Jacob Krüger Jensen

ARABINAN DEFICIENT 1 Is a Putative Arabinosyltransferase Involved in Biosynthesis of Pectic Arabinan in Arabidopsis

The function of a putative glycosyltransferase (At2g35100) was investigated in Arabidopsis (Arabidopsis thaliana). The protein is predicted to be a type 2 membrane protein with a signal anchor. Two independent mutant lines with T-DNA insertion in the ARABINAN DEFICIENT 1 (ARAD1) gene were analyzed. The gene was shown to be expressed in all tissues but particularly in vascular tissues of leaves and stems. Analysis of cell wall polysaccharides isolated from leaves and stems showed that arabinose content was reduced to about 75% and 46%, respectively, of wild-type levels. Immunohistochemical analysis indicated a specific decrease in arabinan with no change in other pectic domains or in glycoproteins. The cellular structure of the stem was also not altered. Isolated rhamnogalacturonan I from mutant tissues contained only about 30% of the wild-type amount of arabinose, confirming the specific deficiency in arabinan. Linkage analysis showed that the small amount of arabinan present in mutant tissue was structurally similar to that of the wild type. Transformation of mutant plants with the ARAD1 gene driven by the 35S promoter led to full complementation of the phenotype, but none of the transformants had more arabinan than the wild-type level. The data suggest that ARAD1 is an arabinan α-1,5-arabinosyltransferase. To our knowledge, the identification of other l-arabinosyltransferases has not been published.

Comparative deep transcriptional profiling of four developing oilseeds

Transcriptome analysis based on deep expressed sequence tag (EST) sequencing allows quantitative comparisons of gene expression across multiple species. Using pyrosequencing, we generated over 7 million ESTs from four stages of developing seeds of Ricinus communis, Brassica napus, Euonymus alatus and Tropaeolum majus, which differ in their storage tissue for oil, their ability to photosynthesize and in the structure and content of their triacylglycerols (TAG). The larger number of ESTs in these 16 datasets provided reliable estimates of the expression of acyltransferases and other enzymes expressed at low levels. Analysis of EST levels from these oilseeds revealed both conserved and distinct species-specific expression patterns for genes involved in the synthesis of glycerolipids and their precursors. Independent of the species and tissue type, ESTs for core fatty acid synthesis enzymes maintained a conserved stoichiometry and a strong correlation in temporal profiles throughout seed development. However, ESTs associated with non-plastid enzymes of oil biosynthesis displayed dissimilar temporal patterns indicative of different regulation. The EST levels for several genes potentially involved in accumulation of unusual TAG structures were distinct. Comparison of expression of members from multi-gene families allowed the identification of specific isoforms with conserved function in oil biosynthesis. In all four oilseeds, ESTs for Rubisco were present, suggesting its possible role in carbon metabolism, irrespective of light availability. Together, these data provide a resource for use in comparative and functional genomics of diverse oilseeds. Expression data for more than 350 genes encoding enzymes and proteins involved in lipid metabolism are available at the ‘ARALIP’ website (http://aralip.plantbiology.msu.edu/).

Identification of a Xylogalacturonan Xylosyltransferase Involved in Pectin Biosynthesis in Arabidopsis

Xylogalacturonan (XGA) is a class of pectic polysaccharide found in plant cell walls. The Arabidopsis thaliana locus At5g33290 encodes a predicted Type II membrane protein, and insertion mutants of the At5g33290 locus had decreased cell wall xylose. Immunological studies, enzymatic extraction of polysaccharides, monosaccharide linkage analysis, and oligosaccharide mass profiling were employed to identify the affected cell wall polymer. Pectic XGA was reduced to much lower levels in mutant than in wild-type leaves, indicating a role of At5g33290 in XGA biosynthesis. The mutated gene was designated xylogalacturonan deficient1 (xgd1). Transformation of the xgd1-1 mutant with the wild-type gene restored XGA to wild-type levels. XGD1 protein heterologously expressed in Nicotiana benthamiana catalyzed the transfer of xylose from UDP-xylose onto oligogalacturonides and endogenous acceptors. The products formed could be hydrolyzed with an XGA-specific hydrolase. These results confirm that the XGD1 protein is a XGA xylosyltransferase. The protein was shown by expression of a fluorescent fusion protein in N. benthamiana to be localized in the Golgi vesicles as expected for a glycosyltransferase involved in pectin biosynthesis.

