Glycosyl transferase GT2 genes mediate the biosynthesis of an unusual (1,3;1,4)-ß-glucan exopolysaccharide in the bacterium Sarcina ventriculi.

Linear, unbranched (1,3;1,4)-ß-glucans (mixed-linkage glucans or MLGs) are commonly found in the cell walls of grasses, but have also been detected in basal land plants, algae, fungi and bacteria. Here we show that two family GT2 glycosyltransferases from the Gram-positive bacterium Sarcina ventriculi are capable of synthesizing MLGs. Immunotransmission electron microscopy demonstrates that MLG is secreted as an exopolysaccharide, where it may play a role in organizing individual cells into packets that are characteristic of Sarcina species. Heterologous expression of these two genes shows that they are capable of producing MLGs in planta, including an MLG that is chemically identical to the MLG secreted from S. ventriculi cells but which has regularly spaced (1,3)-ß-linkages in a structure not reported previously for MLGs. The tandemly arranged, paralogous pair of genes are designated SvBmlgs1 and SvBmlgs2. The data indicate that MLG synthases have evolved different enzymic mechanisms for the incorporation of (1,3)-ß- and (1,4)-ß-glucosyl residues into a single polysaccharide chain. Amino acid variants associated with the evolutionary switch from (1,4)-ß-glucan (cellulose) to MLG synthesis have been identified in the active site regions of the enzymes. The presence of MLG synthesis in bacteria could prove valuable for large-scale production of MLG for medical, food and beverage applications.

Distinct functions of FASCILIN-LIKE ARABINOGALACTAN PROTEINS relate to domain structure.

The role of glycoproteins as key cell surface molecules during development and stress is well established; yet, the relationship between their structural features and functional mechanisms is poorly defined. FASCICLIN-LIKE ARABINOGALACTAN PROTEINs (FLAs), which impact plant growth and development, are an excellent example of a glycoprotein family with a complex multidomain structure. FLAs combine globular fasciclin-like (FAS1) domains with regions that are intrinsically disordered and contain glycomotifs for directing the addition of O-linked arabinogalactan (AG) glycans. Additional posttranslational modifications on FLAs include N-linked glycans in the FAS1 domains, a cleaved signal peptide at the N terminus, and often a glycosylphosphatidylinositol (GPI) anchor signal sequence at the C terminus. The roles of glycosylation, the GPI anchor, and FAS1 domain functions in the polysaccharide-rich extracellular matrix of plants remain unclear, as do the relationships between them. In this study, we examined sequence-structure-function relationships of Arabidopsis (Arabidopsis thaliana) FLA11, demonstrated to have roles in secondary cell wall (SCW) development, by introducing domain mutations and functional specialization through domain swaps with FLA3 and FLA12. We identified FAS1 domains as essential for FLA function, differentiating FLA11/FLA12, with roles in SCW development, from FLA3, specific to flowers and involved in pollen development. The GPI anchor and AG glycosylation co-regulate the cell surface location and release of FLAs into cell walls. The AG glycomotif sequence closest to the GPI anchor (AG2) is a major feature differentiating FLA11 from FLA12. The results of our study show that the multidomain structure of different FLAs influences their subcellular location and biological functions during plant development.

Spatiotemporal deposition of cell wall polysaccharides in oat endosperm during grain development.

