Skip to main content
eScholarship
Open Access Publications from the University of California

Scholarly Works (6 results)

  • Thesis
  • Peer Reviewed
UC Berkeley Electronic Theses and Dissertations (2013)

Glycosyltransferases catalyze the transfer of sugars from activated phosphate sugar substrates to various acceptor molecules ranging from primary metabolites to glycolipids to plant cell walls. The model plant Arabidopsis encodes about 500 glycosyltransferase proteins. Forty-one of these are members of Glycosyltransferase Family 8. These enzymes are thought to function in synthesis of diverse compounds including the disaccharide galactinol, and the cell wall polymer pectin, and the energy storage polysaccharide starch. This thesis will describe evidence that allowed us to assign additional functions of members of this family: five of these enzymes synthesize the cell wall polymer glucuronoxylan, while one synthesizes glycosyl inositol phosphoryl-ceramide sphingolipids.

The eight Plant Glycogenin-like Starch Initiation Proteins (PGSIPs) were named based on their homology to glycogenin, a protein in fungi and animals that initiates glycogen synthesis by glucosylating itself. The PGSIP proteins were proposed to initiate starch synthesis in a similar manner. However, two of these proteins, PGSIP1 and PGSIP3, are highly coexpressed with glucuronoxylan biosynthetic proteins. Mutations in PGSIP1 cause a decrease in cell wall glucuronoxylan content and microsomal xylan:glucuronosyltransferase activity, leading to the re-annotation of five of the PGSIP proteins as Glucuronic Acid Substitution of Xylan (GUX). Heterologously expressed and purified GUX1, GUX2, and GUX4 show xylan glucuronosyltransferase activity in vitro. These proteins transferred glucuronic acid to a xylooligomer acceptor with different patterns, showing that these related proteins have subtle differences in specificity.

Another protein, PGSIP6, is related to both the GUX proteins and the galactinol synthase (GolS) proteins, which transfer galactose onto the acceptor inositol. The phylogenetic placement of PGSIP6 between the GUX and GolS proteins led us to suspect that this enzyme catalyzes the transfer of glucuronic acid to the inositol moiety of glycosyl inositol phosphorylceramides, a class of glycosphingolipids that are abundant in plant plasma membranes. Expression of PGSIP6 in yeast led to production of glucuronic acid linked to inositol phosphorylceramide. Overexpressing PGSIP6 in Nicotiana benthamiana plants led to an increase in inositol phosphorylceramide glucuronosyltransferase activity, while silencing expression of PGSIP6 led to a decrease in activity. PGSIP6 has now been renamed Inositol Phosphorylceramide Glucuronosyltransferase 1 (IPGT1).

