Genome-Wide Analysis of the UDP-Glycosyltransferase Family Reveals Its Roles in Coumarin Biosynthesis and Abiotic Stress in Melilotus albus

Coumarins, natural products abundant in Melilotus albus, confer features in response to abiotic stresses, and are mainly present as glycoconjugates. UGTs (UDP-glycosyltransferases) are responsible for glycosylation modification of coumarins. However, information regarding the relationship between coumarin biosynthesis and stress-responsive UGTs remains limited. Here, a total of 189 MaUGT genes were identified from the M. albus genome, which were distributed differentially among its eight chromosomes. According to the phylogenetic relationship, MaUGTs can be classified into 13 major groups. Sixteen MaUGT genes were differentially expressed between genotypes of Ma46 (low coumarin content) and Ma49 (high coumarin content), suggesting that these genes are likely involved in coumarin biosynthesis. About 73.55% and 66.67% of the MaUGT genes were differentially expressed under ABA or abiotic stress in the shoots and roots, respectively. Furthermore, the functions of MaUGT68 and MaUGT186, which were upregulated under stress and potentially involved in coumarin glycosylation, were characterized by heterologous expression in yeast and Escherichia coli. These results extend our knowledge of the UGT gene family along with MaUGT gene functions, and provide valuable findings for future studies on developmental regulation and comprehensive data on UGT genes in M. albus.

ProXs drive the formation of membraneless organelles in plants

Membraneless organelles (MLOs) are non-membranous structures inside cells that organize cellular space and processes. The recent discovery that MLOs can be assembled via liquid-liquid phase separation (LLPS) advanced our understanding of these structures. However, the proteins that are capable of forming MLOs are largely unknown, especially in plants. In this study, we developed a method to identify proteins that we referred as ProXs (Proteins enriched by b-isoX) in Arabidopsis. Heterologous expression in yeast cells showed that most ProXs were capable of forming MLOs autonomously. We applied this method to several model and crop species including early and higher plants. This allowed us to generate an atlas of ProXs for studying plant MLOs. Analysis of ProXs from different species revealed high degree of conservation, supporting that they play important roles in cellular functions and are positively selected during evolution. Our method will be a valuable tool to characterize novel MLOs from desired cells and the data generated in present study will be instrumental for the plant research community to investigate MLO biology.

Genome-wide Identification and Expression Analysis of NRAMP Transporter Genes in Cucumis sativus and Citrullus lanatus

Natural resistance-associated macrophage proteins (NRAMPs) are able to transport various metal ions across cell membranes, which play an important role in plant normal growth and development. Here, a survey of cucumber (Cucumis sativus) and watermelon (Citrullus lanatus) genomes found a total of five CsNRAMPs and four ClNRAMPs, respectively. Based on the phylogenetic relationships, CsNRAMPs and ClNRAMPs were clustered into three groups (I, II and III). Five orthologous pairs were identified between cucumber and watermelon genome, and they were clustered on the same branch of the phylogenetic tree. The number of introns in CsNRAMPs and ClNRAMPs ranged from 3 to 13 and the genes from group Ι were more fragmented than those in group II. Subsequently, analysis of promoter sequences found that five putative transcription factors could act on NRAMPs. Moreover, CsNRAMPs and ClNRAMPs were differentially regulated by deficiencies of Fe, Mn, Cu or Zn, along with toxicities of Fe, Mn, Cu, Zn or Cd. Functional analysis by heterologous expression in yeast indicated that CsNRAMP4 and ClNRAMP3 participate in Cd transport. Overall, the comprehensive analysis of CsNRAMPs and ClNRAMPs reported herein may pave the way for further investigations examining the regulation and functions of this gene family in cucumber and watermelon.

Palmitvaccenic acid (Δ11-cis-hexadecenoic acid) is synthesized by an OLE1-like desaturase in the arbuscular mycorrhiza fungus Rhizophagus irregularis

Arbuscular mycorrhiza (AM) fungi deliver mineral nutrients to the plant host in exchange for reduced carbon in the form of sugars and lipids. Colonization with AM fungi upregulates a specific host lipid synthesis pathway resulting in the production of fatty acids. The fungus Rhizophagus irregularis accumulates predominantly palmitic acid (16:0) and the unusual palmitvaccenic acid (16:1Δ11cis). Here, we present the isolation and characterization of RiOLE1-LIKE, the desaturase involved in palmitvaccenic acid synthesis, by heterologous expression in yeast and plants. Results are in line with the scenario that RiOLE1-LIKE encodes an acyl-CoA desaturase with substrate specificity for C15-C18 acyl groups, in particular C16. Phylogenetic analysis of RiOLE1-LIKE related sequences revealed that this gene is conserved in AM fungi from the Glomales and Diversisporales, but is absent from non-symbiotic Mortierellaceae and Mucoromycotina fungi, suggesting that 16:1Δ11cis provides a specific function during AM colonization.