Oat (Avena sativa) is a cereal crop whose grains are rich in (1,3;1,4)-ß-D-glucan (mixed-linkage glucan or MLG), a soluble dietary fiber. In our study, we analyzed oat endosperm development in 2 Canadian varieties with differing MLG content and nutritional value. We confirmed that oat undergoes a nuclear type of endosperm development but with a shorter cellularization phase than barley (Hordeum vulgare). Callose and cellulose were the first polysaccharides to be detected in the early anticlinal cell walls at 11 days postemergence (DPE) of the panicle. Other polysaccharides such as heteromannan and homogalacturonan were deposited early in cellularization around 12 DPE after the first periclinal walls are laid down. In contrast to barley, heteroxylan deposition coincided with completion of cellularization and was detected from 14 DPE but was only detectable after demasking. Notably, MLG was the last polysaccharide to be laid down at 18 DPE within the differentiation phase, rather than during cellularization. In addition, differences in the spatiotemporal patterning of MLG were also observed between the 2 varieties. The lower MLG-containing cultivar AC Morgan (3.5% w/w groats) was marked by the presence of a discontinuous pattern of MLG labeling, while labeling in the same walls in CDC Morrison (5.6% w/w groats) was mostly even and continuous. RNA-sequencing analysis revealed higher transcript levels of multiple MLG biosynthetic cellulose synthase-like F (CSLF) and CSLH genes during grain development in CDC Morrison compared with AC Morgan that likely contributes to the increased abundance of MLG at maturity in CDC Morrison. CDC Morrison was also observed to have smaller endosperm cells with thicker walls than AC Morgan from cellularization onwards, suggesting the processes controlling cell size and shape are established early in development. This study has highlighted that the molecular processes influencing MLG content and deposition are more complex than previously imagined.

FLA11 and FLA12 glycoproteins fine-tune stem secondary wall properties in response to mechanical stresses.

Secondary cell walls (SCWs) in stem xylem vessel and fibre cells enable plants to withstand the enormous compressive forces associated with upright growth. It remains unclear if xylem vessel and fibre cells can directly sense mechanical stimuli and modify their SCW during development. We provide evidence that Arabidopsis SCW-specific Fasciclin-Like Arabinogalactan-proteins 11 (FLA11) and 12 (FLA12) are possible cell surface sensors regulating SCW development in response to mechanical stimuli. Plants overexpressing FLA11 (OE-FLA11) showed earlier SCW development compared to the wild-type (WT) and altered SCW properties that phenocopy WT plants under compression stress. By contrast, OE-FLA12 stems showed higher cellulose content compared to WT plants, similar to plants experiencing tensile stress. fla11, OE-FLA11, fla12, and OE-FLA12 plants showed altered SCW responses to mechanical stress compared to the WT. Quantitative polymerase chain reaction (qPCR) and RNA-seq analysis revealed the up-regulation of genes and pathways involved in stress responses and SCW synthesis and regulation. Analysis of OE-FLA11 nst1 nst3 plants suggests that FLA11 regulation of SCWs is reliant on classical transcriptional networks. Our data support the involvement of FLA11 and FLA12 in SCW sensing complexes to fine-tune both the initiation of SCW development and the balance of lignin and cellulose synthesis/deposition in SCWs during development and in response to mechanical stimuli.

UDP-Api/UDP-Xyl synthases affect plant development by controlling the content of UDP-Api to regulate the RG-II-borate complex.

Rhamnogalacturonan-II (RG-II) is structurally the most complex glycan in higher plants, containing 13 different sugars and 21 distinct glycosidic linkages. Two monomeric RG-II molecules can form an RG-II-borate diester dimer through the two apiosyl (Api) residues of side chain A to regulate cross-linking of pectin in the cell wall. But the relationship of Api biosynthesis and RG-II dimer is still unclear. In this study we investigated the two homologous UDP-D-apiose/UDP-D-xylose synthases (AXSs) in Arabidopsis thaliana that synthesize UDP-D-apiose (UDP-Api). Both AXSs are ubiquitously expressed, while AXS2 has higher overall expression than AXS1 in the tissues analyzed. The homozygous axs double mutant is lethal, while heterozygous axs1/+ axs2 and axs1 axs2/+ mutants display intermediate phenotypes. The axs1/+ axs2 mutant plants are unable to set seed and die. By contrast, the axs1 axs2/+ mutant plants exhibit loss of shoot and root apical dominance. UDP-Api content in axs1 axs2/+ mutants is decreased by 83%. The cell wall of axs1 axs2/+ mutant plants is thicker and contains less RG-II-borate complex than wild-type Col-0 plants. Taken together, these results provide direct evidence of the importance of AXSs for UDP-Api and RG-II-borate complex formation in plant growth and development.