The DUF579 domain containing proteins IRX15 and IRX15-L affect xylan synthesis in Arabidopsis

Xylan is the principal hemicellulose in the secondary cell walls of eudicots and in the primary and secondary cell walls of grasses and cereals. The biosynthesis of this important cell wall component has yet to be fully determined although a number of proteins have been shown to be required for xylan synthesis. To discover new genes involved in xylan biosynthesis we explored the psyllium (Plantago ovata Forsk) seed mucilaginous layer through EST profiling. This tissue synthesizes large amounts of a complex heteroxylan over a short period of time. By comparing abundant transcripts in this tissue with abundant transcripts specifically present during secondary cell wall formation in Arabidopsis thaliana, where glucuronoxylan biosynthesis is pronounced, we identified two Arabidopsis genes likely involved in xylan biosynthesis. These genes encode proteins containing a Domain of Unknown Function (DUF) 579 and were designated IRREGULAR XYLEM (IRX) 15 and IRX15-LIKE (IRX15-L). We obtained Arabidopsis T-DNA knockout lines for the two genes and analyzed their lower stems for changes in neutral monosaccharide composition. No changes were observed in each of these mutants, although the irx15 irx15-L double mutant displayed a moderate reduction in stem xylose. Further characterization of the irx15 irx15-L mutant revealed irregular secondary cell wall margins in fiber cells and a lower xylan degree of polymerization. Through these studies we conclude that IRX15 and IRX15-L function in a redundant manner and are involved in xylan biosynthesis.

The Cooperative Activities of CSLD2, CSLD3, and CSLD5 Are Required for Normal Arabidopsis Development

Glycosyltransferases of the Cellulose Synthase Like D (CSLD) subfamily have been reported to be involved in tip growth and stem development in Arabidopsis. The csld2 and csld3 mutants are root hair defective and the csld5 mutant has reduced stem growth. In this study, we produced double and triple knockout mutants of CSLD2, CSLD3, and CSLD5. Unlike the single mutants and the csld2/csld3 double mutant, the csld2/csld5, csld3/csld5, and csld2/ csld3/csld5 mutants were dwarfed and showed severely reduced viability. This demonstrates that the cooperative activities of CSLD2, CSLD3, and CSLD5 are required for normal Arabidopsis development, and that they are involved in important processes besides the specialized role in tip growth. The mutant phenotypes indicate that CSLD2 and CSLD3 have overlapping functions with CSLD5 in early plant development, whereas the CSLD2 and CSLD3 proteins are non-redundant. To determine the biochemical function of CSLD proteins, we used transient expression in tobacco leaves. Microsomes containing heterologously expressed CSLD5 transferred mannose from GDP-mannose onto endogenous acceptors. The same activity was detected when CSLD2 and CSLD3 were co-expressed but not when they were expressed separately. With monosaccharides as exogenous acceptors, microsomal preparations from CSLD5-expressing plants mediated the transfer of mannose from GDP-mannose onto mannose. These results were supported by immunodetection studies that showed reduced levels of a mannan epitope in the cell walls of stem interfascicular fibers and xylem vessels of the csld2/csld3/csld5 mutant.