Cloning and Characterization of Oxidosqualene Cyclases Involved in Taraxasterol, Taraxerol and Bauerenol Triterpene Biosynthesis in Taraxacum coreanum

Triterpenes, consisting of six isoprene units, are one of the largest classes of natural compounds in plants. The genus Taraxacum is in the family Asteraceae and is widely distributed in the Northern Hemisphere. Various triterpenes, especially taraxerol and taraxasterol, are present in Taraxacum plants. Triterpene biosynthesis occurs through the action of oxidosqualene cyclase (OSC), which generates various types of triterpenes from 2,3-oxidosqualene after the rearrangement of the triterpene skeleton. However, no functional characterization of the OSC genes involved in triterpene biosynthesis, except for a lupeol synthase in Taraxacum officinale, has been performed. Taraxacum coreanum, or Korean dandelion, grows in Korea and China. Putative OSC genes in T. coreanum plants were isolated by transcriptome analysis, and four of these (TcOSC1, TcOSC2, TcOSC3 and TcOSC4) were functionally characterized by heterologous expression in yeast. Both TcOSC1 and TcOSC2 were closely related to dammarenediol-II synthases. TcOSC3 and TcOSC4 were strongly grouped with β-amyrin synthases. Functional analysis revealed that TcOSC1 produced several triterpenes, including taraxasterol; Ψ-taraxasterol; α-, β- and δ-amyrin; and dammarenediol-II. TcOSC2 catalyzed the production of bauerenol and another unknown triterpene, TcOSC3 catalyzed the production of β-amyrin. TcOSC4 catalyzed the production of taraxerol. Moreover, we identified taraxasterol, ψ-taraxasterol, taraxerol, lupeol, δ-amyrin, α-amyrin, β-amyrin and bauerenol in the roots and leaves of T. coreanum. Our results suggest that TcOSC1, TcOSC2, TcOSC3 and TcOSC4 are key triterpene biosynthetic enzymes in T. coreanum. These enzymes are novel triterpene synthases involved in the production of taraxasterol, bauerenol and taraxerol.

The Plasmodium falciparum Ca2+-ATPase PfATP6: insensitive to artemisinin, but a potential drug target

The disease malaria, caused by the parasite Plasmodium falciparum, remains one of the most important causes of morbidity and mortality in sub-Saharan Africa. In the absence of an efficient vaccine, the medical treatment of malaria is dependent on the use of drugs. Since artemisinin is a powerful anti-malarial drug which has been proposed to target a particular Ca2+-ATPase (PfATP6) in the parasite, it has been important to characterize the molecular properties of this enzyme. PfATP6 is a 139 kDa protein composed of 1228 amino acids with a 39% overall identity with rabbit SERCA1a (sarcoplasmic/endoplasmic reticulum Ca2+-ATPase 1a). PfATP6 conserves all sequences and motifs that are important for the function and/or structure of a SERCA, such as two high-affinity Ca2+-binding sites, a nucleotide-binding site and a phosphorylation site. We have been successful in isolating PfATP6 after heterologous expression in yeast and affinity chromatography in a pure, active and stable detergent-solubilized form. With this preparation, we have characterized and compared with the eukaryotic SERCA1a isoform the substrate (Ca2+ and ATP) -dependency for PfATP6 activity as well as the specific inhibition/interaction of the protein with drugs. Our data fully confirm that PfATP6 is a SERCA, but with a distinct pharmacological profile: compared with SERCA1a, it has a lower affinity for thapsigargin and much higher affinity for cyclopiazonic acid. On the other hand, we were not able to demonstrate any inhibition by artemisinin and were also not able to monitor any binding of the drug to the isolated enzyme. Thus it is unlikely that PfATP6 plays an important role as a target for artemisinin in the parasite P. falciparum.

Validation of a Candidate Deoxynivalenol-Inactivating UDP-Glucosyltransferase from Barley by Heterologous Expression in Yeast

Resistance to the virulence factor deoxynivalenol (DON) due to formation of DON-3-O-glucoside (D3G) is considered to be an important component of resistance against Fusarium spp. which produce this toxin. Multiple candidate UDP-glycosyltransferase (UGT) genes from different crop plants that are either induced by Fusarium spp. or differentially expressed in cultivars varying in Fusarium disease resistance have been described. However, UGT are encoded by a very large gene family in plants. The study of candidate plant UGT is highly warranted because of the potential relevance for developing Fusarium-spp.-resistant crops. We tested Arabidopsis thaliana genes closely related to a previously identified DON-glucosyltransferase gene by heterologous expression in yeast and showed that gene products with very high sequence similarity can have pronounced differences in detoxification capabilities. We also tested four candidate barley glucosyltransferases, which are highly DON inducible. Upon heterologous expression of full-length cDNAs, only one gene, HvUGT13248, conferred DON resistance. The conjugate D3G accumulated in the supernatant of DON-treated yeast transformants. We also present evidence that the product of the TaUGT3 gene recently proposed to encode a DON-detoxification enzyme of wheat does not protect yeast against DON.