The apiosyltransferase celery UGT94AX1 catalyzes the biosynthesis of the flavone glycoside apiin.

Apiose is a unique branched-chain pentose found in plant glycosides and a key component of the cell wall polysaccharide pectin and other specialized metabolites. More than 1,200 plant-specialized metabolites contain apiose residues, represented by apiin, a distinctive flavone glycoside found in celery (Apium graveolens) and parsley (Petroselinum crispum) in the family Apiaceae. The physiological functions of apiin remain obscure, partly due to our lack of knowledge on apiosyltransferase during apiin biosynthesis. Here, we identified UGT94AX1 as an A. graveolens apiosyltransferase (AgApiT) responsible for catalyzing the last sugar modification step in apiin biosynthesis. AgApiT showed strict substrate specificity for the sugar donor, UDP-apiose, and moderate specificity for acceptor substrates, thereby producing various apiose-containing flavone glycosides in celery. Homology modeling of AgApiT with UDP-apiose, followed by site-directed mutagenesis experiments, identified unique Ile139, Phe140, and Leu356 residues in AgApiT, which are seemingly crucial for the recognition of UDP-apiose in the sugar donor pocket. Sequence comparison and molecular phylogenetic analysis of celery glycosyltransferases suggested that AgApiT is the sole apiosyltransferase-encoding gene in the celery genome. Identification of this plant apiosyltransferase gene will enhance our understanding of the physioecological functions of apiose and apiose-containing compounds.

FLOURY ENDOSPERM 6 mutations enhance the sugary phenotype caused by the loss of ISOAMYLASE1 in barley.

KEY MESSAGE: Barley double mutants in two genes involved in starch granule morphology, HvFLO6 and HvISA1, had impaired starch accumulation and higher grain sugar levels than either single mutant. Starch is a biologically and commercially important glucose polymer synthesized by plants as semicrystalline starch granules (SGs). Because SG morphology affects starch properties, mutants with altered SG morphology may be useful in breeding crops with desirable starch properties, including potentially novel properties. In this study, we employed a simple screen for mutants with altered SG morphology in barley (Hordeum vulgare). We isolated mutants that formed compound SGs together with the normal simple SGs in the endosperm and found that they were allelic mutants of the starch biosynthesis genes ISOAMYLASE1 (HvISA1) and FLOURY ENDOSPERM 6 (HvFLO6), encoding starch debranching enzyme and CARBOHYDRATE-BINDING MODULE 48-containing protein, respectively. We generated the hvflo6 hvisa1 double mutant and showed that it had significantly reduced starch biosynthesis and developed shrunken grains. In contrast to starch, soluble α-glucan, phytoglycogen, and sugars accumulated to higher levels in the double mutant than in the single mutants. In addition, the double mutants showed defects in SG morphology in the endosperm and in the pollen. This novel genetic interaction suggests that hvflo6 acts as an enhancer of the sugary phenotype caused by hvisa1 mutation.

Biochemical Characterization of Parsley Glycosyltransferases Involved in the Biosynthesis of a Flavonoid Glycoside, Apiin.

The flavonoid glycoside apiin (apigenin 7-O-[ß-D-apiosyl-(1→2)-ß-D-glucoside]) is abundant in apiaceous and asteraceous plants, including celery and parsley. Although several enzymes involved in apiin biosynthesis have been identified in celery, many of the enzymes in parsley (Petroselinum crispum) have not been identified. In this study, we identified parsley genes encoding the glucosyltransferase, PcGlcT, and the apiosyltransferase, PcApiT, that catalyze the glycosylation steps of apiin biosynthesis. Their substrate specificities showed that they were involved in the biosynthesis of some flavonoid 7-O-apiosylglucosides, including apiin. The expression profiles of PcGlcT and PcApiT were closely correlated with the accumulation of flavonoid 7-O-apiosylglucosides in parsley organs and developmental stages. These findings support the idea that PcGlcT and PcApiT are involved in the biosynthesis of flavonoid 7-O-apiosylglucosides in parsley. The identification of these genes will elucidate the physiological significance of apiin and the development of apiin production methods.