RNA-Seq Analysis of Developing Nasturtium Seeds (Tropaeolum majus): Identification and Characterization of an Additional Galactosyltransferase Involved in Xyloglucan Biosynthesis

A deep-sequencing approach was pursued utilizing 454 and Illumina sequencing methods to discover new genes involved in xyloglucan biosynthesis. cDNA sequences were generated from developing nasturtium (Tropaeolum majus) seeds, which produce large amounts of non-fucosylated xyloglucan as a seed storage polymer. In addition to known xyloglucan biosynthetic genes, a previously uncharacterized putative xyloglucan galactosyltransferase was identified. Analysis of an Arabidopsis thaliana mutant line defective in the corresponding ortholog (AT5G62220) revealed that this gene shows no redundancy with the previously characterized xyloglucan galactosyltransferase, MUR3, but is required for galactosyl-substitution of xyloglucan at a different position. The gene was termed XLT2 for Xyloglucan L-side chain galactosylTransferase position 2. It represents an enzyme in the same subclade of glycosyltransferase family 47 as MUR3. A double mutant defective in both MUR3 (mur3.1) and XLT2 led to an Arabidopsis plant with xyloglucan that consists essentially of only xylosylated glucosyl units, with no further substitutions.

ARAD proteins associated with pectic Arabinan biosynthesis form complexes when transiently overexpressed in planta

Glycosyltransferase complexes are known to be involved in plant cell wall biosynthesis, as for example in cellulose. It is not known to what extent such complexes are involved in biosynthesis of pectin as well. To address this question, work was initiated on ARAD1 (ARABINAN DEFICIENT 1) and its close homolog ARAD2 of glycosyltransferase family GT47. Using bimolecular fluorescence complementation, Förster resonance energy transfer and non-reducing gel electrophoresis, we show that ARAD1 and ARAD2 are localized in the same Golgi compartment and form homo-and heterodimeric intermolecular dimers when expressed transiently in Nicotiana benthamiana. Biochemical analysis of arad2 cell wall or fractions hereof showed no difference in the monosaccharide composition, when compared with wild type. The double mutant arad1 arad2 had an arad1 cell wall phenotype and overexpression of ARAD2 did not complement the arad1 phenotype, indicating that ARAD1 and ARAD2 are not redundant enzymes. To investigate the cell wall structure of the mutants in detail, immunohistochemical analyses were carried out on arad1, arad2 and arad1 arad2 using the arabinan-specific monoclonal antibody LM13. In roots, the labeling pattern of arad2 was distinct from both that of wild type, arad1 and arad1 arad2. Likewise, in epidermal cell walls of inflorescence stems, LM13 binding differed between arad2 and WILD TYPE, arad1 or arad1 arad2. Altogether, these data show that ARAD2 is associated with arabinan biosynthesis, not redundant with ARAD1, and that the two glycosyltransferases may function in complexes held together by disulfide bridges.

Discovery of diversity in xylan biosynthetic genes by transcriptional profiling of a heteroxylan containing mucilaginous tissue

The exact biochemical steps of xylan backbone synthesis remain elusive. In Arabidopsis, three non-redundant genes from two glycosyltransferase (GT) families, IRX9 and IRX14 from GT43 and IRX10 from GT47, are candidates for forming the xylan backbone. In other plants, evidence exists that different tissues express these three genes at widely different levels, which suggests that diversity in the makeup of the xylan synthase complex exists. Recently we have profiled the transcripts present in the developing mucilaginous tissue of psyllium (Plantago ovata Forsk). This tissue was found to have high expression levels of an IRX10 homolog, but very low levels of the two GT43 family members. This contrasts with recent wheat endosperm tissue profiling that found a relatively high abundance of the GT43 family members. We have performed an in-depth analysis of all GTs genes expressed in four developmental stages of the psyllium mucilagenous layer and in a single stage of the psyllium stem using RNA-Seq. This analysis revealed several IRX10 homologs, an expansion in GT61 (homologs of At3g18170/At3g18180), and several GTs from other GT families that are highly abundant and specifically expressed in the mucilaginous tissue. Our current hypothesis is that the four IRX10 genes present in the mucilagenous tissues have evolved to function without the GT43 genes. These four genes represent some of the most divergent IRX10 genes identified to date. Conversely, those present in the psyllium stem are very similar to those in other eudicots. This suggests these genes are under selective pressure, likely due to the synthesis of the various xylan structures present in mucilage that has a different biochemical role than that present in secondary walls. The numerous GT61 family members also show a wide sequence diversity and may be responsible for the larger number of side chain structures present in the psyllium mucilage.

