonic epithelium associates with poor survival in CRC patients. Therefore, targeting Smad4 in both colonic epithelium and NK cells could provide an excellent opportunity to manage CRC. Toward this end, we showed that dietary intervention with black raspberries (BRBs) increased Smad4 expression in colonic epithelium in patients with FAP or CRC and in the two CRC mouse models. Also, benzoate metabolites of BRBs, such as hippurate, upregulated Smad4 and Gzmb expression that might enhance the cytotoxicity of primary human NK cells. Of note, increased levels of hippurate is a metabolomic marker of a healthy gut microbiota in humans, and hippurate also has antitumor effects. In conclusion, our study suggests a new mechanism for the action of benzoate metabolites derived from plant-based foods. This mechanism could be exploited clinically to upregulate Smad4 in colonic epithelium and NK cells, thereby delaying CRC progression.

Designer biomass for next-generation biorefineries: leveraging recent insights into xylan structure and biosynthesis.

Xylans are the most abundant noncellulosic polysaccharides in lignified secondary cell walls of woody dicots and in both primary and secondary cell walls of grasses. These polysaccharides, which comprise 20-35% of terrestrial biomass, present major challenges for the efficient microbial bioconversion of lignocellulosic feedstocks to fuels and other value-added products. Xylans play a significant role in the recalcitrance of biomass to degradation, and their bioconversion requires metabolic pathways that are distinct from those used to metabolize cellulose. In this review, we discuss the key differences in the structural features of xylans across diverse plant species, how these features affect their interactions with cellulose and lignin, and recent developments in understanding their biosynthesis. In particular, we focus on how the combined structural and biosynthetic knowledge can be used as a basis for biomass engineering aimed at developing crops that are better suited as feedstocks for the bioconversion industry.

The Arabidopsis thiamin-deficient mutant pale green1 lacks thiamin monophosphate phosphatase of the vitamin B1 biosynthesis pathway.

Thiamin diphosphate (TPP, vitamin B1 ) is an essential coenzyme present in all organisms. Animals obtain TPP from their diets, but plants synthesize TPPde novo. We isolated and characterized an Arabidopsis pale green1 (pale1) mutant that contained higher concentrations of thiamin monophosphate (TMP) and less thiamin and TPP than the wild type. Supplementation with thiamin, but not the thiazole and pyrimidine precursors, rescued the mutant phenotype, indicating that the pale1 mutant is a thiamin-deficient mutant. Map-based cloning and whole-genome sequencing revealed that the pale1 mutant has a mutation in At5g32470 encoding a TMP phosphatase of the TPP biosynthesis pathway. We further confirmed that the mutation of At5g32470 is responsible for the mutant phenotypes by complementing the pale1 mutant with constructs overexpressing full-length At5g32470. Most plant TPP biosynthetic enzymes are located in the chloroplasts and cytosol, but At5g32470-GFP localized to the mitochondrion of the root, hypocotyl, mesophyll and guard cells of the 35S:At5g32470-GFP complemented plants. The subcellular localization of a functional TMP phosphatase suggests that the complete vitamin B1 biosynthesis pathway may involve the chloroplasts, mitochondria and cytosol in plants. Analysis of PALE1 promoter-uidA activity revealed that PALE1 is mainly expressed in vascular tissues of Arabidopsis seedlings. Quantitative RT-PCR analysis of TPP biosynthesis genes and genes encoding the TPP-dependent enzymes pyruvate dehydrogenase, α-ketoglutarate dehydrogenase and transketolase revealed that the transcript levels of these genes were upregulated in the pale1 mutant. These results suggest that endogenous levels of TPP may affect the expression of genes involved in TPP biosynthesis and TPP-dependent enzymes.

Systemic Metabolite Changes in Wild-type C57BL/6 Mice Fed Black Raspberries.

