Search

Cytochrome P450 enzymes (CYPs) are widely distributed among various plant groups and constitute approximately 1% of the total number of protein-coding genes. Extensive studies suggest that CYPs are involved in nearly all molecular processes that occur in plants. Over the past two decades, the identification of CYP genes has expanded rapidly, with more than 40,000 CYP genes and 819 CYP families being discovered. Copy number variation is a significant evolutionary characteristic of gene families, yet a systematic characterization of the copy evolution patterns in plant CYP gene families has been lacking, resulting in confusion and challenges in understanding CYP functions. To address these concerns, this review provides comprehensive statistics and analyses of the copy number and diversity of almost all plant CYP gene families, focusing on CYP evolution from Chlorophyta to Dicotyledoneae. Additionally, we examined the subfamily characteristics of certain CYP families with restricted copy changes and identified several CYP subfamilies that play pivotal roles in this event. Furthermore, we analyzed the structural conservation of CYPs across different taxa and compiled a comprehensive database to support plant CYP studies. Our analysis revealed differences in the six core conserved motifs of plant CYP proteins among various clans and plant taxa, while demonstrating similar conservation patterns for the ERR (glutamic acid-arginine-arginine) triad motifs. These findings will significantly facilitate the understanding of plant CYP gene evolution and metabolic diversity and serve as a valuable reference for researchers studying CYP enzymes., (© The Author(s) 2024. Published by Oxford University Press on behalf of Society for Molecular Biology and Evolution.)

Arogenate dehydratase (ADT) is the key limiting enzyme of plant phenylalanine biosynthesis, but some ADTs display a prephenate decarboxylase/dehydratase activity-conferring (PAC) domain. The genome resources of 70 species were employed to identify genes and outline their characteristics, especially the number and type of PAC domain structures. We obtained 522 ADTs, and their size, exon number, amino acid number and putative protein isoelectric point greatly varied from 306 to 2520 bp, 1 to 15, 101 to 839 and 4.37 to 11.18, respectively. We classified the ADTs into Class α (without a PAC domain) (115, 22.0 %), β (with a type I PAC domain) (244, 46.7 %) and γ (with a type II PAC domain) (163, 31.2 %), and their distribution frequencies exhibited large differences among various branches of angiosperms. We found that Class γ members are more conserved than Class β members, although they commonly experienced multiple duplication events and strong purifying selection, which resulted in a small number, and the putative origin order was from Class α to β and then to γ. In addition, the co-occurrence of both Class β and γ members could ensure the survival of angiosperms, while their optimized composition and strategically intertwined regulation may facilitate core eudicot success., Competing Interests: Declaration of competing interest The authors declare no competing interests., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Background: Flavonoids, an important class of specialized metabolites, are synthesized from phenylalanine and present in almost all plant species. Different branches of flavonoid biosynthesis lead to products like flavones, flavonols, anthocyanins, and proanthocyanidins. Dihydroflavonols form the branching point towards the production of non-colored flavonols via flavonol synthase (FLS) and colored anthocyanins via dihydroflavonol 4-reductase (DFR). Despite the wealth of publicly accessible data, there remains a gap in understanding the mechanisms that mitigate competition between FLS and DFR for the shared substrate, dihydroflavonols., Results: An angiosperm-wide comparison of FLS and DFR sequences revealed the amino acids at positions associated with the substrate specificity in both enzymes. A global analysis of the phylogenetic distribution of these amino acid residues revealed that monocots generally possess FLS with Y132 (FLSY) and DFR with N133 (DFRN). In contrast, dicots generally possess FLSH and DFRN, DFRD, and DFRA. DFRA, which restricts substrate preference to dihydrokaempferol, previously believed to be unique to strawberry species, is found to be more widespread in angiosperms and has evolved independently multiple times. Generally, angiosperm FLS appears to prefer dihydrokaempferol, whereas DFR appears to favor dihydroquercetin or dihydromyricetin. Moreover, in the FLS-DFR competition, the dominance of one over the other is observed, with typically only one gene being expressed at any given time., Conclusion: This study illustrates how almost mutually exclusive gene expression and substrate-preference determining residues could mitigate competition between FLS and DFR, delineates the evolution of these enzymes, and provides insights into mechanisms directing the metabolic flux of the flavonoid biosynthesis, with potential implications for ornamental plants and molecular breeding strategies., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Choudhary, Pucker. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Monoterpene synthases (MTSs) catalyze the first committed step in the biosynthesis of monoterpenoids, a class of specialized metabolites with particularly high chemical diversity in angiosperms. In addition to accomplishing a rate enhancement, these enzymes manage the formation and turnover of highly reactive carbocation intermediates formed from a prenyl diphosphate substrate. At each step along the reaction path, a cationic intermediate can be subject to cyclization, migration of a proton, hydride, or alkyl group, or quenching to terminate the sequence. However, enzymatic control of ligand folding, stabilization of specific intermediates, and defined quenching chemistry can maintain the specificity for forming a signature product. This review article will discuss our current understanding of how angiosperm MTSs control the reaction environment. Such knowledge allows inferences about the origin and regulation of chemical diversity, which is pertinent for appreciating the role of monoterpenoids in plant ecology but also for aiding commercial efforts that harness the accumulation of these specialized metabolites for the food, cosmetic, and pharmaceutical industries., (© 2024 The Authors. The Plant Journal published by Society for Experimental Biology and John Wiley & Sons Ltd.)

Next-generation sequencing has resulted in an explosion of available data, much of which remains unstudied in terms of biochemical function; yet, experimental characterization of these sequences has the potential to provide unprecedented insight into the evolution of enzyme activity. One way to make inroads into the experimental study of the voluminous data available is to engage students by integrating teaching and research in a college classroom such that eventually hundreds or thousands of enzymes may be characterized. In this study, we capitalize on this potential to focus on SABATH methyltransferase enzymes that have been shown to methylate the important plant hormone, salicylic acid (SA), to form methyl salicylate. We analyze data from 76 enzymes of flowering plant species in 23 orders and 41 families to investigate how widely conserved substrate preference is for SA methyltransferase orthologs. We find a high degree of conservation of substrate preference for SA over the structurally similar metabolite, benzoic acid, with recent switches that appear to be associated with gene duplication and at least three cases of functional compensation by paralogous enzymes. The presence of Met in active site position 150 is a useful predictor of SA methylation preference in SABATH methyltransferases but enzymes with other residues in the homologous position show the same substrate preference. Although our dense and systematic sampling of SABATH enzymes across angiosperms has revealed novel insights, this is merely the "tip of the iceberg" since thousands of sequences remain uncharacterized in this enzyme family alone., (© The Author(s) 2022. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.)

The prevalence of phylogenetic constraints in Rubisco evolution has been emphasised recently by (Bouvier et al., 2021), who argued that phylogenetic inheritance limits Rubisco adaptation much more than the biochemical trade-off between specificity, CO 2 affinity and turn-over. In this Opinion, we have critically examined how a phylogenetic signal can be computed with Rubisco kinetic properties and phylogenetic trees, and we arrive at a different conclusion. In particular, Rubisco's adaptation is partly driven by C 4 vs. C 3 photosynthetic conditions in Angiosperms, apparent phylogenetic signals being mostly due to either homoplasy, computation artefacts or the use of nearly identical sister species. While phylogenetic inheritance of an ancestral enzyme form probably has some role in Rubisco's adaptation landscape, it is a minor player, at least compared to microenvironmental conditions such as CO 2 and O 2 concentrations., (Copyright © 2021 Elsevier GmbH. All rights reserved.)