Targeted mutation of barley (1,3;1,4)-ß-glucan synthases reveals complex relationships between the storage and cell wall polysaccharide content.

Barley (Hordeum vulgare L) grain is comparatively rich in (1,3;1,4)-ß-glucan, a source of fermentable dietary fibre that protects against various human health conditions. However, low grain (1,3;1,4)-ß-glucan content is preferred for brewing and distilling. We took a reverse genetics approach, using CRISPR/Cas9 to generate mutations in members of the Cellulose synthase-like (Csl) gene superfamily that encode known (HvCslF6 and HvCslH1) and putative (HvCslF3 and HvCslF9) (1,3;1,4)-ß-glucan synthases. Resultant mutations ranged from single amino acid (aa) substitutions to frameshift mutations causing premature stop codons, and led to specific differences in grain morphology, composition and (1,3;1,4)-ß-glucan content. (1,3;1,4)-ß-Glucan was absent in the grain of cslf6 knockout lines, whereas cslf9 knockout lines had similar (1,3;1,4)-ß-glucan content to wild-type (WT). However, cslf9 mutants showed changes in the abundance of other cell-wall-related monosaccharides compared with WT. Thousand grain weight (TGW), grain length, width and surface area were altered in cslf6 knockouts, and to a lesser extent TGW in cslf9 knockouts. cslf3 and cslh1 mutants had no effect on grain (1,3;1,4)-ß-glucan content. Our data indicate that multiple members of the CslF/H family fulfil important functions during grain development but, with the exception of HvCslF6, do not impact the abundance of (1,3;1,4)-ß-glucan in mature grain.

Evolution of Sequence-Diverse Disordered Regions in a Protein Family: Order within the Chaos.

Approaches for studying the evolution of globular proteins are now well established yet are unsuitable for disordered sequences. Our understanding of the evolution of proteins containing disordered regions therefore lags that of globular proteins, limiting our capacity to estimate their evolutionary history, classify paralogs, and identify potential sequence-function relationships. Here, we overcome these limitations by using new analytical approaches that project representations of sequence space to dissect the evolution of proteins with both ordered and disordered regions, and the correlated changes between these. We use the fasciclin-like arabinogalactan proteins (FLAs) as a model family, since they contain a variable number of globular fasciclin domains as well as several distinct types of disordered regions: proline (Pro)-rich arabinogalactan (AG) regions and longer Pro-depleted regions. Sequence space projections of fasciclin domains from 2019 FLAs from 78 species identified distinct clusters corresponding to different types of fasciclin domains. Clusters can be similarly identified in the seemingly random Pro-rich AG and Pro-depleted disordered regions. Sequence features of the globular and disordered regions clearly correlate with one another, implying coevolution of these distinct regions, as well as with the N-linked and O-linked glycosylation motifs. We reconstruct the overall evolutionary history of the FLAs, annotated with the changing domain architectures, glycosylation motifs, number and length of AG regions, and disordered region sequence features. Mapping these features onto the functionally characterized FLAs therefore enables their sequence-function relationships to be interrogated. These findings will inform research on the abundant disordered regions in protein families from all kingdoms of life.

Recent advances in Cannabis sativa genomics research.

Cannabis (Cannabis sativa L.) is one of the oldest cultivated plants purported to have unique medicinal properties. However, scientific research of cannabis has been restricted by the Single Convention on Narcotic Drugs of 1961, an international treaty that prohibits the production and supply of narcotic drugs except under license. Legislation governing cannabis cultivation for research, medicinal and even recreational purposes has been relaxed recently in certain jurisdictions. As a result, there is now potential to accelerate cultivar development of this multi-use and potentially medically useful plant species by application of modern genomics technologies. Whilst genomics has been pivotal to our understanding of the basic biology and molecular mechanisms controlling key traits in several crop species, much work is needed for cannabis. In this review we provide a comprehensive summary of key cannabis genomics resources and their applications. We also discuss prospective applications of existing and emerging genomics technologies for accelerating the genetic improvement of cannabis.