    Cover page: Cell Wall- and Glycolipid-related Functions of Arabidopsis Glycosyltransferase Family Eight Proteins
    • Article
    • Peer Reviewed
    UC Berkeley Previously Published Works (2017)
    Sphingolipids are a major component of plant plasma membranes and endomembranes, and mediate a diverse range of biological processes. Study of the highly glycosylated glycosyl inositol phosphorylceramide (GIPC) sphingolipids has been slow as a result of challenges associated with the extractability of GIPCs, and their functions in the plant remain poorly characterized. We recently discovered an Arabidopsis GIPC glucuronosyltransferase, INOSITOL PHOSPHORYLCERAMIDE GLUCURONOSYLTRANSFERASE 1 (IPUT1), which is the first enzyme in the GIPC glycosylation pathway. Plants homozygous for the iput1 loss-of-function mutation were unobtainable, and so the developmental effects of reduced GIPC glucuronosylation could not be analyzed in planta. Using a pollen-specific rescue construct, we have here isolated homozygous iput1 mutants. The iput1 mutants show severe dwarfism, compromised pollen tube guidance, and constitutive activation of salicyclic acid-mediated defense pathways. The mutants also possess reduced GIPCs, increased ceramides, and an increased incorporation of short-chain fatty acids and dihydroxylated bases into inositol phosphorylceramides and GIPCs. The assignment of a direct role for GIPC glycan head groups in the impaired processes in iput1 mutants is complicated by the vast compensatory changes in the sphingolipidome; however, our results reveal that the glycosylation steps of GIPC biosynthesis are important regulated components of sphingolipid metabolism. This study corroborates previously suggested roles for GIPC glycans in plant growth and defense, suggests important roles for them in reproduction and demonstrates that the entire sphingolipidome is sensitive to their status.
      • Article
      • Peer Reviewed
      UC Berkeley Previously Published Works (2021)
      Plant endosymbiosis relies on the development of specialized membranes that encapsulate the endosymbiont and facilitate nutrient exchange. However, the identity and function of lipids within these membrane interfaces is largely unknown. Here, we identify GLUCOSAMINE INOSITOL PHOSPHORYLCERAMIDE TRANSFERASE1 (GINT1) as a sphingolipid glycosyltransferase highly expressed in Medicago truncatula root nodules and roots colonized by arbuscular mycorrhizal (AM) fungi and further demonstrate that this enzyme functions in the synthesis of N-acetyl-glucosamine-decorated glycosyl inositol phosphoryl ceramides (GIPCs) in planta. MtGINT1 expression was developmentally regulated in symbiotic tissues associated with the development of symbiosome and periarbuscular membranes. RNAi silencing of MtGINT1 did not affect overall root growth but strongly impaired nodulation and AM symbiosis, resulting in the senescence of symbiosomes and arbuscules. Our results indicate that, although M. truncatula root sphingolipidome predominantly consists of hexose-decorated GIPCs, local reprogramming of GIPC glycosylation by MtGINT1 is required for the persistence of endosymbionts within the plant cell.
        Cover page: Reprogramming sphingolipid glycosylation is required for endosymbiont persistence in Medicago truncatula
        • Article
        • Peer Reviewed
        UC Berkeley Previously Published Works (2018)
        Glycosylinositol phosphorylceramides (GIPCs), which have a ceramide core linked to a glycan headgroup of varying structures, are the major sphingolipids in the plant plasma membrane. Recently, we identified the major biosynthetic genes for GIPC glycosylation in Arabidopsis (Arabidopsis thaliana) and demonstrated that the glycan headgroup is essential for plant viability. However, the function of GIPCs and the significance of their structural variation are poorly understood. Here, we characterized the Arabidopsis glycosyltransferase GLUCOSAMINE INOSITOLPHOSPHORYLCERAMIDE TRANSFERASE1 (GINT1) and showed that it is responsible for the glycosylation of a subgroup of GIPCs found in seeds and pollen that contain GlcNAc and GlcN [collectively GlcN(Ac)]. In Arabidopsis gint1 plants, loss of the GlcN(Ac) GIPCs did not affect vegetative growth, although seed germination was less sensitive to abiotic stress than in wild-type plants. However, in rice, where GlcN(Ac) containing GIPCs are the major GIPC subgroup in vegetative tissue, loss of GINT1 was seedling lethal. Furthermore, we could produce, de novo, "rice-like" GlcN(Ac) GIPCs in Arabidopsis leaves, which allowed us to test the function of different sugars in the GIPC headgroup. This study describes a monocot GIPC biosynthetic enzyme and shows that its Arabidopsis homolog has the same biochemical function. We also identify a possible role for GIPCs in maintaining cell-cell adhesion.
          Creative Commons 'BY-NC-ND' version 4.0 license
          • Article
          • Peer Reviewed
          UC Berkeley Previously Published Works (2016)
          Glycosylinositol phosphorylceramides (GIPCs) are a class of glycosylated sphingolipids found in plants, fungi, and protozoa. These lipids are abundant in the plant plasma membrane, forming ∼25% of total plasma membrane lipids. Little is known about the function of the glycosylated headgroup, but two recent studies have indicated that they play a key role in plant signaling and defense. Here, we show that a member of glycosyltransferase family 64, previously named ECTOPICALLY PARTING CELLS1, is likely a Golgi-localized GIPC-specific mannosyl-transferase, which we renamed GIPC MANNOSYL-TRANSFERASE1 (GMT1). Sphingolipid analysis revealed that the Arabidopsis thaliana gmt1 mutant almost completely lacks mannose-carrying GIPCs. Heterologous expression of GMT1 in Saccharomyces cerevisiae and tobacco (Nicotiana tabacum) cv Bright Yellow 2 resulted in the production of non-native mannosylated GIPCs. gmt1 displays a severe dwarfed phenotype and a constitutive hypersensitive response characterized by elevated salicylic acid and hydrogen peroxide levels, similar to that we previously reported for the Golgi-localized, GIPC-specific, GDP-Man transporter GONST1 (Mortimer et al., 2013). Unexpectedly, we show that gmt1 cell walls have a reduction in cellulose content, although other matrix polysaccharides are unchanged.
            • Article
            • Peer Reviewed
            UC Berkeley Previously Published Works (2018)
            Pectin is a major component of primary cell walls and performs a plethora of functions crucial for plant growth, development and plant-defense responses. Despite the importance of pectic polysaccharides their biosynthesis is poorly understood. Several genes have been implicated in pectin biosynthesis by mutant analysis, but biochemical activity has been shown for very few. We used reverse genetics and biochemical analysis to study members of Glycosyltransferase Family 92 (GT92) in Arabidopsis thaliana. Biochemical analysis gave detailed insight into the properties of GALS1 (Galactan synthase 1) and showed galactan synthase activity of GALS2 and GALS3. All proteins are responsible for adding galactose onto existing galactose residues attached to the rhamnogalacturonan-I (RG-I) backbone. Significant GALS activity was observed with galactopentaose as acceptor but longer acceptors are favored. Overexpression of the GALS proteins in Arabidopsis resulted in accumulation of unbranched β-1, 4-galactan. Plants in which all three genes were inactivated had no detectable β-1, 4-galactan, and surprisingly these plants exhibited no obvious developmental phenotypes under standard growth conditions. RG-I in the triple mutants retained branching indicating that the initial Gal substitutions on the RG-I backbone are added by enzymes different from GALS.
              Cover page: The Three Members of the Arabidopsis Glycosyltransferase Family 92 are Functional β-1,4-Galactan Synthases
              Top