A temperature-sensitive allele of a putative mRNA splicing helicase down-regulates many cell wall genes and causes radial swelling in Arabidopsis thaliana.

The putative RNA helicase encoded by the Arabidopsis gene At1g32490 is a homolog of the yeast splicing RNA helicases Prp2 and Prp22. We isolated a temperature-sensitive allele (rsw12) of the gene in a screen for root radial swelling mutants. Plants containing this allele grown at the restrictive temperature showed weak radial swelling, were stunted with reduced root elongation, and contained reduced levels of cellulose. The role of the protein was further explored by microarray analysis. By using both fold change cutoffs and a weighted gene coexpression network analysis (WGCNA) to investigate coexpression of genes, we found that the radial swelling phenotype was not linked to genes usually associated with primary cell wall biosynthesis. Instead, the mutation has strong effects on expression of secondary cell wall related genes. Many genes potentially associated with secondary walls were present in the most significant WGCNA module, as were genes coding for arabinogalactans and proteins with GPI anchors. The proportion of up-regulated genes that possess introns in rsw12 was above that expected if splicing was unrelated to the activity of the RNA helicase, suggesting that the helicase does indeed play a role in splicing in Arabidopsis. The phenotype may be due to a change in the expression of one or more genes coding for cell wall proteins.

Systems biology and metabolic modelling unveils limitations to polyhydroxybutyrate accumulation in sugarcane leaves; lessons for C4 engineering.

In planta production of the bioplastic polyhydroxybutyrate (PHB) is one important way in which plant biotechnology can address environmental problems and emerging issues related to peak oil. However, high biomass C4 plants such as maize, switch grass and sugarcane develop adverse phenotypes including stunting, chlorosis and reduced biomass as PHB levels in leaves increase. In this study, we explore limitations to PHB accumulation in sugarcane chloroplasts using a systems biology approach, coupled with a metabolic model of C4 photosynthesis. Decreased assimilation was evident in high PHB-producing sugarcane plants, which also showed a dramatic decrease in sucrose and starch content of leaves. A subtle decrease in the C/N ratio was found which was not associated with a decrease in total protein content. An increase in amino acids used for nitrogen recapture was also observed. Based on the accumulation of substrates of ATP-dependent reactions, we hypothesized ATP starvation in bundle sheath chloroplasts. This was supported by mRNA differential expression patterns. The disruption in ATP supply in bundle sheath cells appears to be linked to the physical presence of the PHB polymer which may disrupt photosynthesis by scattering photosynthetically active radiation and/or physically disrupting thylakoid membranes.

The use of an acetoacetyl-CoA synthase in place of a ß-ketothiolase enhances poly-3-hydroxybutyrate production in sugarcane mesophyll cells.

Engineering the production of polyhydroxyalkanoates (PHAs) into high biomass bioenergy crops has the potential to provide a sustainable supply of bioplastics and energy from a single plant feedstock. One of the major challenges in engineering C4 plants for the production of poly[(R)-3-hydroxybutyrate] (PHB) is the significantly lower level of polymer produced in the chloroplasts of mesophyll (M) cells compared to bundle sheath (BS) cells, thereby limiting the full PHB yield-potential of the plant. In this study, we provide evidence that the access to substrate for PHB synthesis may limit polymer production in M chloroplasts. Production of PHB in M cells of sugarcane is significantly increased by replacing ß-ketothiolase, the first enzyme in the bacterial PHA pathway, with acetoacetyl-CoA synthase. This novel pathway enabled the production of PHB reaching an average of 6.3% of the dry weight of total leaf biomass, with levels ranging from 3.6 to 11.8% of the dry weight (DW) of individual leaves. These yields are more than twice the level reported in PHB-producing sugarcane containing the ß-ketothiolase and illustrate the importance of producing polymer in mesophyll plastids to maximize yield. The molecular weight of the polymer produced was greater than 2 × 10(6)  Da. These results are a major step forward in engineering a high biomass C4 grass for the commercial production of PHB.

Factors affecting polyhydroxybutyrate accumulation in mesophyll cells of sugarcane and switchgrass.