Cell wall modification by the xyloglucan endotransglucosylase/hydrolase XTH19 influences freezing tolerance after cold and sub-zero acclimation.

Freezing triggers extracellular ice formation leading to cell dehydration and deformation during a freeze-thaw cycle. Many plant species increase their freezing tolerance during exposure to low, non-freezing temperatures, a process termed cold acclimation. In addition, exposure to mild freezing temperatures after cold acclimation evokes a further increase in freezing tolerance (sub-zero acclimation). Previous transcriptome and proteome analyses indicate that cell wall remodelling may be particularly important for sub-zero acclimation. In the present study, we used a combination of immunohistochemical, chemical and spectroscopic analyses to characterize the cell walls of Arabidopsis thaliana and characterized a mutant in the XTH19 gene, encoding a xyloglucan endotransglucosylase/hydrolase (XTH). The mutant showed reduced freezing tolerance after both cold and sub-zero acclimation, compared to the Col-0 wild type, which was associated with differences in cell wall composition and structure. Most strikingly, immunohistochemistry in combination with 3D reconstruction of centres of rosette indicated that epitopes of the xyloglucan-specific antibody LM25 were highly abundant in the vasculature of Col-0 plants after sub-zero acclimation but absent in the XTH19 mutant. Taken together, our data shed new light on the potential roles of cell wall remodelling for the increased freezing tolerance observed after low temperature acclimation.

Blue Light Regulates Secondary Cell Wall Thickening via MYC2/MYC4 Activation of the NST1-Directed Transcriptional Network in Arabidopsis.

Secondary cell walls (SCWs) are formed in some specific types of plant cells, providing plants with mechanical strength. During plant growth and development, formation of secondary cell walls is regulated by various developmental and environmental signals. The underlying molecular mechanisms are poorly understood. In this study, we analyzed the blue light receptor cryptochrome1 (cry1) mutant of Arabidopsis thaliana for its SCW phenotypes. During inflorescence stem growth, SCW thickening in the vasculature was significantly affected by blue light. cry1 plants displayed a decline of SCW thickening in fiber cells, while CRY1 overexpression led to enhanced SCW formation. Transcriptome analysis indicated that the reduced SCW thickening was associated with repression of the NST1-directed transcription regulatory networks. Further analyses revealed that the expression of MYC2/MYC4 that is induced by blue light activates the transcriptional network underlying SCW thickening. The activation is caused by direct binding of MYC2/MYC4 to the NST1 promoter. This study demonstrates that SCW thickening in fiber cells is regulated by a blue light signal that is mediated through MYC2/MYC4 activation of NST1-directed SCW formation in Arabidopsis.

Functional Characterization of a Glycosyltransferase from the Moss Physcomitrella patens Involved in the Biosynthesis of a Novel Cell Wall Arabinoglucan.

Mixed-linkage (1,3;1,4)-ß-glucan (MLG), an abundant cell wall polysaccharide in the Poaceae, has been detected in ascomycetes, algae, and seedless vascular plants, but not in eudicots. Although MLG has not been reported in bryophytes, a predicted glycosyltransferase from the moss Physcomitrella patens (Pp3c12_24670) is similar to a bona fide ascomycete MLG synthase. We tested whether Pp3c12_24670 encodes an MLG synthase by expressing it in wild tobacco (Nicotiana benthamiana) and testing for release of diagnostic oligosaccharides from the cell walls by either lichenase or (1,4)-ß-glucan endohydrolase. Lichenase, an MLG-specific endohydrolase, showed no activity against cell walls from transformed N. benthamiana, but (1,4)-ß-glucan endohydrolase released oligosaccharides that were distinct from oligosaccharides released from MLG by this enzyme. Further analysis revealed that these oligosaccharides were derived from a novel unbranched, unsubstituted arabinoglucan (AGlc) polysaccharide. We identified sequences similar to the P. patens AGlc synthase from algae, bryophytes, lycophytes, and monilophytes, raising the possibility that other early divergent plants synthesize AGlc. Similarity of P. patens AGlc synthase to MLG synthases from ascomycetes, but not those from Poaceae, suggests that AGlc and MLG have a common evolutionary history that includes loss in seed plants, followed by a more recent independent origin of MLG within the monocots.