Nepenthes ampullaria is a unique carnivorous tropical pitcher plant with the detritivorous capability of sequestering nutrients from leaf litter apart from being insectivorous. The changes in the protein composition and protease activity of its pitcher fluids during the early opening of pitchers (D0 and D3C) were investigated via a proteomics approach and a controlled protein depletion experiment (D3L). A total of 193 proteins were identified. Common proteins such as pathogenesis-related protein, proteases (Nep [EC:3.4.23.12], SCP [EC:3.4.16.-]), peroxidase [EC:1.11.1.7], GDSL esterase/lipase [EC:3.1.1.-], and purple acid phosphatase [EC:3.1.3.2] were found in high abundance in the D0 pitchers and were replenished in D3L samples. This reflects their importance for biological processes upon pitcher opening. Meanwhile, prey-inducible chitinases [EC:3.2.1.14] were found in D0 but not in D3C and D3L samples, which suggests their degradation in the absence of prey. Protease activity assays demonstrated the replenishment of proteases in D3L with similar levels of proteolytic activities to that of D3C samples. This supports a feedback mechanism and signaling in the molecular regulation of endogenous protein secretion, turnover, and activity in Nepenthes pitcher fluids. Furthermore, we also discovered several new enzymes (XTH [EC:2.4.1.207], PAE [EC:3.1.1.98]) with possible functions in cell wall degradation that could contribute to the detritivory habit of N. ampullaria., (© 2021 Scandinavian Plant Physiology Society.)

Serotonin (Ser) and melatonin (Mel) serve as master regulators of plant growth and development by influencing diverse cellular processes. The enzymes namely, tryptophan decarboxylase ( TDC ) and tryptamine 5-hydroxylase ( T5H ) catalyse the formation of Ser from tryptophan. Subsequently, serotonin N-acetyl transferase ( SNAT ) and acetyl-serotonin methyltransferase ( ASMT ) form Mel from Ser. Plant genomes harbour multiple genes for each of these four enzymes, all of which have not been identified. Therefore, to delineate information regarding these four gene families, we carried out a genome-wide analysis of the genes involved in Ser and Mel biosynthesis in Arabidopsis, tomato, rice and sorghum. Phylogenetic analysis unravelled distinct evolutionary relationships among these genes from different plants. Interestingly, no gene family except ASMTs showed monocot- or dicot-specific clustering of respective proteins. Further, we observed tissue-specific, developmental and stress/hormone-mediated variations in the expression of the four gene families. The light/dark cycle also affected their expression in agreement with our quantitative reverse transcriptase-PCR (qRT-PCR) analysis. Importantly, we found that miRNAs (miR6249a and miR-1846e) regulated the expression of Ser and Mel biosynthesis under light and stress by influencing the expression of OsTDC5 and OsASMT18 , respectively. Thus, this study may provide opportunities for functional characterization of suitable target genes of the Ser and Mel pathway to decipher their exact roles in plant physiology.

Starch phosphorylase is a member of the GT35-glycogen-phosphorylase superfamily. Glycogen phosphorylases have been researched in animals thoroughly when compared to plants. Genetic evidence signifies the integral role of plastidial starch phosphorylase (PHO1) in starch biosynthesis in model plants. The counterpart of PHO1 is PHO2, which specifically resides in cytosol and is reported to lack L80 peptide in the middle region of proteins as seen in animal and maltodextrin forms of phosphorylases. The function of this extra peptide varies among species and ranges from the substrate of proteasomes to modulate the degradation of PHO1 in Solanum tuberosum to a non-significant effect on biochemical activity in Oryza sativa and Hordeum vulgare . Various regulatory functions, e.g., phosphorylation, protein-protein interactions, and redox modulation, have been reported to affect the starch phosphorylase functions in higher plants. This review outlines the current findings on the regulation of starch phosphorylase genes and proteins with their possible role in the starch biosynthesis pathway. We highlight the gaps in present studies and elaborate on the molecular mechanisms of phosphorylase in starch metabolism. Moreover, we explore the possible role of PHO1 in crop improvement.

In this study, we aimed to investigate the chemical components and biological activities of Musella lasiocarpa , a special flower that is edible and has functional properties. The crude methanol extract and its four fractions (petroleum ether, ethyl acetate, n-butanol, and aqueous fractions) were tested for their total antioxidant capacity, followed by their α-glucosidase, acetylcholinesterase, and xanthine oxidase inhibitory activities. Among the samples, the highest total phenolic and total flavonoid contents were found in the ethyl acetate (EtOAc) fraction (224.99 mg GAE/g DE) and crude methanol extract (187.81 mg QE/g DE), respectively. The EtOAc fraction of Musella lasiocarpa exhibited the strongest DPPH· scavenging ability, ABTS· + scavenging ability, and α-glucosidase inhibitory activity with the IC 50 values of 22.17, 12.10, and 125.66 μg/mL, respectively. The EtOAc fraction also showed the strongest ferric reducing antioxidant power (1513.89 mg FeSO 4 /g DE) and oxygen radical absorbance capacity ability (524.11 mg Trolox/g DE), which were higher than those of the control BHT. In contrast, the aqueous fraction demonstrated the highest acetylcholinesterase inhibitory activity (IC 50 = 10.11 μg/mL), and the best xanthine oxidase inhibitory ability (IC 50 = 5.23 μg/mL) was observed from the crude methanol extract as compared with allopurinol (24.85 μg/mL). The HPLC-MS/MS and GC-MS analyses further revealed an impressive arsenal of compounds, including phenolic acids, fatty acids, esters, terpenoids, and flavonoids, in the most biologically active EtOAc fraction. Taken together, this is the first report indicating the potential of Musella lasiocarpa as an excellent natural source of antioxidants with possible therapeutic, nutraceutical, and functional food applications.

A common morphological feature of typical angiosperms is the patterning of lateral organs along primary axes of asymmetry-a proximodistal, a mediolateral, and an adaxial-abaxial axis. Angiosperm leaves usually have distinct adaxial-abaxial identity, which is required for the development of a flat shape. By contrast, many unifacial leaves, consisting of only the abaxial side, show a flattened morphology. This implicates a unique mechanism that allows leaf flattening independent of adaxial-abaxial identity. In this study, we report a role for auxin in outgrowth of unifacial leaves. In two closely related unifacial-leaved species of Juncaceae, Juncus prismatocarpus with flattened leaves, and Juncus wallichianus with transversally radialized leaves, the auxin-responsive gene GLYCOSIDE HYDROLASE3 displayed spatially different expression patterns within leaf primordia. Treatment of J. prismatocarpus seedlings with exogenous auxin or auxin transport inhibitors, which disturb endogenous auxin distribution, eliminated leaf flatness, resulting in a transversally radialized morphology. These treatments did not affect the radialized morphology of leaves of J. wallichianus. Moreover, elimination of leaf flatness by these treatments accompanied dysregulated expression of genetic factors needed to specify the leaf central-marginal polarity in J. prismatocarpus. The findings imply that lamina outgrowth of unifacial leaves relies on proper placement of auxin, which might induce initial leaf flattening and subsequently act to specify leaf polarity, promoting further flattening growth of leaves., (© American Society of Plant Biologists 2021. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Contrary to animals, little is known in plants about enzymes able to produce fatty acid epoxides. In our attempt to find and characterize a new fatty acid epoxygenase in Arabidopsis thaliana, data mining brought our attention on CYP77B1. Modification of the N-terminus was necessary to get enzymatic activity after heterologous expression in yeast. The common plant fatty acid C18:2 was converted into the diol 12,13-dihydroxy-octadec-cis-9-enoic acid when incubated with microsomes of yeast expressing modified CYP77B1 and AtEH1, a soluble epoxide hydrolase. This diol originated from the hydrolysis by AtEH1 of the epoxide 12,13-epoxy-octadec-cis-9-enoic acid produced by CYP77B1. A spatio-temporal study of CYP77B1 expression performed with RT-qPCR revealed the highest level of transcripts in flower bud while, in open flower, the enzyme was mainly present in pistil. CYP77B1 promoter-driven GUS expression confirmed reporter activities in pistil and also in stamens and petals. In silico co-regulation data led us to hypothesize that CYP77B1 could be involved in cutin synthesis but when flower cutin of loss-of-function mutants cyp77b1 was analyzed, no difference was found compared to cutin of wild type plants. Phylogenetic analysis showed that CYP77B1 is strictly conserved in flowering plants, suggesting a specific function in this lineage., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Background: The CIPKs are a group of plant-specific Ser/Thr protein kinases acting in response to calcium signaling, which plays an important role in the physiological and developmental adaptation of plants to adverse environments. However, the functions of halophyte-derived CIPKs are still poorly understood, that limits a potential application of CIPKs from halophytes for improving the tolerance of glycophytes to abiotic stresses., Results: In this study, we characterized the NtCIPK11 gene from the halophyte Nitraria tangutorum and subsequently analyzed its role in salt and drought stress tolerance, using Arabidopsis as a transgenic model system. NtCIPK11 expression was upregulated in N. tangutorum root, stem and blade tissues after salt or drought treatment. Overexpressing NtCIPK11 in Arabidopsis improved seed germination on medium containing different levels of NaCl. Moreover, the transgenic plants grew more vigorously under salt stress and developed longer roots under salt or drought conditions than the WT plants. Furthermore, NtCIPK11 overexpression altered the transcription of genes encoding key enzymes involved in proline metabolism in Arabidopsis exposed to salinity, however, which genes showed a relatively weak expression in the transgenic Arabidopsis undergoing mannitol treatment, a situation that mimics drought stress. Besides, the proline significantly accumulated in NtCIPK11-overexpressing plants compared with WT under NaCl treatment, but that was not observed in the transgenic plants under drought stress caused by mannitol application., Conclusions: We conclude that NtCIPK11 promotes plant growth and mitigates damage associated with salt stress by regulating the expression of genes controlling proline accumulation. These results extend our understanding on the function of halophyte-derived CIPK genes and suggest that NtCIPK11 can serve as a candidate gene for improving the salt and drought tolerance of glycophytes through genetic engineering.

