Spatio‐temporal characterization of polyhydroxybutyrate accumulation in sugarcane

Abstract

SummaryWe 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.

EVALUATION OF OIL PALM LEAF-SPECIFIC PROMOTER (LSP1) ACTIVITY FOR EXPRESSING PHB GENES IN Arabidopsis thaliana

“…The number of granules increased in line with the maturity of the leaves indicating accumulation of the PHB polymer throughout the developmental stage of the A. thaliana plant (Figures 5b to 5d). This result is in accordance with other studies which also indicated that the accumulation of PHB granule being the lowest in the youngest leaves and increasing with the leaf age (Purnell et al, 2007;Somleva et al, 2008;Petrasovits et al, 2012). The PHB fluorescence granules were abundantly distributed throughout the leaf of palisade chlorophyll rather than sponge mesophyll.…”

“…The expression patterns of all the PHB genes were similar to findings from other studies (Purnell et al, 2007;Somleva et al, 2008;Parveez et al, 2015) where they found that in the PHB producing plants, the expression of phbC was the lowest among the three genes even though the genes were controlled by the same promoter.…”

“…PHB production in R. eutropha is catalysed by three enzymes namely β-ketothiolase, acetoacetyl-CoA reductase and PHB synthase, which are encoded by the genes phbA, phbB and phbC, respectively. Nowadays, production of PHB in plants have been demonstrated in many plant species such as A. thaliana (Poirier et al, 1992;Nawrath et al, 1994), tobacco (Nakashita et al, 1999), corn (Zhong et al, 2003), tamarix (Endo et al, 2006) and sugar cane (Petrasovits et al, 2007). The production of PHB in plants would reduce the production cost because it does not require expensive fermentation equipment and substrates (Scheller and Conrad, 2005).…”

Expression of bacterial genes in transgenic tobacco: methods, applications and future prospects

“…Recombinant bacteria were originally used to produce PHB, but production costs were too high compared to production of synthetic plastics (Choi and Lee, 1999). As an alternative, researchers have attempted to produce PHB in transgenic plants including Arabidopsis thaliana (Poirier et al 1992;Bohmert et al 2000), Brassica napus (Houmiel et al 1999), corn (Poirier and Gruys, 2002), potato (Bohmert et al 2002), sugarcane Purnell et al 2007), andtobacco (Bohmert et al 2002;Lössl et al 2003;Arai et al 2004).…”

High Levels of Bioplastic Are Produced in Fertile Transplastomic Tobacco Plants Engineered with a Synthetic Operon for the Production of Polyhydroxybutyrate

“…PHB synthesis in biomass crops of industrial interest such as switchgrass (Panicum virgatum; Somleva et al, 2008), sugarcane (Saccharum spp. hybrids; Petrasovits et al, 2007;Purnell et al, 2007), and corn stover (leaves and stock of Zea mays; Poirier and Gruys, 2002) have also been reported. Further work, however, is needed to increase levels in these crops for commercial purposes.…”