The cell wall polysaccharides of a photosynthetic relative of apicomplexans, Chromera velia.

Chromerids are a group of alveolates, found in corals, that show peculiar morphological and genomic features. These organisms are evolutionary placed in-between symbiotic dinoflagellates and parasitic apicomplexans. There are two known species of chromerids: Chromera velia and Vitrella brassicaformis. Here, the biochemical composition of the C. velia cell wall was analyzed. Several polysaccharides adorn this structure, with glucose being the most abundant monosaccharide (approx. 80%) and predominantly 4-linked (approx. 60%), suggesting that the chromerids cell wall is mostly cellulosic. The presence of cellulose was cytochemically confirmed with calcofluor white staining of the algal cell. The remaining wall polysaccharides, assuming structures are similar to those of higher plants, are indicative of a mixture of galactans, xyloglucans, heteroxylans, and heteromannans. The present work provides, for the first time, insights into the outermost layers of the photosynthetic alveolate C. velia.

A Pipeline towards the Biochemical Characterization of the Arabidopsis GT14 Family.

Glycosyltransferases (GTs) catalyze the synthesis of glycosidic linkages and are essential in the biosynthesis of glycans, glycoconjugates (glycolipids and glycoproteins), and glycosides. Plant genomes generally encode many more GTs than animal genomes due to the synthesis of a cell wall and a wide variety of glycosylated secondary metabolites. The Arabidopsis thaliana genome is predicted to encode over 573 GTs that are currently classified into 42 diverse families. The biochemical functions of most of these GTs are still unknown. In this study, we updated the JBEI Arabidopsis GT clone collection by cloning an additional 105 GT cDNAs, 508 in total (89%), into Gateway-compatible vectors for downstream characterization. We further established a functional analysis pipeline using transient expression in tobacco (Nicotiana benthamiana) followed by enzymatic assays, fractionation of enzymatic products by reversed-phase HPLC (RP-HPLC) and characterization by mass spectrometry (MS). Using the GT14 family as an exemplar, we outline a strategy for identifying effective substrates of GT enzymes. By addition of UDP-GlcA as donor and the synthetic acceptors galactose-nitrobenzodiazole (Gal-NBD), ß-1,6-galactotetraose (ß-1,6-Gal4) and ß-1,3-galactopentose (ß-1,3-Gal5) to microsomes expressing individual GT14 enzymes, we verified the ß-glucuronosyltransferase (GlcAT) activity of three members of this family (AtGlcAT14A, B, and E). In addition, a new family member (AT4G27480, 248) was shown to possess significantly higher activity than other GT14 enzymes. Our data indicate a likely role in arabinogalactan-protein (AGP) biosynthesis for these GT14 members. Together, the updated Arabidopsis GT clone collection and the biochemical analysis pipeline present an efficient means to identify and characterize novel GT catalytic activities.

N-linked Glycan Micro-heterogeneity in Glycoproteins of Arabidopsis.