The most paradigmatic examples of molecular evolution under positive selection involve genes related to the immune system. Recently, different chloroplastic factors have been shown to be important for plant defenses, among them, the α- and β-subunits of the ATP synthase. The β-subunit has been reported to interact with several viral proteins while both proteins have been implicated with sensitivity to tentoxin, a phytotoxin produced by the widespread fungus Alternaria alternata. Given the relation of both protein to virulence factors, we studied whether these proteins are evolving under positive selection. To this end, we used the dN/dS ratio to examine possible sites under positive selection in several Angiosperm clades. After examining 79 plant genera and 1232 species, we found three times more sites under pervasive diversifying selection in the N-terminal region of the β-subunit compared to the α-subunit, supporting previous results which identified this region as responsible for interacting with viral proteins. Moreover, we found the site 83 of β-subunit under positive selection in several plant genera, a site clearly related to the sensitivity to tentoxin according to biochemistry assays, which possibly reflects the selective pressure of the non-host specific tentoxin across various Angiosperm clades.

We characterized an Na + transporter SvHKT1;1 from a halophytic turf grass, Sporobolus virginicus . SvHKT1;1 mediated inward and outward Na + transport in Xenopus laevis oocytes and did not complement K + transporter-defective mutant yeast. SvHKT1;1 did not complement athkt1;1 mutant Arabidopsis , suggesting its distinguishable function from other typical HKT1 transporters. The transcript was abundant in the shoots compared with the roots in S. virginicus and was upregulated by severe salt stress (500 mM NaCl), but not by lower stress. SvHKT1;1 -expressing Arabidopsis lines showed higher shoot Na + concentrations and lower salt tolerance than wild type (WT) plants under nonstress and salt stress conditions and showed higher Na + uptake rate in roots at the early stage of salt treatment. These results suggested that constitutive expression of SvHKT1;1 enhanced Na + uptake in root epidermal cells, followed by increased Na + transport to shoots, which led to reduced salt tolerance. However, Na + concentrations in phloem sap of the SvHKT1;1 lines were higher than those in WT plants under salt stress. Based on this result, together with the induction of the SvHKT1;1 transcription under high salinity stress, it was suggested that SvHKT1;1 plays a role in preventing excess shoot Na + accumulation in S. virginicus .

Iridoids are plant-derived terpenoids with a rich array of bioactivities. The key step in iridoid skeleton formation is the reduction of 8-oxogeranial by certain members of the progesterone 5β-reductase/iridoid synthase (PRISE) family of short-chain alcohol dehydrogenases. Other members of the PRISE family have previously been implicated in the biosynthesis of the triterpenoid class of cardenolides, which requires the reduction of progesterone. Here, we explore the occurrence and activity of PRISE across major lineages of plants. We observed trace activities toward either 8-oxogeranial or progesterone in all PRISEs, including those from nonseed plants and green algae. Phylogenetic analysis, coupled with enzymatic assays, show that these activities appear to have become specialized in specific angiosperm lineages. This broad analysis of the PRISE family provides insight into how these enzymes evolved in plants and also suggests that iridoid synthase activity is an ancestral trait in all land plants, which might have contributed to the rise of iridoid metabolites.

Light-dependent protochlorophyllide oxidoreductase (LPOR) catalyzes the reduction of protochlorophyllide to chlorophyllide, which is a key reaction for angiosperm development. Dark operative light-independent protochlorophyllide oxidoreductase (DPOR) is the other enzyme able to catalyze this reaction, however, it is not present in angiosperms. LPOR, which evolved later than DPOR, requires light to trigger the reaction. The ancestors of angiosperms lost DPOR genes and duplicated the LPORs, however, the LPOR evolution in angiosperms has not been yet investigated. In the present study, we built a phylogenetic tree using 557 nucleotide sequences of LPORs from both bacteria and plants to uncover the evolution of LPOR. The tree revealed that all modern sequences of LPOR diverged from a single sequence ∼1.36 billion years ago. The LPOR gene was then duplicated at least 10 times in angiosperms, leading to the formation of two or even more LPOR isoforms in multiple species. In the case of Arabidopsis thaliana, AtPORA and AtPORB originated in one duplication event, in contrary to the isoform AtPORC, which diverged first. We performed biochemical characterization of these isoforms in vitro, revealing differences in the lipid-driven properties. The results prone us to hypothesize that duplication events of LPOR gave rise to the isoforms having different lipid-driven activity, which may predispose them for functioning in different locations in plastids. Moreover, we showed that LPOR from Synechocystis operated in the lipid-independent manner, revealing differences between bacterial and plant LPORs. Based on the presented results, we propose a novel classification of LPOR enzymes based on their biochemical properties and phylogenetic relationships., (© 2020 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

Background: Nitrogen is an indispensable nutrient for plant growth. It is used and transported in the form of amino acids in living organisms. Transporting amino acids to various parts of plants requires relevant transport proteins, such as amino acid permeases (AAPs), which were our focus in this study., Results: We found that 5 AAP genes were present in Chlorophyte species and more AAP genes were predicted in Bryophyta and Lycophytes. Two main groups were defined and group I comprised 5 clades. Our phylogenetic analysis indicated that the origin of clades 2, 3, and 4 is Gymnospermae and that these clades are closely related. The members of clade 1 included Chlorophyta to Gymnospermae. Group II, as a new branch consisting of non-seed plants, is first proposed in our research. Our results also indicated that the AAP family was already present in Chlorophyta and then expanded accompanying the development of vasculature. Concurrently, the AAP family experienced multiple duplication events that promoted the generation of new functions and differentiation of sub-functions., Conclusions: Our findings suggest that the AAP gene originated in Chlorophyta, and some non-seed AAP genes clustered in one group. A second group, which contained plants of all evolutionary stages, indicated the evolution of AAPs. These new findings can be used to guide future research.

