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.

Enhanced polyhydroxybutyrate production in transgenic sugarcane.

Polyhydroxybutyrate (PHB) is a bacterial polyester that has properties similar to some petrochemically produced plastics. Plant-based production has the potential to make this biorenewable plastic highly competitive with petrochemical-based plastics. We previously reported that transgenic sugarcane produced PHB at levels as high as 1.8% leaf dry weight without penalty to biomass accumulation, suggesting scope for improving PHB production in this species. In this study, we used different plant and viral promoters, in combination with multigene or single-gene constructs to increase PHB levels. Promoters tested included the maize and rice polyubiquitin promoters, the maize chlorophyll A/B-binding protein promoter and a Cavendish banana streak badnavirus promoter. At the seedling stage, the highest levels of polymer were produced in sugarcane plants when the Cavendish banana streak badnavirus promoter was used. However, in all cases, this promoter underwent silencing as the plants matured. The rice Ubi promoter enabled the production of PHB at levels similar to the maize Ubi promoter. The maize chlorophyll A/B-binding protein promoter enabled the production of PHB to levels as high as 4.8% of the leaf dry weight, which is approximately 2.5 times higher than previously reported levels in sugarcane. This is the first time that this promoter has been tested in sugarcane. The highest PHB-producing lines showed phenotypic differences to the wild-type parent, including reduced biomass and slight chlorosis.

Production of polyhydroxybutyrate in sugarcane.

We report here the production of the bacterial polyester, polyhydroxybutyrate (PHB), in the crop species sugarcane (Saccharum spp. hybrids). The PHB biosynthesis enzymes of Ralstonia eutropha [beta-ketothiolase (PHAA), acetoacetyl-reductase (PHAB) and PHB synthase (PHAC)] were expressed in the cytosol or targeted to mitochondria or plastids. PHB accumulated in cytosolic lines at trace amounts, but was not detected in mitochondrial lines. In plastidic lines, PHB accumulated in leaves to a maximum of 1.88% of dry weight without obvious deleterious effects. Epifluorescence and electron microscopy of leaf sections from these lines revealed that PHB granules were visible in plastids of most cell types, except mesophyll cells. The concentration of PHB in culm internodes of plastidic lines was substantially lower than in leaves. Western blot analysis of these lines indicated that expression of the PHB biosynthesis proteins was not limiting in culm internodes. Epifluorescence microscopy of culm internode sections from plastidic lines showed that PHB granules were visible in most cell types, except photosynthetic cortical cells in the rind, and that the lower PHB concentration in culm internodes was probably a result of dilution of PHB-containing cells by the large number of cells with little or no PHB. We discuss strategies for producing PHB in mitochondria and mesophyll cell plastids, and for increasing PHB yields in culms.

Spatio-temporal characterization of polyhydroxybutyrate accumulation in sugarcane.

We report here the results from a glasshouse trial of several transgenic sugarcane (Saccharum spp. hybrids) lines accumulating the bacterial polyester polyhydroxybutyrate (PHB) in plastids. The aims of the trial were to characterize the spatio-temporal pattern of PHB accumulation at a whole-plant level, to identify factors limiting PHB production and to determine whether agronomic performance was affected adversely by PHB accumulation. Statistical analysis showed that a vertical PHB concentration gradient existed throughout the plant, the polymer concentration being lowest in the youngest leaves and increasing with leaf age. In addition, there was a horizontal gradient along the length of a leaf, with the PHB concentration increasing from the youngest part of the leaf (the base) to the oldest (the tip). The rank order of the lines did not change over time. Moreover, there was a uniform spatio-temporal pattern of relative PHB accumulation among the lines, despite the fact that they showed marked differences in absolute PHB concentration. Molecular analysis revealed that the expression of the transgenes encoding the PHB biosynthesis enzymes was apparently coordinated, and that there were good correlations between PHB concentration and the abundance of the PHB biosynthesis enzymes. The maximum recorded PHB concentration, 1.77% of leaf dry weight, did not confer an agronomic penalty. The plant height, total aerial biomass and culm-internode sugar content were not affected relative to controls. Although moderate PHB concentrations were achieved in leaves, the maximum total-plant PHB yield was only 0.79% (11.9 g PHB in 1.51 kg dry weight). We combine the insights from our statistical and molecular analyses to discuss possible strategies for increasing the yield of PHB in sugarcane.