INTRODUCTION: Freeze-dried black raspberries (BRBs) elicit chemopreventive effects against colorectal cancer in humans and in rodents. The objective of this study was to investigate potential BRB-caused metabolite changes using wild-type (WT) C57BL/6 mice. METHODS AND RESULTS: WT mice were fed either control diet or control diet supplemented with 5% BRBs for 8 wk. A nontargeted metabolomic analysis was conducted on colonic mucosa, liver, and fecal specimens collected from both diet groups. BRBs significantly changed the levels of 41 colonic mucosa metabolites, 40 liver metabolites, and 34 fecal metabolites compared to control diet-fed mice. BRBs reduced 34 lipid metabolites in colonic mucosa and increased levels of amino acids in liver. One metabolite, 3-[3-(sulfooxy) phenyl] propanoic acid, might be a useful biomarker of BRB consumption. In addition, BRB powder was found to contain 30-fold higher levels of linolenate compared to control diets. Consistently, multiple omega-3 polyunsaturated fatty acids (ω-3 PUFAs), including stearidonate, docosapentaenoate (ω-3 DPA), eicosapentaenoate (EPA), and docosahexaenoate (DHA), were significantly elevated in livers of BRB-fed mice. CONCLUSION: The data from the current study suggest that BRBs produce systemic metabolite changes in multiple tissue matrices, supporting our hypothesis that BRBs may serve as both a chemopreventive agent and a beneficial dietary supplement.

Loss of free fatty acid receptor 2 enhances colonic adenoma development and reduces the chemopreventive effects of black raspberries in ApcMin/+ mice.

We previously showed that black raspberries (BRBs) have beneficial effects in human colorectal cancer and a mouse model of colorectal cancer (ApcMin/+). The current study investigated the role of free fatty acid receptor 2 (FFAR2) in colon carcinogenesis and whether the FFAR2 signaling pathway contributes to BRB-mediated chemoprevention in mice. FFAR2 (also named GPR43) is a member of the G-protein-coupled receptor family that is expressed in leukocytes and colon. ApcMin/+ and ApcMin/+-FFAR2-/- mice were given a control diet or the control diet supplemented with 5% BRBs for 8 weeks. FFAR2 deficiency promoted colonic polyp development, with 100% incidence and increased polyp number and size. The ApcMin/+ mice developed colonic tubular adenoma, whereas the ApcMin/+-FFAR2-/- mice developed colonic tubular adenoma with high-grade dysplasia. FFAR2 deficiency also enhanced the cAMP-PKA-CREB-HDAC pathway, downstream of FFAR2 signaling, and increased activation of the Wnt pathway, and raised the percentage of GR-1+ neutrophils in colonic lamina propria (LP) and increased infiltration of GR-1+ neutrophils into colonic polyps. BRBs suppressed colonic polyp development and inhibited the cAMP-PKA-CREB-HDAC and Wnt pathways in the ApcMin/+ mice but not the ApcMin/+-FFAR2-/- mice. They also increased the percentage of GR-1+ neutrophils and cytokine secretion in colonic LP and decreased the infiltration of GR-1+ neutrophils and IL-1ß expression in colon polyps of ApcMin/+ mice but not ApcMin/+-FFAR2-/- mice. These results suggest that loss of FFAR2 drives colon tumorigenesis and that BRBs require functional FFAR2 to be chemopreventive. BRBs have the potential to modulate the host immune system, thereby enhancing the antitumor immune microenvironment.

HDAC10 promotes lung cancer proliferation via AKT phosphorylation.

Histone deacetylase 10 (HDAC10) is a member of the class II HDACs, and its role in cancer is emerging. In this study, we found that HDAC10 is highly expressed in lung cancer tissues. It resides mainly in the cytoplasm of lung cancer cells but resides in the nucleus of adjacent normal cells. Further examinations revealed that HDAC10 resides in the cytoplasm in multiple lung cancer cell lines, including the A549, H358 and H460 cell lines, but mainly resides in the nucleus of normal lung epithelial 16HBE cells. A leucine-rich motif, R505L506L507C508V509A510L511, was identified as its nuclear localization signal (NLS), and a mutant (Mut-505-511) featuring mutations to A at each of its original R and L positions was found to be nuclear-localization defective. Functional analysis revealed that HDAC10 promoted lung cancer cell growth and that its knockdown induced cell cycle arrest and apoptosis. Mechanistic studies showed that HDAC10 knockdown significantly decreased the phosphorylation of AKT at Ser473 and that AKT expression significantly rescued the cell cycle arrest and apoptosis elicited by HDAC10 knockdown. A co-immunoprecipitation assay suggested that HDAC10 interacts with AKT and that inhibition of HDAC10 activity decreases its interaction with and phosphorylation of AKT. Finally, we confirmed that HDAC10 promoted lung cancer proliferation in a mouse model. Our study demonstrated that HDAC10 localizes and functions in the cytoplasm of lung cancer cells, thereby underscoring its potential role in the diagnosis and treatment of lung cancer.