Oilseed crop oils contain a variety of unsaturated fatty acids that are synthesized and regulated by fatty acid desaturases (FADs). In this study, 14 FAD3 (ω3 desaturase) protein sequences from oilseeds are analyzed and presented through the application of several computational tools. The results indicated a close relationship between Brassica napus and Camelina sativa, as well as between Salvia hispanica and Perilla frutescens FAD3s, due to a high similarity in codon preferences in codon usage clusters and the phylogenetic tree. The cis-acting element results reveal that the seed-specific promoter region of BnFAD3 contains the critical conserved boxes such as HSE and ABRE, which are involved in responsiveness to heat stress and abscisic acid. The presence of the aforementioned conserved boxes may increase cold acclimation as well as tolerance to drought and high salinity. Omega(ω)3 desaturases contain a Skn-1 motif which is a cis-acting regulatory element required involved in endosperm development. In oilseed FAD3s, leucine is the most repeated amino acid in FAD3 proteins. The study conveyed that B. napus, Camelina sativa, Linum usitatissimum, Vernicia fordii, Gossypium hirsutum, S. hispanica, Cannabis sativa, and P. frutescens have retention signal KXKXX/XKXX at their c-terminus sites, which is one of the most important characteristics of FADs. Additionally, it was found that BnFAD3 is a transmembrane protein that can convert ω6 to ω3 fatty acids and may simultaneously act as a potassium ion channel in the ER.

Rubisco specificity factor (S c/o ), a measure of the relative capacities of an enzyme to catalyze carboxylation and oxygenation of ribulose-1,5-bisphosphate, determines the extent of photosynthetic CO 2 assimilation and photorespiratory CO 2 release. The current model of C3 photosynthesis, the Farquhar-von Caemmerer-Berry (FvCB) model, requires a species-specific S c/o . However, S c/o values have never been reported in conifers, likely because in vitro kinetic analysis of conifer Rubisco presents difficulties. To estimate the S c/o of conifers and compare it with angiosperm S c/o , we measured changes in leaf CO 2 compensation points (Γ) in response to O 2 partial pressure for a variety of leaves, with different rates of day respiration (R day ) and maximum Rubisco carboxylation (V cmax ) in gymnosperms (Ginkgo biloba), conifers (Metasequoia glyptostroboides and Cryptomeria japonica), and angiosperms (Nicotiana tabacum and Phaseolus vulgaris). As predicted by the FvCB model, the slope of a linear function of Γ vs O 2 partial pressure, d, increased alongside increasing R day /V cmax . The S c/o was obtainable from this relationship between d and R day /V cmax , because the d values at R day /V cmax = 0 corresponded to α/S c/o , where α was the photorespiratory CO 2 release rate per Rubisco oxygenation rate (generally assumed to be 0.5). The calculated S c/o values of N. tabacum and P. vulgaris exhibited good agreement with those reported by in vitro studies. The S c/o values of both conifers were similar to those of the two angiosperm species. In contrast, the S c/o value of G. biloba was significantly lower than those of the other four studied species. These results suggest that our new method for S c/o estimation is applicable to C3 plants, including those for which in vitro kinetic analysis is difficult. Furthermore, results also suggest that conifer S c/o does not differ significantly from that of C3 angiosperms, assuming α remains unchanged.

Background: Clinopodium gracile (Benth.) Matsum (C. gracile) is an annual herb with pharmacological properties effective in the treatment of various diseases, including hepatic carcinoma. Triterpenoid saponins are crucial bioactive compounds in C. gracile. However, the molecular understanding of the triterpenoid saponin biosynthesis pathway remains unclear., Results: In this study, we performed RNA sequencing (RNA-Seq) analysis of the flowers, leaves, roots, and stems of C. gracile plants using the BGISEQ-500 platform. The assembly of transcripts from all four types of tissues generated 128,856 unigenes, of which 99,020 were mapped to several public databases for functional annotation. Differentially expressed genes (DEGs) were identified via the comparison of gene expression levels between leaves and other tissues (flowers, roots, and stems). Multiple genes encoding pivotal enzymes, such as squalene synthase (SS), or transcription factors (TFs) related to triterpenoid saponin biosynthesis were identified and further analyzed. The expression levels of unigenes encoding important enzymes were verified by quantitative real-time PCR (qRT-PCR). Different chemical constituents of triterpenoid saponins were identified by Ultra-Performance Liquid Chromatography coupled with quadrupole time-of-flight mass spectrometry (UPLC/Q-TOF-MS)., Conclusions: Our results greatly extend the public transcriptome dataset of C. gracile and provide valuable information for the identification of candidate genes involved in the biosynthesis of triterpenoid saponins and other important secondary metabolites.

Boraginales (the forget-me-not order) is a core group within the lamiids clade. However, until now, no genome from Boraginales has been reported, and published transcriptomes are also rare. Here, we report the first Boraginales species de novo genome (i.e. Echium plantagineum genome) and seven other Boraginales species transcriptomes to probe three issues: (i) Boraginales' phylogenetic position within the lamiids clade; (ii) potential whole genome duplications (WGDs) in Boraginales; and (iii) candidate key enzyme genes in the alkannin/shikonin core pathway. The results showed that: (i) Boraginales was most probably closer to the Solanales/Gentianales clade than the Lamiales clade, at least based on the single-copy orthologous genes from genome/transcriptome data; (ii) after the gamma (γ) event, Boraginaceae (classified into the Boraginales I clade) probably underwent at least two rounds of WGD, whereas Heliotropiaceae and Ehretiaceae (classified into the Boraginales II clade) probably underwent only one round of WGD; and (iii) several candidate key enzyme genes in the alkannin/shikonin core pathway were inferred, e.g. genes corresponding to geranyl cyclase, naphthol hydroxylase and O-acyl transferase., (© 2019 John Wiley & Sons Ltd.)

Dimethylsulfoniopropionate (DMSP) is one of the most abundant sulfur metabolites in marine environments. The biosynthesis of DMSP and its degradation to dimethylsulfide are important links in the planetary sulfur cycle. Herein, the first complete description of a DMSP biosynthetic pathway is provided by the in vitro reconstitution of four enzymes from Streptomyces mobaraensis. The isolation of DMSP from S. mobaraensis cells grown at high salinity confirmed that this actinobacterium is indeed is a DMSP-producing organism. The described DMSP biosynthesis follows the same route as that previously described for angiosperm plants. Despite this chemical congruence, limited sequence similarity between plant and bacterial enzymes suggests that the two biosynthetic activities emerged by convergent evolution., (© 2019 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The plant kingdom supports an extraordinary chemical diversity, with terpenoids representing a particularly diversified class of secondary (or specialized) metabolites. Volatile and semi-volatile terpenoids in the C10-C20 range are often formed in specialized cell types and secretory structures. In the angiosperm lineage, glandular trichomes play an important role in enabling the biosynthesis and storage (or in some cases secretion) of functionalized terpenoids. The 'decoration' of a terpenoid scaffold with functional groups changes its physical and chemical properties, and can therefore affect the perception of a specific metabolite by other organisms. Because of the ecological implications (e.g. plant-herbivore interactions) and commercial relevance (e.g. volatiles used in the flavor and fragrance industries), terpenoid functionalization has been researched extensively. Recent successes in the cloning and functional evaluation of genes as well as the structural and biochemical characterization of enzyme catalysts have laid the foundation for an improved understanding of how pathways toward functionalized monoterpenes may have evolved. In this review, we will focus on an up-to-date account of functionalization reactions present in glandular trichomes., (© The Author(s) 2019. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

