The plant glycosyltransferase clone collection for functional genomics

Abstract

SUMMARYThe glycosyltransferases (GTs) are an important and functionally diverse family of enzymes involved in glycan and glycoside biosynthesis. Plants have evolved large families of GTs which undertake the array of glycosylation reactions that occur during plant development and growth. Based on the Carbohydrate-Active enZymes (CAZy) database, the genome of the reference plant Arabidopsis thaliana codes for over 450 GTs, while the rice genome (Oryza sativa) contains over 600 members. Collectively, GTs from these reference plants can be classified into over 40 distinct GT families. Although these enzymes are involved in many important plant specific processes such as cell-wall and secondary metabolite biosynthesis, few have been functionally characterized. We have sought to develop a plant GTs clone resource that will enable functional genomic approaches to be undertaken by the plant research community. In total, 403 (88%) of CAZy defined Arabidopsis GTs have been cloned, while 96 (15%) of the GTs coded by rice have been cloned. The collection resulted in the update of a number of Arabidopsis GT gene models. The clones represent full-length coding sequences without termination codons and are Gateway â compatible. To demonstrate the utility of this JBEI GT Collection, a set of efficient particle bombardment plasmids (pBullet) was also constructed with markers for the endomembrane. The utility of the pBullet collection was demonstrated by localizing all members of the Arabidopsis GT14 family to the Golgi apparatus or the endoplasmic reticulum (ER). Updates to these resources are available at the JBEI GT Collection website http://www.addgene.org/.

The diversion of 2‐C‐methyl‐d‐erythritol‐2,4‐cyclodiphosphate from the 2‐C‐methyl‐d‐erythritol 4‐phosphate pathway to hemiterpene glycosides mediates stress responses in Arabidopsis thaliana

“…Glycosyltransferases form a large and diverse family of enzymes with hundreds of members in Arabidopsis. Recent, large scale characterizations of the subcellular localizations of glycosyltransferases indicate they are primarily located in the Golgi apparatus, followed by the cytosol, the ER, and then the plasma membrane (Tanz et al, 2013;Lao et al, 2014). They are generally understood to be nonplastid targeted enzymes (Li et al, 2001), supporting the assertion that some if not all of the conversions reported here require export of intermediates out of the plastid.…”

Loss of Inositol Phosphorylceramide Sphingolipid Mannosylation Induces Plant Immune Responses and Reduces Cellulose Content in Arabidopsis

“…To investigate the subcellular localization of GMT1, the GMT1 coding sequence was fused to YFP at the C terminus under the control of the CaMV 35S promoter. This construct was cotransformed with the Golgi marker a-mannosidase-ECFP into onion epidermal cells by particle bombardment (Lao et al, 2014). The localization of the fusion proteins was examined by fluorescence confocal microscopy.…”

MUCILAGE-RELATED10 Produces Galactoglucomannan That Maintains Pectin and Cellulose Architecture in Arabidopsis Seed Mucilage

“…For transient expression in N. benthamiana, we created a 35S:MUCI10-YFP construct by introducing the pDONR MUCI10 clone obtained from the Joint BioEnergy Institute GT collection (Lao et al, 2014) into the pEarleyGate101 vector (Earley et al, 2006) using the LR Clonase II reaction according to the Life Technologies protocol. Constructs were verified by sequencing.…”