N-glycosylation is one of the most common protein post-translational modifications in eukaryotes and has a relatively conserved core structure between fungi, animals and plants. In plants, the biosynthesis of N-glycans has been extensively studied with all the major biosynthetic enzymes characterized. However, few studies have applied advanced mass spectrometry to profile intact plant N-glycopeptides. In this study, we use hydrophilic enrichment, high-resolution tandem mass spectrometry with complementary and triggered fragmentation to profile Arabidopsis N-glycopeptides from microsomal membranes of aerial tissues. A total of 492 N-glycosites were identified from 324 Arabidopsis proteins with extensive N-glycan structural heterogeneity revealed through 1110 N-glycopeptides. To demonstrate the precision of the approach, we also profiled N-glycopeptides from the mutant (xylt) of ß-1,2-xylosyltransferase, an enzyme in the N-glycan biosynthetic pathway. This analysis represents the most comprehensive and unbiased collection of Arabidopsis N-glycopeptides revealing an unsurpassed level of detail on the micro-heterogeneity present in N-glycoproteins of Arabidopsis. Data are available via ProteomeXchange with identifier PXD006270.

Effects of Excess Manganese on the Xylem Sap Protein Profile of Tomato (Solanum lycopersicum) as Revealed by Shotgun Proteomic Analysis.

Metal toxicity is a common problem in crop species worldwide. Some metals are naturally toxic, whereas others such as manganese (Mn) are essential micro-nutrients for plant growth but can become toxic when in excess. Changes in the composition of the xylem sap, which is the main pathway for ion transport within the plant, is therefore vital to understanding the plant's response(s) to metal toxicity. In this study we have assessed the effects of exposure of tomato roots to excess Mn on the protein profile of the xylem sap, using a shotgun proteomics approach. Plants were grown in nutrient solution using 4.6 and 300 µM MnCl2 as control and excess Mn treatments, respectively. This approach yielded 668 proteins reliably identified and quantified. Excess Mn caused statistically significant (at p ≤ 0.05) and biologically relevant changes in relative abundance (≥2-fold increases or ≥50% decreases) in 322 proteins, with 82% of them predicted to be secretory using three different prediction tools, with more decreasing than increasing (181 and 82, respectively), suggesting that this metal stress causes an overall deactivation of metabolic pathways. Processes most affected by excess Mn were in the oxido-reductase, polysaccharide and protein metabolism classes. Excess Mn induced changes in hydrolases and peroxidases involved in cell wall degradation and lignin formation, respectively, consistent with the existence of alterations in the cell wall. Protein turnover was also affected, as indicated by the decrease in proteolytic enzymes and protein synthesis-related proteins. Excess Mn modified the redox environment of the xylem sap, with changes in the abundance of oxido-reductase and defense protein classes indicating a stress scenario. Finally, results indicate that excess Mn decreased the amounts of proteins associated with several signaling pathways, including fasciclin-like arabinogalactan-proteins and lipids, as well as proteases, which may be involved in the release of signaling peptides and protein maturation. The comparison of the proteins changing in abundance in xylem sap and roots indicate the existence of tissue-specific and systemic responses to excess Mn. Data are available via ProteomeXchange with identifier PXD021973.

The cotton ß-galactosyltransferase 1 (GalT1) that galactosylates arabinogalactan proteins participates in controlling fiber development.

Arabinogalactan proteins (AGPs) are highly glycosylated proteins that play pivotal roles in diverse developmental processes in plants. Type-II AG glycans, mostly O-linked to the hydroxyproline residues of the protein backbone, account for up to 95% w/w of the AGP, but their functions are still largely unclear. Cotton fibers are extremely elongated single-cell trichomes on the seed epidermis; however, little is known of the molecular basis governing the regulation of fiber cell development. Here, we characterized the role of a CAZy glycosyltransferase 31 (GT31) family member, GhGalT1, in cotton fiber development. The fiber length of the transgenic cotton overexpressing GhGalT1 was shorter than that of the wild type, whereas in the GhGalT1-silenced lines there was a notable increase in fiber length compared with wild type. The carbohydrate moieties of AGPs were altered in fibers of GhGalT1 transgenic cotton. The galactose: arabinose ratio of AG glycans was higher in GhGalT1 overexpression fibers, but was lower in GhGalT1-silenced lines, compared with that in the wild type. Overexpression of GhGalT1 upregulates transcript levels of a broad range of cell wall-related genes, especially the fasciclin-like AGP (FLA) backbone genes. An enzyme activity assay demonstrated that GhGalT1 is a ß-1,3-galactosyltransferase (ß-1,3-GalT) involved in biosynthesis of the ß-1,3-galactan backbone of the type-II AG glycans of AGPs. We also show that GhGalT1 can form homo- and heterodimers with other cotton GT31 family members to facilitate AG glycan assembly of AGPs. Thus, our data demonstrate that GhGalT1 influences cotton fiber development via controlling the glycosylation of AGPs, especially FLAs.