Structural features of free N-glycans in α1,3/4-fucosidase-deficient Arabidopsis thaliana: deletion of α1,3/4-fucosidase activity induced accumulation of plant complex type GN1 free N-glycans.

Deletion of α-1,3/4-fucosidase activity in Arabidopsis thaliana resulted in the accumulation of GN1-type free N-glycans with the Lewis a epitope (GN1-FNG). This suggests that the release of α-fucose residue(s) may trigger rapid degradation of the plant complex-type (PCT) GN1-FNG. The fact that PCT-GN1-FNG has rarely been detected to date is probably due to its easier degradation compared with PCT-GN2-FNG.

Degradation pathway of plant complex-type N-glycans: identification and characterization of a key α1,3-fucosidase from glycoside hydrolase family 29.

Plant complex-type N-glycans are characterized by the presence of α1,3-linked fucose towards the proximal N-acetylglucosamine residue and ß1,2-linked xylose towards the ß-mannose residue. These glycans are ultimately degraded by the activity of several glycoside hydrolases. However, the degradation pathway of plant complex-type N-glycans has not been entirely elucidated because the gene encoding α1,3-fucosidase, a glycoside hydrolase acting on plant complex-type N-glycans, has not yet been identified, and its substrate specificity remains to be determined. In the present study, we found that AtFUC1 (an Arabidopsis GH29 α-fucosidase) is an α1,3-fucosidase acting on plant complex-type N-glycans. This fucosidase has been known to act on α1,4-fucoside linkage in the Lewis A epitope of plant complex-type N-glycans. We found that this glycoside hydrolase specifically acted on GlcNAcß1-4(Fucα1-3)GlcNAc, a degradation product of plant complex-type N-glycans, by sequential actions of vacuolar α-mannosidase, ß1,2-xylosidase, and endo-ß-mannosidase. The AtFUC1-deficient mutant showed no distinct phenotypic plant growth features; however, it accumulated GlcNAcß1-4(Fucα1-3)GlcNAc, a substrate of AtFUC1. These results showed that AtFUC1 is an α1,3-fucosidase acting on plant complex-type N-glycans and elucidated the degradation pathway of plant complex-type N-glycans.

Biochemical characterization of rhamnosyltransferase involved in biosynthesis of pectic rhamnogalacturonan I in plant cell wall.

The pectin in plant cell walls consists of three domains: homogalacturonan, rhamnogalacturonan (RG)-I, and RG-II. It is predicted that around 50 different glycosyltransferases are required for their biosynthesis. Among these, the activities of only a few glycosyltransferases have been detected because pectic oligosaccharides are not readily available for use as substrates. In this study, fluorogenic pyridylaminated RG-I-backbone oligosaccharides (PA-RGs) with 3-14 degrees of polymerization (DP) were prepared. Using these oligosaccharides, the activity of RG-I:rhamnosyltransferase (RRT), involved in the biosynthesis of the RG-I backbone diglycosyl repeating units (-4GalUAα1-2Rhaα1-), was detected from the microsomes of azuki bean epicotyls. RRT was found to prefer longer acceptor substrates, PA-RGs with a DP > 7, and it does not require any metal ions for its activity. RRT is located in the Golgi and endoplasmic reticulum. The activity of RRT coincided with epicotyl growth, suggesting that RG-I biosynthesis is involved in plant growth.

Semisynthesis of a post-translationally modified protein by using chemical cleavage and activation of an expressed fusion polypeptide.

Herein, we describe a new semisynthetic strategy of a post-translationally modified protein in which the middle region is glycosylated. We designed a single-plasmid coding for a fusion polypeptide, which can provide both an N-terminal α-thioester and a C-terminal cysteine peptide of a target glycoprotein by using chemical-cleavage and activation methods. The use of these resultant peptide derivatives resulted in the successful synthesis of N-glycosylated-interleukin 13.

Expression of an endo-rhamnogalacturonase from Aspergillus aculeatus enhances release of Arabidopsis transparent mucilage.