We conducted biochemical and physiological experiments to investigate the mode of action of tiafenacil (Terrad'or™), a new protoporphyrinogen IX oxidase (PPO)-inhibiting pyrimidinedione herbicide. Analysis of the half-maximal inhibitory concentration (IC 50 ) against recombinant PPO enzymes from various plant species, including amaranth (Amaranthus tuberculatus), soybean (Glycine max), arabidopsis (Arabidopsis thaliana), and rapeseed (Brassica napus), showed that tiafenacil had an IC 50 of 22 to 28 nM, similar to the pyrimidinedione herbicides butafenacil and saflufenacil and the N-phenylphthalimide herbicide flumioxazin. By contrast, tiafenacil exhibited 3- to 134-fold lower IC 50 values than the diphenyl ether herbicides fomesafen, oxyfluorfen, and acifluorfen. Tiafenacil is non-selective and is herbicidal to both dicots and monocots, such as the weeds velvetleaf (Abutilon theophrasti), amaranth, and barnyardgrass (Echinochloa crus-galli) as well as the crops soybean, rapeseed, rice (Oryza sativa), and maize (Zea mays) at concentrations ranging from 1 to 50 μM. Treatment of plant tissue with tiafenacil in darkness resulted in the accumulation of protoporphyrin IX. Subsequent exposure to light increased the content of malondialdehyde and significantly decreased the Fv/Fm values of chlorophyll fluorescence. The results suggest that tiafenacil is a new PPO-inhibiting pyrimidinedione herbicide., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Cell walls are crucial for the integrity and function of all land plants and are of central importance in human health, livestock production, and as a source of renewable bioenergy. Many enzymes that mediate the biosynthesis of cell wall polysaccharides are encoded by members of the large cellulose synthase ( CesA ) gene superfamily. Here, we analyzed 29 sequenced genomes and 17 transcriptomes to revise the phylogeny of the CesA gene superfamily in angiosperms. Our results identify ancestral gene clusters that predate the monocot-eudicot divergence and reveal several novel evolutionary observations, including the expansion of the Poaceae-specific cellulose synthase-like CslF family to the graminids and restiids and the characterization of a previously unreported eudicot lineage, CslM , that forms a reciprocally monophyletic eudicot-monocot grouping with the CslJ clade. The CslM lineage is widely distributed in eudicots, and the CslJ clade, which was thought previously to be restricted to the Poales, is widely distributed in monocots. Our analyses show that some members of the CslJ lineage, but not the newly identified CslM genes, are capable of directing (1,3;1,4)-β-glucan biosynthesis, which, contrary to current dogma, is not restricted to Poaceae., (© 2018 American Society of Plant Biologists. All rights reserved.)

Glycoside Hydrolase 3 (GH3) is a phytohormone-responsive family of proteins found in many plant species. These proteins contribute to the biological activity of indolacetic acid (IAA), jasmonic acid (JA), and salicylic acid (SA). They also affect plant growth and developmental processes as well as some types of stress. In this study, GH3 genes were identified in 48 plant species, including algae, mosses, ferns, gymnosperms, and angiosperms. No GH3 representative protein was found in algae, but we identified 4 genes in mosses, 19 in ferns, 7 in gymnosperms, and several in angiosperms. The results showed that GH3 proteins are mainly present in seed plants. Phylogenetic analysis of all GH3 proteins showed three separate clades. Group I was related to JA adenylation, group II was related to IAA adenylation, and group III was separated from group II, but its function was not clear. The structure of the GH3 proteins indicated highly conserved sequences in the plant kingdom. The analysis of JA adenylation in relation to gene expression of GH3 in potato ( Solanum tuberosum ) showed that StGH3.12 greatly responded to methyl jasmonate (MeJA) treatment. The expression levels of StGH3.1 , StGH3.11 , and StGH3.12 were higher in the potato flowers, and StGH3.11 expression was also higher in the stolon. Our research revealed the evolution of the GH3 family, which is useful for studying the precise function of GH3 proteins related to JA adenylation in S. tuberosum when the plants are developing and under biotic stress.

Sweetpotato is a significant crop which is widely cultivated particularly in the developing countries with high and stable yield. However, drought stress is a major limiting factor that antagonistically influences the crop's productivity. Dehydration stress caused by drought causes aggregation of reactive oxygen species (ROS) in plants, and aldose reductases are first-line safeguards against ROS caused by oxidative stress. In the present study, we generated transgenic sweetpotato plants expressing aldose reductase, XvAld1 isolated from Xerophyta viscosa under the control of a stress-inducible promoter via Agrobacterium-mediated transformation. Our results demonstrated that the transgenic sweetpotato lines displayed significant enhanced tolerance to simulated drought stress and enhanced recuperation after rehydration contrasted with wild-type plants. In addition, the transgenic plants exhibited improved photosynthetic efficiency, higher water content and more proline accumulation under dehydration stress conditions compared with wild-type plants. These results demonstrate that exploiting the XvAld1 gene is not only a compelling and attainable way to improve sweetpotato tolerance to drought stresses without causing any phenotypic imperfections but also a promising gene candidate for more extensive crop improvement.

The plant hormone abscisic acid (ABA) plays a crucial role in regulating plant responses to environmental stresses. Interplay of several different proteins including the PYR/PYL/RCAR receptors, A-group PP2C protein phosphatases, SnRK2 protein kinases, and downstream transcription factors regulates ABA signalling. We report here the identification of a family of ABA-induced transcription repressors (AITRs) that act as feedback regulators in ABA signalling. We found that the expression of all the 6 Arabidopsis AITR genes was induced by exogenously ABA, and their expression levels were decreased in ABA biosynthesis mutant aba1-5. BLAST searches showed that AITRs are exclusively present in angiosperms. When recruited to the promoter region of a reporter gene by a fused DNA binding domain, all AITRs inhibited reporter gene expression in transfected protoplasts. In Arabidopsis, aitr mutants showed reduced sensitivity to ABA and to stresses such as salt and drought. Quantitative RT-PCR analysis demonstrated that the ABA-induced response of PP2C and some PYR/PYL/RCAR genes was reduced in AITR5 transgenic plants but increased in an aitr2 aitr5 aitr6 triple mutant. These results provide important new insights into the regulation of ABA signalling in plants, and such information may lead to the production of plants with enhanced resistance to environmental stresses., (© 2017 John Wiley & Sons Ltd.)

The native Putranjiva roxburghii family 1 glycoside hydrolase enzyme showed β-D-fucosidase activity in addition to β-D-glucosidase and β-D-galactosidase activities reported in our previous study. A single step concanvalin A affinity chromatography for native PRGH1 improved the yield and reduced the purification time. The PRGH1 gene was cloned and overexpressed in E. coli. The full length gene contained an ORF of 1617 bp encoding a polypeptide of 538 amino acids. The amino acid sequence of PRGH1 showed maximum similarities to β-glucosidases and myrosinases. Both native and recombinant protein showed maximum hydrolytic activity for pNP-Fuc followed by pNP-Glc and pNP-Gal. Significant enzyme activity was also observed for cellobiose, however it decreased with increase in chain-length for glycan substrates. The enzyme showed significant resistant to D-glucose concentration up to 500 mM. Mutational studies confirmed the predicted catalytic acid/base Glu173 and nucleophile Glu389 as key residues for its activity. Moreover, Glu446 and Asn172 played essential role in substrate binding by interacting with the -1 subsite of substrates. Bioinformatic analysis suggested the possible reasons for the broad substrate specificity and other properties of the enzyme. PRGH1 had high sequence similarity towards S-glucosidase and may be involved in defence. The broad specificity, catalytic efficiency and thermostability make PRGH1 potentially an important industrial enzyme., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

The early evolution of angiosperms was marked by a number of innovations of the reproductive cycle including an accelerated fertilization process involving faster transport of sperm to the egg via a pollen tube. Fast pollen tube growth rates in angiosperms are accompanied by a hard shank-soft tip pollen tube morphology. A critical actor in that morphology is the wall-embedded enzyme pectin methylesterase (PME), which in type II PMEs is accompanied by a co-transcribed inhibitor, PMEI. PMEs convert the esterified pectic tip wall to a stiffer state in the subapical flank by pectin de-esterification. It is hypothesized that rapid and precise targeting of PME activity was gained with the origin of type II genes, which are derived and have only expanded since the origin of vascular plants. Pollen-active PMEs have yet to be reported in early-divergent angiosperms or gymnosperms. Gene expression studies in Nymphaea odorata found transcripts from four type II VGD1-like and 16 type I AtPPME1-like homologs that were more abundant in pollen and pollen tubes than in vegetative tissues. The near full-length coding sequence of one type II PME (NoPMEII-1) included at least one PMEI domain. The identification of possible VGD1 homologs in an early-diverging angiosperm suggests that the refined control of PMEs that mediate de-esterification of pectins near pollen tube tips is a conserved feature across angiosperms. The recruitment of type II PMEs into a pollen tube elongation role in angiosperms may represent a key evolutionary step in the development of faster growing pollen tubes., (Copyright © 2017 Elsevier Inc. All rights reserved.)