Transformation and transposon mutagenesis of Leifsonia xyli subsp. xyli, causal organism of ratoon stunting disease of sugarcane.

Conditions have been developed for genetic transformation and insertional mutagenesis in Leifsonia xyli subsp. xyli (Lxx), the causal organism of ratoon stunting disease (RSD), one of the most damaging and intractable diseases of sugarcane internationally. Transformation frequencies ranged from 1 to 10 colony forming units (CFU)/microg of plasmid DNA using Clavibacter/Escherichia coli shuttle vectors pCG188, pDM302, and pDM306 and ranged from 50 to 500 CFU/microg using cosmid cloning vectors pLAFR3 and pLAFR5-km. The transformation/transposition frequency was 0 to 70 CFU/microg of DNA, using suicide vectors pUCD623 and pSUP2021 containing transposable elements Tn4431 and Tn5, respectively. It was necessary to grow Lxx in media containing 0.1% glycine for electroporation and to amplify large plasmids in a dam-/dcm- E. coli strain and purify the DNA by anion exchange. To keep selection pressure at an optimum, the transformants were grown on nitrocellulose filters (0.2-microm pore size) on media containing the appropriate antibiotics. Transposon Tn4431 containing a promoterless lux operon from Vibrio fischeri and a tetracycline-resistance gene was introduced on the suicide vector pUCD623. All but 1% of the putative transposon mutants produce light, indicating transposition into functional Lxx genes. Southern blot analysis of these transformants indicates predominantly single transposon insertions at unique sites. The cosmid cloning vector pLAFR5-km was stably maintained in Lxx. The development of a transformation and transposon mutagenesis system opens the way for molecular analysis of pathogenicity determinants in Lxx.

Establishment of a functional genomics platform for Leifsonia xyli subsp. xyli.

Leifsonia xyli subsp. xyli, the causal agent of ratoon stunting disease in sugarcane, is a xylem-limited, nutritionally fastidious, slow growing, gram-positive coryneform bacterium. Because of the difficulties in growing this bacterium in pure culture, little is known about the molecular mechanisms of pathogenesis. Currently, the genome sequence of L. xyli subsp. xyli is being completed by the Agronomical and Environmental Genomes group from the Organization for Nucleotide Sequencing and Analysis in Brazil. To complement this work, we produced 712 Lxx::Tn4431 transposon mutants and sequenced flanking regions from 383 of these, using a rapid polymerase chain reaction-based approach. Tn4431 insertions appeared to be widespread throughout the L. xyli subsp. xyli genome; however, there were regions that had significantly higher concentrations of insertions. The Tn4431 mutant library was screened for individuals unable to colonize sugarcane, and one noncolonizing mutant was found. The mutant contained a transposon insertion disrupting two open reading frames (ORF), one of which had homology to an integral membrane protein from Mycobacterium leprae. Sequencing of the surrounding regions revealed two operons, pro and cyd, both of which are believed to play roles in disease. Complementation studies were carried out using the noncolonizing Lxx::Tn4431 mutant. The noncolonizing mutant was transformed with a cosmid containing 40 kbp of wild-type sequence, which included the two ORF disrupted in the mutant, and several transformants were subsequently able to colonize sugarcane. However, analysis of each of these transformants, before and after colonization, suggests that they have all undergone various recombinant events, obscuring the roles of these ORF in L. xyli subsp. xyli pathogenesis.

Protein blotting protocol for beginners.

The transfer and immobilization of biological macromolecules onto solid nitrocellulose or nylon (polyvinylidene difluoride (PVDF)) membranes subsequently followed by specific detection is referred to as blotting. DNA blots are called Southerns after the inventor of the technique, Edwin Southern. By analogy, RNA blots are referred to as northerns and protein blots as westerns (Burnette, Anal Biochem 112:195-203, 1981). With few exceptions, western blotting involves five steps, namely, sample collection, preparation, separation, immobilization, and detection. In this chapter, protocols for the entire process from sample collection to detection are described.