Black raspberries suppress colonic adenoma development in ApcMin/+ mice: relation to metabolite profiles.

Freeze-dried black raspberries (BRBs) have demonstrated chemopreventive effects in a dietary intervention trial with human colorectal cancer patients. The aim of this study was to investigate BRB-caused metabolite changes using the Apc(Min/+) mouse as a model of human colorectal cancer. Wild-type (WT) mice were fed control diet, and Apc(Min/+) mice were fed either control diet or control diet supplemented with 5% BRBs for 8 weeks. Colonic and intestinal polyp size and number were measured. A non-targeted metabolomic analysis was conducted on colonic mucosa, liver and fecal specimens. Eight weeks of BRB treatment significantly decreased intestinal and colonic polyp number and size in Apc(Min/+) mice. The apc gene mutation significantly changed 52 metabolites in colonic mucosa associated with increased amino acid and decreased lipid metabolites, as well as 39 liver and 8 fecal metabolites. BRBs significantly reversed 23 apc-regulated metabolites, including 13 colonic mucosa, 8 liver and 2 fecal metabolites that were involved in amino acid, glutathione, lipid and nucleotide metabolism. Of these, changes in eight metabolites were linearly correlated with decreased colonic polyp number and size in BRB-treated Apc(Min/+) mice. Elevated levels of putrescine and linolenate in Apc(Min/+) mice were significantly decreased by BRBs. Ornithine decarboxylase expression, the key enzyme in putrescine generation, was fully suppressed by BRBs. These results suggest that BRBs produced beneficial effects against colonic adenoma development in Apc(Min/+) mice and modulated multiple metabolic pathways. The metabolite changes produced by BRBs might potentially reflect the BRB-mediated chemopreventive effects in colorectal cancer patients.

Functional evidence for the critical amino-terminal conserved domain and key amino acids of Arabidopsis 4-HYDROXY-3-METHYLBUT-2-ENYL DIPHOSPHATE REDUCTASE.

The plant 4-HYDROXY-3-METHYLBUT-2-ENYL DIPHOSPHATE REDUCTASE (HDR) catalyzes the last step of the methylerythritol phosphate pathway to synthesize isopentenyl diphosphate and its allyl isomer dimethylallyl diphosphate, which are common precursors for the synthesis of plastid isoprenoids. The Arabidopsis (Arabidopsis thaliana) genomic HDR transgene-induced gene-silencing lines are albino, variegated, or pale green, confirming that HDR is essential for plants. We used Escherichia coli isoprenoid synthesis H (Protein Data Bank code 3F7T) as a template for homology modeling to identify key amino acids of Arabidopsis HDR. The predicted model reveals that cysteine (Cys)-122, Cys-213, and Cys-350 are involved in iron-sulfur cluster formation and that histidine (His)-152, His-241, glutamate (Glu)-242, Glu-243, threonine (Thr)-244, Thr-312, serine-379, and asparagine-381 are related to substrate binding or catalysis. Glu-242 and Thr-244 are conserved only in cyanobacteria, green algae, and land plants, whereas the other key amino acids are absolutely conserved from bacteria to plants. We used site-directed mutagenesis and complementation assay to confirm that these amino acids, except His-152 and His-241, were critical for Arabidopsis HDR function. Furthermore, the Arabidopsis HDR contains an extra amino-terminal domain following the transit peptide that is highly conserved from cyanobacteria, and green algae to land plants but not existing in the other bacteria. We demonstrated that the amino-terminal conserved domain was essential for Arabidopsis and cyanobacterial HDR function. Further analysis of conserved amino acids in the amino-terminal conserved domain revealed that the tyrosine-72 residue was critical for Arabidopsis HDR. These results suggest that the structure and reaction mechanism of HDR evolution have become specific for oxygen-evolving photosynthesis organisms and that HDR probably evolved independently in cyanobacteria versus other prokaryotes.