The potential of alternative oxidase (AOX) genes to develop functional markers for plant breeding programs has been emphasized. In this sense, it is essential to have a reliable classification system, which could aid in the selection of candidate AOX genes from different species. In the case of angiosperms AOX, a robust classification system is required because this enzyme is encoded by variable gene numbers (1-6 genes) with variable AOX subfamilies and subtypes. Thus, in this protocol, we present a detailed guideline to application of a classification scheme of AOX based on specific amino acids and phylogeny. We believe that this classification protocol provides an easier and practical way of classifying new angiosperm AOX genes besides that it can help to standardize AOX gene names used in AOX research community.

Gene duplication could be beneficial by functional division but might increase the risk of genetic load. The dynamics of duplicated paralogs number could involve recombination, positive selection, and functional divergence. Duplication of DIHYDROFLAVONOL 4-REDUCTASE (DFR) has been reported in several organisms and may have been retained by escape from adaptive conflict (EAC). In this study, we screened the angiosperm DFR gene focusing on a diversified genus Scutellaria to investigate how these duplicated genes are retained. We deduced that gene duplication involved multiple independent events in angiosperms, but the duplication of DFR was before the divergence of Scutellaria. Asymmetric positive selective pressures resulted in different evolutionary rates between the duplicates. Different numbers of regulatory elements, differential codon usages, radical amino acid changes, and differential gene expressions provide evidences of functional divergence between the two DFR duplicates in Scutellaria, implying adaptive subfunctionalization between duplicates. The discovery of pseudogenes accompanying a reduced replacement rate in one DFR paralogous gene suggested possibly leading to "loss of function" due to dosage imbalance after the transient adaptive subfunctionalization in the early stage of duplication. Notwithstanding, episodic gene duplication and functional divergence may be relevant to the diversification of ecological function of DFR gene in Scutellaria.

This study determined whether phosphoenolpyruvate carboxykinase (PEPCK) and phosphoenolpyruvate carboxylase (PEPC) are phosphorylated in the flesh of a range of fruits. This was done by incubating fruit flesh with 32 P[P] (where 32 P[P] = 32 PO 4 3- ), then PEPCK and PEPC were immunoprecipitated from extracts using specific antisera. The incorporation of 32 P[P] into these enzymes was then determined by autoradiography of SDS-PAGE gels. Both enzymes were subject to phosphorylation in vivo in the flesh of grape, tomato, cherry and plum. PEPCK was also subject to phosphorylation in vivo in developing grape seeds. Proteolytic cleavage of PEPCK showed that it was phosphorylated at a site(s) located on its N-terminal extension. Potentially phosphorylation of these enzymes could contribute to the coordinate regulation of their activities in the flesh of fruits and in developing seeds., (Copyright © 2016 Elsevier Masson SAS. All rights reserved.)

The NADPH-dependent cytochrome P450 oxidoreductase (POR) is the obligate electron donor to eukaryotic microsomal cytochromes P450 enzymes. The number of PORs within plant species is limited to one to four isoforms, with the most common being two PORs per plant. These enzymes provide electrons to a huge number of different cytochromes P450s (from 50 to several hundred within one plant). Within the eudicotyledons, PORs can be divided into two major clades, POR 1 and POR 2. Based on our own sequencing analysis and publicly available data, we have identified 45 PORs from the angiosperm order Apiales. These were subjected to a phylogenetic analysis along with 237 other publicly available (NCBI and oneKP) POR sequences found within the clade Asterids. Here, we show that the order Apiales only harbor members of the POR 2 clade, which are further divided into two distinct subclades. This is in contrast to most other eudicotyledon orders that have both POR 1 and POR 2. This suggests that through gene duplications and one gene deletion, Apiales only contain members of the POR 2 clade. Three POR 2 isoforms from Thapsia garganica L., Apiaceae, were all full-length in an Illumina root transcriptome dataset (available from the SRA at NCBI). All three genes were shown to be functional upon reconstitution into nanodiscs, confirming that none of the isoforms are pseudogenes., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Genes for the plastid-encoded RNA polymerase (PEP) persist in the plastid genomes of all photosynthetic angiosperms. However, three unrelated lineages (Annonaceae, Passifloraceae and Geraniaceae) have been identified with unusually divergent open reading frames (ORFs) in the conserved region of rpoA, the gene encoding the PEP α subunit. We used sequence-based approaches to evaluate whether these genes retain function. Both gene sequences and complete plastid genome sequences were assembled and analyzed from each of the three angiosperm families. Multiple lines of evidence indicated that the rpoA sequences are likely functional despite retaining as low as 30% nucleotide sequence identity with rpoA genes from outgroups in the same angiosperm order. The ratio of non-synonymous to synonymous substitutions indicated that these genes are under purifying selection, and bioinformatic prediction of conserved domains indicated that functional domains are preserved. One of the lineages (Pelargonium, Geraniaceae) contains species with multiple rpoA-like ORFs that show evidence of ongoing inter-paralog gene conversion. The plastid genomes containing these divergent rpoA genes have experienced extensive structural rearrangement, including large expansions of the inverted repeat. We propose that illegitimate recombination, not positive selection, has driven the divergence of rpoA.

The pectin matrix of the angiosperm cell wall is regulated in both synthesis and modification and greatly influences the direction and extent of cell growth. Pathogens, herbivory and mechanical stresses all influence this pectin matrix and consequently plant form and function. The cell wall-associated kinases (WAKs) bind to pectin and regulate cell expansion or stress responses depending upon the state of the pectin. This review explores the WAKs in the context of cell wall biology and signal transduction pathways., (© The Author 2015. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Nepenthesins are aspartic proteases secreted by carnivorous pitcher plants of the genus Nepenthes. They significantly differ in sequence from other plant aspartic proteases. This difference, which provides more cysteine residues in the structure of nepenthesins, may contribute to their unique stability profile. Recombinantly produced nepenthesin 1 (rNep1) from N. gracilis in complex with pepstatin A was crystallized under two different crystallization conditions using a newly formulated low-pH crystallization screen. The diffraction data were processed to 2.9 and 2.8 Å resolution, respectively. The crystals belonged to space group P212121, with unit-cell parameters a = 86.63, b = 95.90, c = 105.40 Å, α = β = γ = 90° and a = 86.28, b = 97.22, c = 103.78 Å, α = β = γ = 90°, respectively. Matthews coefficient and solvent-content calculations suggest the presence of two molecules of rNep1 in the asymmetric unit. Here, the details of the crystallization experiment and analysis of the X-ray data are reported.

During the diversification of angiosperms, seeds have evolved structural, chemical, molecular and physiologically developing changes that specially affect the nucellus and endosperm. All through seed evolution, programmed cell death (PCD) has played a fundamental role. However, examples of PCD during seed development are limited. The present review examines PCD in integuments, nucellus, suspensor and endosperm in those representative examples of seeds studied to date., (© 2015 Institute of Botany, Chinese Academy of Sciences.)