Mucilage is a gelatinous and sticky hydrophilic polysaccharide released from epidermal cells of seed coat after the hydration of mature seeds and is composed primarily of unbranched rhamnogalacturonan I (RG-I). In this study, we produced a recombinant endo-RG-I hydrolase from Aspergillus aculeatus (AaRhgA) in the fission yeast Schizosaccharomyces pombe and examined its substrate preference for pyridylaminated (PA) RG-I with the various degrees of polymerization (DP). Recombinant AaRhgA requires PA-RG-I with a DP of 10 or higher for its hydrolase activity. We heterologously expressed the AarhgA gene under the strong constitutive promoter, cauliflower mosaic virus 35S promoter, in Arabidopsis thaliana. In a series of biochemical analyses of each mucilage fraction released from the water-imbibed seeds of the transgenic plants, we found the enhanced deposition of the transparent mucilage layer that existed in the peripheral regions of the adherent mucilage and was not stained with ruthenium red. This study demonstrated the feasibility of manipulating the mucilage organization by heterologous expression of the endo-RG-I hydrolase.

Lanthanum Supplementation Alleviates Tomato Root Growth Suppression under Low Light Stress.

Supplementation with rare earth elements (REEs) such as lanthanum and cerium has been shown to promote plant elongation and/or increase crop yields. On the other hand, there are reports that REE supplementation of plants has no such effect. The appropriate modes for REE utilization and the underlying mechanism are not fully understood. In this study, we investigated how REE supplementation of plants under low light stress affects plant growth and gene expression. Under low light stress conditions, tomato root elongation was observed to be reduced by about half. This suppression of root elongation was found to be considerably alleviated by 20 mM lanthanum ion supplementation. This effect was plant-species-dependent and nutrient-condition-dependent. Under low light stress, the expression of the genes for phytochrome-interacting factor, which induces auxin synthesis, and several auxin-synthesis-related proteins were markedly upregulated by lanthanum ion supplementation. Thus, we speculate that REE supplementation of plants results in auxin-induced cell elongation and alleviates growth suppression under stress conditions.

Cobtorin target analysis reveals that pectin functions in the deposition of cellulose microfibrils in parallel with cortical microtubules.

Cellulose and pectin are major components of primary cell walls in plants, and it is believed that their mechanical properties are important for cell morphogenesis. It has been hypothesized that cortical microtubules guide the movement of cellulose microfibril synthase in a direction parallel with the microtubules, but the mechanism by which this alignment occurs remains unclear. We have previously identified cobtorin as an inhibitor that perturbs the parallel relationship between cortical microtubules and nascent cellulose microfibrils. In this study, we searched for the protein target of cobtorin, and we found that overexpression of pectin methylesterase and polygalacturonase suppressed the cobtorin-induced cell-swelling phenotype. Furthermore, treatment with polygalacturonase restored the deposition of cellulose microfibrils in the direction parallel with cortical microtubules, and cobtorin perturbed the distribution of methylated pectin. These results suggest that control over the properties of pectin is important for the deposition of cellulose microfibrils and/or the maintenance of their orientation parallel with the cortical microtubules.

Effects of L-arginine on solubilization and purification of plant membrane proteins.

Biochemical analysis of membrane proteins is problematic at the level of solubilization and/or purification because of their hydrophobic nature. Here, we developed methods for efficient solubilization and purification of membrane proteins using L-arginine. The addition of 100 mM of basic amino acids (L-arginine, L-lysine, and L-ornithine) to a detergent-containing solubilization buffer enhanced solubilization (by 2.6-4.3 fold) of a model membrane protein-polygalacturonic acid synthase. Of all the amino acids, arginine was the most effective additive for solubilization of this membrane protein. Arginine addition also resulted in the best solubilization of other plant membrane proteins. Next, we examined the effects of arginine on purification of a model membrane protein. In anion-exchange chromatography, the addition of arginine to the loading and elution buffers resulted in a greater recovery of a membrane protein. In ultrafiltration, the addition of arginine to a protein solution significantly improved the recovery of a membrane protein. These results were thought to be due to the properties of arginine that prevent aggregation of hydrophobic proteins. Taken together, the results of our study showed that arginine is useful for solubilization and purification of aggregate-prone membrane proteins.