The Three Members of the Arabidopsis Glycosyltransferase Family 92 are Functional ß-1,4-Galactan Synthases.

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 ß-1, 4-galactan. Plants in which all three genes were inactivated had no detectable ß-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.

Pipeline to Identify Hydroxyproline-Rich Glycoproteins.

Intrinsically disordered proteins (IDPs) are functional proteins that lack a well-defined three-dimensional structure. The study of IDPs is a rapidly growing area as the crucial biological functions of more of these proteins are uncovered. In plants, IDPs are implicated in plant stress responses, signaling, and regulatory processes. A superfamily of cell wall proteins, the hydroxyproline-rich glycoproteins (HRGPs), have characteristic features of IDPs. Their protein backbones are rich in the disordering amino acid proline, they contain repeated sequence motifs and extensive posttranslational modifications (glycosylation), and they have been implicated in many biological functions. HRGPs are evolutionarily ancient, having been isolated from the protein-rich walls of chlorophyte algae to the cellulose-rich walls of embryophytes. Examination of HRGPs in a range of plant species should provide valuable insights into how they have evolved. Commonly divided into the arabinogalactan proteins, extensins, and proline-rich proteins, in reality, a continuum of structures exists within this diverse and heterogenous superfamily. An inability to accurately classify HRGPs leads to inconsistent gene ontologies limiting the identification of HRGP classes in existing and emerging omics data sets. We present a novel and robust motif and amino acid bias (MAAB) bioinformatics pipeline to classify HRGPs into 23 descriptive subclasses. Validation of MAAB was achieved using available genomic resources and then applied to the 1000 Plants transcriptome project (www.onekp.com) data set. Significant improvement in the detection of HRGPs using multiple-k-mer transcriptome assembly methodology was observed. The MAAB pipeline is readily adaptable and can be modified to optimize the recovery of IDPs from other organisms.

KNS4/UPEX1: A Type II Arabinogalactan ß-(1,3)-Galactosyltransferase Required for Pollen Exine Development.

Pollen exine is essential for protection from the environment of the male gametes of seed-producing plants, but its assembly and composition remain poorly understood. We previously characterized Arabidopsis (Arabidopsis thaliana) mutants with abnormal pollen exine structure and morphology that we named kaonashi (kns). Here we describe the identification of the causal gene of kns4 that was found to be a member of the CAZy glycosyltransferase 31 gene family, identical to UNEVEN PATTERN OF EXINE1, and the biochemical characterization of the encoded protein. The characteristic exine phenotype in the kns4 mutant is related to an abnormality of the primexine matrix laid on the surface of developing microspores. Using light microscopy with a combination of type II arabinogalactan (AG) antibodies and staining with the arabinogalactan-protein (AGP)-specific ß-Glc Yariv reagent, we show that the levels of AGPs in the kns4 microspore primexine are considerably diminished, and their location differs from that of wild type, as does the distribution of pectin labeling. Furthermore, kns4 mutants exhibit reduced fertility as indicated by shorter fruit lengths and lower seed set compared to the wild type, confirming that KNS4 is critical for pollen viability and development. KNS4 was heterologously expressed in Nicotiana benthamiana, and was shown to possess ß-(1,3)-galactosyltransferase activity responsible for the synthesis of AG glycans that are present on both AGPs and/or the pectic polysaccharide rhamnogalacturonan I. These data demonstrate that defects in AGP/pectic glycans, caused by disruption of KNS4 function, impact pollen development and viability in Arabidopsis.