Light-load resistance exercise increases muscle protein synthesis and hypertrophy signaling in elderly men

The present study investigated whether well-tolerated light-load resistance exercise (LL-RE) affects skeletal muscle fractional synthetic rate (FSR) and anabolic intracellular signaling as a way to counteract age-related loss of muscle mass. Untrained healthy elderly (>65-yr-old) men were subjected to 13 h of supine rest. After 2.5 h of rest, unilateral LL-RE, consisting of leg extensions (10 sets, 36 repetitions) at 16% of 1 repetition maximum (RM), was conducted. Subsequently, the subjects were randomized to oral intake of 4 g of whey protein per hour (PULSE, n = 10), 28 g of whey protein at 0 h and 12 g of whey protein at 7 h postexercise (BOLUS, n = 10), or 4 g of maltodextrin per hour (placebo, n = 10). Quadriceps muscle biopsies were taken at 0, 3, 7, and 10 h postexercise from the resting and the exercised leg of each subject. Myofibrillar FSR and activity of select targets from the mechanistic target of rapamycin complex 1-signaling cascade were analyzed from the biopsies. LL-RE increased myofibrillar FSR compared with the resting leg throughout the 10-h postexercise period. Phosphorylated (T308) AKT expression increased in the exercised leg immediately after exercise. This increase persisted in the placebo group only. Levels of phosphorylated (T37/46) eukaryotic translation initiation factor 4E-binding protein 1 increased throughout the postexercise period in the exercised leg in the placebo and BOLUS groups and peaked at 7 h. In all three groups, phosphorylated (T56) eukaryotic elongation factor 2 decreased in response to LL-RE. We conclude that resistance exercise at only 16% of 1 RM increased myofibrillar FSR, irrespective of nutrient type and feeding pattern, which indicates an anabolic effect of LL-RE in elderly individuals. This finding was supported by increased signaling for translation initiation and translation elongation in response to LL-RE.

In the grass species Brachypodium distachyon, the production of mixed-linkage (1,3;1,4)-β-glucan (MLG) occurs in the Golgi apparatus

Mixed-linkage (1,3;1,4)-β-glucan (MLG) is a glucose polymer with beneficial effects on human health and high potential for the agricultural industry. MLG is present predominantly in the cell wall of grasses and is synthesized by cellulose synthase-like F or H families of proteins, with CSLF6 being the best-characterized MLG synthase. Although the function of this enzyme in MLG production has been established, the site of MLG synthesis in the cell is debated. It has been proposed that MLG is synthesized at the plasma membrane, as occurs for cellulose and callose; in contrast, it has also been proposed that MLG is synthesized in the Golgi apparatus, as occurs for other matrix polysaccharides of the cell wall. Testing these conflicting possibilities is fundamentally important in the general understanding of the biosynthesis of the plant cell wall. Using immuno-localization analyses with MLG-specific antibody in Brachypodium and in barley, we found MLG present in the Golgi, in post-Golgi structures and in the cell wall. Accordingly, analyses of a functional fluorescent protein fusion of CSLF6 stably expressed in Brachypodium demonstrated that the enzyme is localized in the Golgi. We also established that overproduction of MLG causes developmental and growth defects in Brachypodium as also occur in barley. Our results indicated that MLG production occurs in the Golgi similarly to other cell wall matrix polysaccharides, and supports the broadly applicable model in grasses that tight mechanisms control optimal MLG accumulation in the cell wall during development and growth. This work addresses the fundamental question of where mixed linkage (1,3;1,4)-β-glucan (MLG) is synthesized in plant cells. By analyzing the subcellular localization of MLG and MLG synthase in an endogenous system, we demonstrated that MLG synthesis occurs at the Golgi in Brachypodium and barley. A growth inhibition due to overproduced MLG in Brachypodium supports the general applicability of the model that a tight control of the cell wall polysaccharides accumulation is needed to maintain growth homeostasis during development.