Wolfberry genomes and the evolution of Lycium (Solanaceae).

Wolfberry Lycium, an economically important genus of the Solanaceae family, contains approximately 80 species and shows a fragmented distribution pattern among the Northern and Southern Hemispheres. Although several herbaceous species of Solanaceae have been subjected to genome sequencing, thus far, no genome sequences of woody representatives have been available. Here, we sequenced the genomes of 13 perennial woody species of Lycium, with a focus on Lycium barbarum. Integration with other genomes provides clear evidence supporting a whole-genome triplication (WGT) event shared by all hitherto sequenced solanaceous plants, which occurred shortly after the divergence of Solanaceae and Convolvulaceae. We identified new gene families and gene family expansions and contractions that first appeared in Solanaceae. Based on the identification of self-incompatibility related-gene families, we inferred that hybridization hotspots are enriched for genes that might be functioning in gametophytic self-incompatibility pathways in wolfberry. Extremely low expression of LOCULE NUBER (LC) and COLORLESS NON-RIPENING (CNR) orthologous genes during Lycium fruit development and ripening processes suggests functional diversification of these two genes between Lycium and tomato. The existence of additional flowering locus C-like MADS-box genes might correlate with the perennial flowering cycle of Lycium. Differential gene expression involved in the lignin biosynthetic pathway between Lycium and tomato likely illustrates woody and herbaceous differentiation. We also provide evidence that Lycium migrated from Africa into Asia, and subsequently from Asia into North America. Our results provide functional insights into Solanaceae origins, evolution and diversification.

UDP-glucose pyrophosphorylase is rate limiting in vegetative and reproductive phases in Arabidopsis thaliana.

UDP-glucose pyrophosphorylase (UGPase) is an important enzyme in the metabolism of UDP-glucose, a precursor for the synthesis of carbohydrate cell wall components, such as cellulose and callose. The Arabidopsis thaliana genome contains two putative genes encoding UGPase, AtUGP1 and AtUGP2. These genes are expressed in all organs. In order to determine the role of UGPase in vegetative and reproductive organs, we employed a reverse genetic approach using the T-DNA insertion mutants, atugp1 and atugp2. Despite a significant decrease in UGPase activity in both the atugp1 and atugp2 single mutants, no decrease in normal growth and reproduction was observed. In contrast, the atugp1/atugp2 double mutant displayed drastic growth defects and male sterility. At the reproductive phase, in the anthers of atugp1/atugp2, pollen mother cells developed normally, but callose deposition around microspores was absent. Genes coding for enzymes at the subsequent steps in the cellulose and callose synthesis pathway were also down-regulated in the double mutant. Taken together, these results demonstrate that the AtUGP1 and AtUGP2 genes are functionally redundant and UGPase activity is essential for both vegetative and reproductive phases in Arabidopsis. Importantly, male fertility was not restored in the double knockout mutant by an application of external sucrose, whereas vegetative growth was comparable in size with that of the wild type. In contrast, an application of external UDP-glucose recovered male fertility in the double mutant, suggesting that control of UGPase in carbohydrate metabolism is different in the vegetative phase as compared with the reproductive phase in A. thaliana.

Rice expression atlas in reproductive development.

Gene expression throughout the reproductive process in rice (Oryza sativa) beginning with primordia development through pollination/fertilization to zygote formation was analyzed. We analyzed 25 stages/organs of rice reproductive development including early microsporogenesis stages with 57,381 probe sets, and identified around 26,000 expressed probe sets in each stage. Fine dissection of 25 reproductive stages/organs combined with detailed microarray profiling revealed dramatic, coordinated and finely tuned changes in gene expression. A decrease in expressed genes in the pollen maturation process was observed in a similar way with Arabidopsis and maize. An almost equal number of ab initio predicted genes and cloned genes which appeared or disappeared coordinated with developmental stage progression. A large number of organ-/stage-specific genes were identified; notably 2,593 probe sets for developing anther, including 932 probe sets corresponding to ab initio predicted genes. Analysis of cell cycle-related genes revealed that several cyclin-dependent kinases (CDKs), cyclins and components of SCF E3 ubiquitin ligase complexes were expressed specifically in reproductive organs. Cell wall biosynthesis or degradation protein genes and transcription factor genes expressed specifically in reproductive stages were also newly identified. Rice genes homologous to reproduction-related genes in other plants showed expression profiles both consistent and inconsistent with their predicted functions. The rice reproductive expression atlas is likely to be the most extensive and most comprehensive data set available, indispensable for unraveling functions of many specific genes in plant reproductive processes that have not yet been thoroughly analyzed.