BACKGROUND: Polyhydroxyalkanoates are linear biodegradable polyesters produced by bacteria as a carbon store and used to produce a range of bioplastics. Widespread polyhydroxyalkanoate production in C4 crops would decrease petroleum dependency by producing a renewable supply of biodegradable plastics along with residual biomass that could be converted into biofuels or energy. Increasing yields to commercial levels in biomass crops however remains a challenge. Previously, lower accumulation levels of the short side chain polyhydroxyalkanoate, polyhydroxybutyrate (PHB), were observed in the chloroplasts of mesophyll (M) cells compared to bundle sheath (BS) cells in transgenic maize (Zea mays), sugarcane (Saccharum sp.), and switchgrass (Panicum virgatum L.) leading to a significant decrease in the theoretical yield potential. Here we explore various factors which might affect polymer accumulation in mesophyll cells, including targeting of the PHB pathway enzymes to the mesophyll plastid and their access to substrate. RESULTS: The small subunit of Rubisco from pea effectively targeted the PHB biosynthesis enzymes to both M and BS chloroplasts of sugarcane and switchgrass. PHB enzyme activity was retained following targeting to M plastids and was equivalent to that found in the BS plastids. Leaf total fatty acid content was not affected by PHB production. However, when fatty acid synthesis was chemically inhibited, polymer accumulated in M cells. CONCLUSIONS: In this study, we provide evidence that access to substrate and neither poor targeting nor insufficient activity of the PHB biosynthetic enzymes may be the limiting factor for polymer production in mesophyll chloroplasts of C4 plants.

Chemical inhibition of acetyl coenzyme A carboxylase as a strategy to increase polyhydroxybutyrate yields in transgenic sugarcane.

Polyhydroxybutyrate (PHB) is a naturally occurring bacterial polymer that can be used as a biodegradable replacement for some petrochemical-derived plastics. Polyhydroxybutyrate is produced commercially by fermentation, but to reduce production costs, efforts are underway to produce it in engineered plants, including sugarcane. However, PHB levels in this high-biomass crop are not yet commercially viable. Chemical ripening with herbicides is a strategy used to enhance sucrose production in sugarcane and was investigated here as a tool to increase PHB production. Class A herbicides inhibit ACCase activity and thus reduce fatty acid biosynthesis, with which PHB production competes directly for substrate. Treatment of PHB-producing transgenic sugarcane plants with 100 µM of the class A herbicide fluazifop resulted in a fourfold increase in PHB content in the leaves, which peaked ten days post-treatment. The minimum effective concentration of herbicide required to maximize PHB production was 30 µM for fluazifop and 70 µM for butroxydim when applied to saturation. Application of a range of class A herbicides from the DIM and FOP groups consistently resulted in increased PHB yields, particularly in immature leaf tissue. Butroxydim or fluazifop treatment of mature transgenic sugarcane grown under glasshouse conditions increased the total leaf biomass yield of PHB by 50%-60%. Application of an ACCase inhibitor in the form of a class A herbicide to mature sugarcane plants prior to harvest is a promising strategy for improving overall PHB yield. Further testing is required on field-grown transgenic sugarcane to more precisely determine the effectiveness of this strategy.

Co-utilization of acidified glycerol pretreated-sugarcane bagasse for microbial oil production by a novel Rhodosporidium strain.

Acidified glycerol pretreatment is very effective to deconstruct lignocellulosics for producing glucose. Co-utilization of pretreated biomass and residual glycerol to bioproducts could reduce the costs associated with biomass wash and solvent recovery. In this study, a novel strain Rhodosporidium toruloides RP 15, isolated from sugarcane bagasse, was selected and tested for coconversion of pretreated biomass and residual glycerol to microbial oils. In the screening trails, Rh. toruloides RP 15 demonstrated the highest oil production capacity on glucose, xylose, and glycerol among the 10 strains. At the optimal C:N molar ratio of 140:1, this strain accumulated 56.7, 38.3, and 54.7% microbial oils based on dry cell biomass with 30 g/L glucose, xylose, and glycerol, respectively. Furthermore, sugarcane bagasse medium containing 32.6 g/L glucose from glycerol-pretreated bagasse and 23.4 g/L glycerol from pretreatment hydrolysate were used to produce microbial oils by Rh. toruloides RP 15. Under the preliminary conditions without pH control, this strain produced 7.7 g/L oil with an oil content of 59.8%, which was comparable or better than those achieved with a synthetic medium. In addition, this strain also produced 3.5 mg/L carotenoid as a by-product. It is expected that microbial oil production can be significantly improved through process optimization.