Carotenoids are essential isoprenoid pigments produced by plants, algae, fungi and bacteria. Lycopene cyclase (LYC) commonly cyclize carotenoids, which is an important branching step in the carotenogenesis, at one or both end of the backbone. Plants have two types of LYC (β-LCY and ϵ-LCY). In this study, plant LYCs were analyzed. Based on domain analysis, all LYCs accommodate lycopene cyclase domain (Pf05834). Furthermore, motif analysis indicated that motifs were conserved among the plants. On the basis of phylogenetic analysis, β-LCYs and ϵ-LCYs were classified in β and ϵ groups. Monocot and dicot plants separated from each other in the phylogenetic tree. Subsequently, Oryza sativa Japonica Group and Zea mays of LYCs as monocot plants and Vitis vinifera and Solanum lycopersicum of LYCs as dicot plants were analyzed. According to nucleotide diversity analysis of β-LCY and ϵ-LCY genes, nucleotide diversities were found to be π: 0.30 and π: 0.25, respectively. The result highlighted β-LCY genes showed higher nucleotide diversity than ϵ-LCY genes. LYCs interacting genes and their co-expression partners were also predicted using String server. The obtained data suggested the importance of LYCs in carotenoid metabolism. 3D modeling revealed that depicted structures were similar in O. sativa, Z mays, S. lycopersicum, and V. vinifera β-LCYs and ϵ-LCYs. Likewise, the predicted binding sites were highly similar between O. sativa, Z mays, S. lycopersicum, and V. vinifera LCYs. Most importantly, analysis elucidated the V/IXGXGXXGXXXA motif for both type of LYC (β-LCY and ϵ-LCY). This motif related to Rossmann fold domain and probably provides a flat platform for binding of FAD in O. sativa, Z mays, S. lycopersicum, and V. vinifera β-LCYs and ϵ-LCYs with conserved structure. In addition to lycopene cyclase domain, the V/IXGXGXXGXXXA motif can be used for exploring LYCs proteins and to annotate the function of unknown proteins containing lycopene cyclase domain. Overall results indicated that a high degree of conserved signature were observed in plant LYCs., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

The RNA-directed DNA methylation (RdDM) pathway can be divided into three phases: 1) small interfering RNA biogenesis, 2) de novo methylation, and 3) chromatin modification. To determine the degree of conservation of this pathway we searched for key genes among land plants. We used OrthoMCL and the OrthoMCL Viridiplantae database to analyze proteomes of species in bryophytes, lycophytes, monilophytes, gymnosperms, and angiosperms. We also analyzed small RNA size categories and, in two gymnosperms, cytosine methylation in ribosomal DNA. Six proteins were restricted to angiosperms, these being NRPD4/NRPE4, RDM1, DMS3 (defective in meristem silencing 3), SHH1 (SAWADEE homeodomain homolog 1), KTF1, and SUVR2, although we failed to find the latter three proteins in Fritillaria persica, a species with a giant genome. Small RNAs of 24 nt in length were abundant only in angiosperms. Phylogenetic analyses of Dicer-like (DCL) proteins showed that DCL2 was restricted to seed plants, although it was absent in Gnetum gnemon and Welwitschia mirabilis. The data suggest that phases (1) and (2) of the RdDM pathway, described for model angiosperms, evolved with angiosperms. The absence of some features of RdDM in F. persica may be associated with its large genome. Phase (3) is probably the most conserved part of the pathway across land plants. DCL2, involved in virus defense and interaction with the canonical RdDM pathway to facilitate methylation of CHH, is absent outside seed plants. Its absence in G. gnemon, and W. mirabilis coupled with distinctive patterns of CHH methylation, suggest a secondary loss of DCL2 following the divergence of Gnetales., (© The Author(s) 2015. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.)

Histone modifications, such as methylation and demethylation, are crucial mechanisms altering chromatin structure and gene expression. Recent biochemical and molecular studies have uncovered a group of histone demethylases called Jumonji C (JmjC) domain proteins. However, their evolutionary history and patterns have not been examined systematically. Here, we report extensive analyses of eukaryotic JmjC genes and define 14 subfamilies, including the Lysine-Specific Demethylase3 (KDM3), KDM5, JMJD6, Putative-Lysine-Specific Demethylase11 (PKDM11), and PKDM13 subfamilies, shared by plants, animals, and fungi. Other subfamilies are detected in plants and animals but not in fungi (PKDM12) or in animals and fungi but not in plants (KDM2 and KDM4). PKDM7, PKDM8, and PKDM9 are plant-specific groups, whereas Jumonji, AT-Rich Interactive Domain2, KDM6, and PKDM10 are animal specific. In addition to known domains, most subfamilies have characteristic conserved amino acid motifs. Whole-genome duplication (WGD) was likely an important mechanism for JmjC duplications, with four pairs from an angiosperm-wide WGD and others from subsequent WGDs. Vertebrates also experienced JmjC duplications associated with the vertebrate ancestral WGDs, with additional mammalian paralogs from tandem duplication and possible transposition. The sequences of paralogs have diverged in both known functional domains and other regions, showing evidence of selection pressure. The increases of JmjC copy number and the divergences in sequence and expression might have contributed to the divergent functions of JmjC genes, allowing the angiosperms and vertebrates to adapt to a great number of ecological niches and contributing to their evolutionary successes., (© 2015 American Society of Plant Biologists. All Rights Reserved.)

Small RNA-mediated chromatin modification is a conserved feature of eukaryotes. In flowering plants, the short interfering (si)RNAs that direct transcriptional silencing are abundant and subfunctionalization has led to specialized machinery responsible for synthesis and action of these small RNAs. In particular, plants possess polymerase (Pol) IV and Pol V, multi-subunit homologs of the canonical DNA-dependent RNA Pol II, as well as specialized members of the RNA-dependent RNA Polymerase (RDR), Dicer-like (DCL), and Argonaute (AGO) families. Together these enzymes are required for production and activity of Pol IV-dependent (p4-)siRNAs, which trigger RNA-directed DNA methylation (RdDM) at homologous sequences. p4-siRNAs accumulate highly in developing endosperm, a specialized tissue found only in flowering plants, and are rare in nonflowering plants, suggesting that the evolution of flowers might coincide with the emergence of specialized RdDM machinery. Through comprehensive identification of RdDM genes from species representing the breadth of the land plant phylogeny, we describe the ancient origin of Pol IV and Pol V, suggesting that a nearly complete and functional RdDM pathway could have existed in the earliest land plants. We also uncover innovations in these enzymes that are coincident with the emergence of seed plants and flowering plants, and recent duplications that might indicate additional subfunctionalization. Phylogenetic analysis reveals rapid evolution of Pol IV and Pol V subunits relative to their Pol II counterparts and suggests that duplicates were retained and subfunctionalized through Escape from Adaptive Conflict. Evolution within the carboxy-terminal domain of the Pol V largest subunit is particularly striking, where illegitimate recombination facilitated extreme sequence divergence., (© The Author 2015. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.)

Carnivorous plants use different morphological features to attract, trap and digest prey, mainly insects. Plants from the genus Nepenthes possess specialized leaves called pitchers that function as pitfall-traps. These pitchers are filled with a digestive fluid that is generated by the plants themselves. In order to digest caught prey in their pitchers, Nepenthes plants produce various hydrolytic enzymes including aspartic proteases, nepenthesins (Nep). Knowledge about the generation and induction of these proteases is limited. Here, by employing a FRET (fluorescent resonance energy transfer)-based technique that uses a synthetic fluorescent substrate an easy and rapid detection of protease activities in the digestive fluids of various Nepenthes species was feasible. Biochemical studies and the heterologously expressed Nep II from Nepenthes mirabilis proved that the proteolytic activity relied on aspartic proteases, however an acid-mediated auto-activation mechanism was necessary. Employing the FRET-based approach, the induction and dynamics of nepenthesin in the digestive pitcher fluid of various Nepenthes plants could be studied directly with insect (Drosophila melanogaster) prey or plant material. Moreover, we observed that proteolytic activity was induced by the phytohormone jasmonic acid but not by salicylic acid suggesting that jasmonate-dependent signaling pathways are involved in plant carnivory.