Diversity of Pectin Rhamnogalacturonan I Rhamnosyltransferases in Glycosyltransferase Family 106.

Rhamnogalacturonan I (RG-I) comprises approximately one quarter of the pectin molecules in land plants, and the backbone of RG-I consists of a repeating sequence of [2)-α-L-Rha(1-4)-α-D-GalUA(1-] disaccharide. Four Arabidopsis thaliana genes encoding RG-I rhamnosyltransferases (AtRRT1 to AtRRT4), which synthesize the disaccharide repeats, have been identified in the glycosyltransferase family (GT106). However, the functional role of RG-I in plant cell walls and the evolutional history of RRTs remains to be clarified. Here, we characterized the sole ortholog of AtRRT1-AtRRT4 in liverwort, Marchantia polymorpha, namely, MpRRT1. MpRRT1 had RRT activity and genetically complemented the AtRRT1-deficient mutant phenotype in A. thaliana. However, the MpRRT1-deficient M. polymorpha mutants showed no prominent morphological changes and only an approximate 20% reduction in rhamnose content in the cell wall fraction compared to that in wild-type plants, suggesting the existence of other RRT gene(s) in the M. polymorpha genome. As expected, we detected RRT activities in other GT106 family proteins such as those encoded by MpRRT3 in M. polymorpha and FRB1/AtRRT8 in A. thaliana, the deficient mutant of which affects cell adhesion. Our results show that RRT genes are more redundant and diverse in GT106 than previously thought.

Rhamnogalacturonan I galactosyltransferase: Detection of enzyme activity and its hyperactivation.

Rhamnogalacturonan I (RG-I), one of the pectic components of the plant cell wall, is composed of a backbone of repeating disaccharide units of rhamnose and galacturonic acid, and side chains, such as galactans, arabinans, and arabinogalactans. The activity of RG-I galactosyltransferase, which transfers galactosyl residues to rhamnosyl residues in the RG-I backbone, has not been detected until now. Here, we detected galactosyltransferase activity in azuki bean epicotyls using fluorogenic RG-I oligosaccharide acceptors. This enzyme prefers oligosaccharides with a degree of polymerization more than 9. The enzyme activity was detected in the Golgi apparatus, which is the site of pectin synthesis. In vitro hyperactivation of this enzyme was also observed. Moreover, enzyme activity was increased up to 40-fold in the presence of cationic surfactants or polyelectrolytes.

Practical preparation of UDP-apiose and its applications for studying apiosyltransferase.

UDP-apiose, a donor substrate of apiosyltransferases, is labile because of its intramolecular self-cyclization ability, resulting in the formation of apiofuranosyl-1,2-cyclic phosphate. Therefore, stabilization of UDP-apiose is indispensable for its availability and identifying and characterizing the apiosyltransferases involved in the biosynthesis of apiosylated sugar chains and glycosides. Here, we established a method for stabilizing UDP-apiose using bulky cations as counter ions. Bulky cations such as triethylamine effectively suppressed the degradation of UDP-apiose in solution. The half-life of UDP-apiose was increased to 48.1 ±â€¯2.4 h at pH 6.0 and 25 °C using triethylamine as a counter cation. UDP-apiose coordinated with a counter cation enabled long-term storage under freezing conditions. UDP-apiose was utilized as a donor substrate for apigenin 7-O-ß-D-glucoside apiosyltransferase to produce the apiosylated glycoside apiin. This apiosyltransferase assay will be useful for identifying genes encoding apiosyltransferases.

Various spatiotemporal expression profiles of anther-expressed genes in rice.