Arabidopsis dynamin-like protein DRP1A: a null mutant with widespread defects in endocytosis, cellulose synthesis, cytokinesis, and cell expansion.

Dynamin-related proteins are large GTPases that deform and cause fission of membranes. The DRP1 family of Arabidopsis thaliana has five members of which DRP1A, DRP1C, and DRP1E are widely expressed. Likely functions of DRP1A were identified by studying rsw9, a null mutant of the Columbia ecotype that grows continuously but with altered morphology. Mutant roots and hypocotyls are short and swollen, features plausibly originating in their cellulose-deficient walls. The reduction in cellulose is specific since non-cellulosic polysaccharides in rsw9 have more arabinose, xylose, and galactose than those in wild type. Cell plates in rsw9 roots lack DRP1A but still retain DRP1E. Abnormally placed and often incomplete cell walls are preceded by abnormally curved cell plates. Notwithstanding these division abnormalities, roots and stems add new cells at wild-type rates and organ elongation slows because rsw9 cells do not grow as long as wild-type cells. Absence of DRP1A reduces endocytotic uptake of FM4-64 into the cytoplasm of root cells and the hypersensitivity of elongation and radial swelling in rsw9 to the trafficking inhibitor monensin suggests that impaired endocytosis may contribute to the development of shorter fatter roots, probably by reducing cellulose synthesis.

A mutation in an Arabidopsis ribose 5-phosphate isomerase reduces cellulose synthesis and is rescued by exogenous uridine.

The Arabidopsis radial swelling mutant rsw10 showed ballooning of root trichoblasts, a lower than wild-type level of cellulose and altered levels of some monosaccharides in non-cellulosic polysaccharides. Map-based cloning showed that the mutated gene (At1g71100) encodes a ribose 5-phosphate isomerase (RPI) and that the rsw10 mutation replaces a conserved glutamic acid residue with lysine. Although RPI is intimately involved with many biochemical pathways, media supplementation experiments suggest that the visible phenotype results from a defect in the production of pyrimidine-based sugar-nucleotide compounds, most likely uridine 5'-diphosphate-glucose, the presumed substrate of cellulose synthase. Two of three RPI sequences in the nuclear genome are cytoplasmic, while the third has a putative chloroplast transit sequence. The sequence encoding both cytoplasmic enzymes could complement the mutation when expressed behind the CaMV 35S promoter, while fusion of the RSW10 promoter region to the GUS reporter gene established that the gene is expressed in many aerial tissues as well as the roots. The prominence of the rsw10 phenotype in roots probably reflects RSW10 being the only cytosolic RPI in this tissue and the gene encoding the plastid RPI being relatively weakly expressed. We could not, however, detect a decrease in total RPI activity in root extracts. The rsw10 phenotype is prominent near the root tip where cells undergo division, endoreduplication and cell expansion and so are susceptible to a restriction in de novo pyrimidine production. The two cytosolic RPIs probably arose in an ancient duplication event, their present expression patterns representing subfunctionalization of the expression of the original ancestral gene.

Genes encoding ADP-ribosylation factors in Arabidopsis thaliana L. Heyn.; genome analysis and antisense suppression.

Vesicle trafficking delivers proteins to intracellular and extracellular compartments, cellulose synthase to the plasma membrane, and non-cellulosic polysaccharides to the cell wall. The Arabidopsis genome potentially encodes 19 proteins with sequence similarities to ARFs (ADP-ribosylation factors) and its relatives such as ARLs (ARF-like proteins). ARFs are essential for vesicle coating and uncoating in all eukaryotic cells, while ARLs play more diverse roles. Nine proteins, six of them highly similar, are possible ARFs, three are putative ARL orthologues and the remainder were designated ARF-related proteins. The functions of the six highly similar, putative ARFs in whole plant development were probed by suppressing their expression with antisense. Antisense plants were severely stunted because cell production rate and final cell size were both reduced. Changed time-to-flowering, apical dominance, and fertility may reflect alterations to hormonal and other signalling pathways with which ARFs may interact. No gross changes in targeting or compartmentalization were seen in antisense plants containing GFP targeted to the ER and Golgi and changes in cell wall composition were limited to increases in some non-cellulosic polysaccharides and a relatively small decrease in cellulose. The reasons why these effects are less drastic than the effects on endomembranes and wall composition that are seen in short-term experiments with brefeldin A and with dominant negative ARF mutants are discussed.