The alternative oxidase (AOX) is an integral monotopic membrane protein located on the inner surface of the inner mitochondrial membrane. Branching from the traditional respiratory chain at the quinone pool, AOX is responsible for cyanide-resistant respiration in plants and fungi, heat generation in thermogenic plants, and survival of parasites, such as Trypanosoma brucei, in the human host. A recently solved AOX structure provides insight into its active site, thereby facilitating rational phytopathogenic and antiparasitic drug design. Here, we describe expression of recombinant AOX using two different expression systems. Purification protocols for the production of highly pure and stable AOX protein in sufficient quantities to facilitate further kinetic, biophysical, and structural analyses are also described.

A classification scheme based on protein phylogenies and sequence harmony method was used to clarify the taxonomic distribution and evolutionary history of the alternative oxidase (AOX) in angiosperms. A large data set analyses showed that AOX1 and AOX2 subfamilies were distributed into 4 phylogenetic clades: AOX1a-c/1e, AOX1d, AOX2a-c and AOX2d. High diversity in AOX family compositions was found. While the AOX2 subfamily was not detected in monocots, the AOX1 subfamily has expanded (AOX1a-e) in the large majority of these plants. In addition, Poales AOX1b and 1d were orthologous to eudicots AOX1d and then renamed as AOX1d1 and 1d2. AOX1 or AOX2 losses were detected in some eudicot plants. Several AOX2 duplications (AOX2a-c) were identified in eudicot species, mainly in the asterids. The AOX2b originally identified in eudicots in the Fabales order (soybean, cowpea) was divergent from AOX2a-c showing some specific amino acids with AOX1d and then it was renamed as AOX2d. AOX1d and AOX2d seem to be stress-responsive, facultative and mutually exclusive among species suggesting a complementary role with an AOX1(a) in stress conditions. Based on the data collected, we present a model for the evolutionary history of AOX in angiosperms and highlight specific areas where further research would be most beneficial., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Juncus acutus arises as possible hyperaccumulator specie, tolerating exogenous Zn concentrations as high as 60 mM. Zinc concentrations here detected in seedlings germinated in the presence high Zn concentrations, were above the described upper toxic levels for higher plants. Even at the highest Zn concentration, growth inhibition only accounted to approximately 30% of control seedlings biomass, presenting an EC₅₀ value in the range of 10-20 mM of metal. PSII quantum yields showed a marked decline, reflection of changes in the thylakoid structure on the PSII electron donor sites. In fact, the electron transport rate was severely affected by Zn in seedlings exposed to higher Zn concentrations leading to a decrease in their maximum electronic transport rate and consequently presenting lower light saturation and lower photosynthetic efficiencies. Although light absorption capacity was not affected by Zn exposure and uptake, energy trapping flux in the photosynthetic apparatus and transport throughout the electronic chain was severely impaired. This lack of efficiency is related with non-functional Zn-chlorophylls formation. There was a strong linear correlation between exogenous Zn concentration applied and the concentration actually verified in the seedlings tissue with the concentration of both ZnChl a and b. There was also a gradual loss of connectivity between the antennae of the PSII units being this more evident at the higher Zn concentrations and thus impairing the energetic transport. The reduction in light harvesting efficiency, leads inevitably to the accumulation of redox energy inside the cells. To counteract ROS generation, all anti-oxidant enzymatic activities (except catalase) showed a proportional response to exogenous and in vivo Zn concentrations. Not only this plant appears to be highly tolerant to high Zn concentrations, but also it can overcome efficiently the damage produced during this uptake by efficiently dissipating the excessive cellular redox potential accumulated, essentially due to Zn incorporation into the chlorophyll molecule., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Allopolyploidization in plants entails the merger of two divergent nuclear genomes, typically with only one set (usually maternal) of parental plastidial and mitochondrial genomes and with an altered cytonuclear stoichiometry. Thus, we might expect cytonuclear coevolution to be an important dimension of allopolyploid evolution. Here, we investigate cytonuclear coordination for the key chloroplast protein rubisco (ribulose 1,5-bisphosphate carboxylase/oxygenase), which is composed of nuclear-encoded, small subunits (SSUs) and plastid-encoded, large subunits. By studying gene composition and diversity as well as gene expression in four model allopolyploid lineages, Arabidopsis, Arachis, Brassica, and Nicotiana, we demonstrate that paralogous nuclear-encoded rbcS genes within diploids are subject to homogenization via gene conversion and that such concerted evolution via gene conversion characterizes duplicated genes (homoeologs) at the polyploid level. Many gene conversions in the polyploids are intergenomic with respect to the diploid progenitor genomes, occur in functional domains of the homoeologous SSUs, and are directionally biased, such that the maternal amino acid states are favored. This consistent preferential maternal-to-paternal gene conversion is mirrored at the transcriptional level, with a uniform transcriptional bias of the maternal-like rbcS homoeologs. These data, repeated among multiple diverse angiosperm genera for an important photosynthetic enzyme, suggest that cytonuclear coevolution may be mediated by intergenomic gene conversion and altered transcription of duplicated, now homoeologous nuclear genes., (© The Author 2014. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.)

A surface- and vertical subsurface-flow-constructed wetland were designed to study the response of chlorophyll and antioxidant enzymes to elevated UV radiation in three types of wetland plants (Canna indica, Phragmites austrail, and Typha augustifolia). Results showed that (1) chlorophyll content of C. indica, P. austrail, and T. augustifolia in the constructed wetland was significantly lower where UV radiation was increased by 10 and 20 % above ambient solar level than in treatment with ambient solar UV radiation (p < 0.05). (2) The malondialdehyde (MDA) content, guaiacol peroxidase (POD), superoxide dismutase (SOD), and catalase (CAT) activities of wetland plants increased with elevated UV radiation intensity. (3) The increased rate of MDA, SOD, POD, and CAT activities of C. indica, P. australis, and T. angustifolia by elevated UV radiation of 10 % was higher in vertical subsurface-flow-constructed wetland than in surface-flow-constructed wetland. The sensitivity of MDA, SOD, POD, and CAT activities of C. indica, P. austrail, and T. augustifolia to the elevated UV radiation was lower in surface-flow-constructed wetland than in the vertical subsurface-flow-constructed wetland, which was related to a reduction in UV radiation intensity through the dissolved organic carbon and suspended matter in the water. C. indica had the highest SOD and POD activities, which implied it is more sensitive to enhanced UV radiation. Therefore, different wetland plants had different antioxidant enzymes by elevated UV radiation, which were more sensitive in vertical subsurface-flow-constructed wetland than in surface-flow-constructed wetland.

Although the S-like ribonucleases (RNases) share sequence homology with the S-RNases involved in the self-incompatibility mechanism in plants, they are not associated with this mechanism. They usually function in stress responses in non-carnivorous plants and in carnivory in carnivorous plants. In this study, we clarified the structures of the S-like RNases of Aldrovanda vesiculosa, Nepenthes bicalcarata and Sarracenia leucophylla, and compared them with those of other plants. At ten positions, amino acid residues are conserved or almost conserved only for carnivorous plants (six in total). In contrast, two positions are specific to non-carnivorous plants. A phylogenetic analysis revealed that the S-like RNases of the carnivorous plants form a group beyond the phylogenetic relationships of the plants. We also prepared and characterized recombinant S-like RNases of Dionaea muscipula, Cephalotus follicularis, A. vesiculosa, N. bicalcarata and S. leucophylla, and RNS1 of Arabidopsis thaliana. The recombinant carnivorous plant enzymes showed optimum activities at about pH 4.0. Generally, poly(C) was digested less efficiently than poly(A), poly(I) and poly(U). The kinetic parameters of the recombinant D. muscipula enzyme (DM-I) and A. thaliana enzyme RNS1 were similar. The k cat/K m of recombinant RNS1 was the highest among the enzymes, followed closely by that of recombinant DM-I. On the other hand, the k cat/K m of the recombinant S. leucophylla enzyme was the lowest, and was ~1/30 of that for recombinant RNS1. The magnitudes of the k cat/K m values or k cat values for carnivorous plant S-like RNases seem to correlate negatively with the dependency on symbionts for prey digestion.