The male gametophyte and tapetum play different roles during anther development although they are differentiated from the same cell lineage, the L2 layer. Until now, it has not been possible to delineate their transcriptomes due to technical difficulties in separating the two cell types. In the present study, we characterized the separated transcriptomes of the rice microspore/pollen and tapetum using laser microdissection (LM)-mediated microarray. Spatiotemporal expression patterns of 28,141 anther-expressed genes were classified into 20 clusters, which contained 3,468 (12.3%) anther-enriched genes. In some clusters, synchronous gene expression in the microspore and tapetum at the same developmental stage was observed as a novel characteristic of the anther transcriptome. Noteworthy expression patterns are discussed in connection with gene ontology (GO) categories and gene annotations, which are related to important biological events in anther development, such as pollen maturation, pollen germination, pollen tube elongation and pollen wall formation.

Molecular characterization of second tomato α1,3/4-fucosidase (α-Fuc'ase Sl-2), a member of glycosyl hydrolase family 29 active toward the core α1,3-fucosyl residue in plant N-glycans.

In a previous study, we molecular-characterized a tomato (Solanum lycopersicum) α1, 3/4-fucosidase (α-Fuc'ase Sl-1) encoded in a tomato gene (Solyc03g006980), indicating that α-Fuc'ase Sl-1 is involved in the turnover of Lea epitope-containing N-glycans. In this study, we have characterized another tomato gene (Solyc11g069010) encoding α1, 3/4-fucosidase (α-Fuc'ase Sl-2), which is also active toward the complex type N-glycans containing Lea epitope(s). The baculovirus-insect cell expression system was used to express that α-Fuc'ase Sl-2 with anti-FLAG tag, and the expression product (rFuc'ase Sl-2), was found as a 65 kDa protein using SDS-PAGE and has an optimum pH of around 5.0. Similarly to rFuc'ase Sl-1, rFuc'ase Sl-2 hydrolyzed the non-reducing terminal α1, 3-fucose residue on LNFP III and α1, 4-fucose residues of Lea epitopes on plant complex type N-glycans, but not the core α1, 3-fucose residue on Manß1-4GlcNAcß1-4(Fucα1-3)GlcNAc or Fucα1-3GlcNAc. However, we found that both α-Fuc'ases Sl-1 and Sl-2 were specifically active toward α1, 3-fucose residue on GlcNAcß1-4(Fucα1-3)GlcNAc, indicating that the non-substituted ß-GlcNAc linked to the proximal GlcNAc residue of the core tri-saccharide moiety of plant specific N-glycans must be a pre-requisite for α-Fuc'ase activity. A 3 D modelled structure of the catalytic sites of α-Fuc'ase Sl-2 suggested that Asp192 and Glu236 may be important for binding to the α1, 3/4 fucose residue.

Pectin RG-I rhamnosyltransferases represent a novel plant-specific glycosyltransferase family.

Pectin is one of the three key cell wall polysaccharides in land plants and consists of three major structural domains: homogalacturonan, rhamnogalacturonan I (RG-I) and RG-II. Although the glycosyltransferase required for the synthesis of the homogalacturonan and RG-II backbone was identified a decade ago, those for the synthesis of the RG-I backbone, which consists of the repeating disaccharide unit [→2)-α-L-Rha-(1 → 4)-α-D-GalUA-(1→], have remained unknown. Here, we report the identification and characterization of Arabidopsis RG-I:rhamnosyltransferases (RRTs), which transfer the rhamnose residue from UDP-ß-L-rhamnose to RG-I oligosaccharides. RRT1, which is one of the four Arabidopsis RRTs, is a single-spanning transmembrane protein, localized to the Golgi apparatus. RRT1 was highly expressed during formation of the seed coat mucilage, which is a specialized cell wall with abundant RG-I. Loss-of-function mutation in RRT1 caused a reduction in the level of RG-I in the seed coat mucilage. The RRTs belong to a novel glycosyltransferase family, now designated GT106. This is a large plant-specific family, and glycosyltransferases in this family seem to have plant-specific roles, such as biosynthesis of plant cell wall polysaccharides.