PHA Production in Plant Peroxisomes

Polyhydroxyalkanoates (PHAs) are a group of bacterial carbon storage polymers which possess diverse plastic-like properties similar to polypropylene and polyethylene. However, unlike their petroleum-derived counterparts, these biorenewable polyesters are fully biodegradable. A wide variety of bacteria synthesise PHAs, with unique monomer units that differ from one another in the R side group attached to the hydroxyalkanoate core. In the Gram-negative bacterium Ralstonia eutropha, polyhydroxybutyrate (PHB), the simplest form of PHA, is produced by a three enzyme pathway. This pathway is driven by the successive activities of three enzymes, a β-ketothiolase, acetoacetyl-CoA reductase and PHA synthase, which are commonly referred to as PhaARe, PhaBRe and PhaCRe. PhaARe condenses two molecules of acetyl-CoA to give acetoacetyl-CoA which is subsequently reduced to hydroxybutyryl-CoA through the action of PhaBRe using one molecule of NADPH. Finally, this hydroxybutyryl-CoA monomer is polymerised to form PHB by PhaCRe. Through expression of such bacterial transgenes, PHB has previously been produced in a number of different plant species. Current research is focused on improving PHA production, including PHB, in the high biomass crop sugarcane (Saccharum sp interspecific hybrids) because this could lead to cost-competitive commercial production of this class of bioplastics. This thesis work is part of a wider research project in plant metabolic engineering where plant cell organelles are being studied to find optimal ways to produce high levels of PHAs and other bioplastic precursors. In this respect, one possibility is to focus on PHA production in plant peroxisomes. In plants, peroxisomes are known to be involved in the catabolism of straight chain fatty acids through the β-oxidation cycle. The peroxisomal fatty acid β-oxidation cycle generates a wealth of substrates for PHA biosynthesis. To further evaluate PHA production in plant peroxisomes, the three enzyme R. eutropha PHA biosynthetic pathway was engineered into peroxisomes in transgenic sugarcane plants. Initially, transient expression analysis of green fluorescent protein (GFP) fusion proteins was used to determine minimal peroxisomal targeting signal type 1 sequences sufficient to target each PHA biosynthetic enzyme. Peroxisomal-targeted PhaARe, PhaBRe and PhaCRe were then constitutively expressed in sugarcane using the maize polyubiquitin promoter. In the resulting transgenic lines, PHB was produced up to 1.6% dry weight in mature leaf tissue, which is on par with previous results when the same R. eutropha PHA pathway under the control of the same promoter was targeted to plastids. PHB biosynthesis at this level was not accompanied by any obvious growth effects. Polymer accumulated throughout most leaf cell types and subcellular localisation studies revealed polymer accumulating in both peroxisomes and vacuoles. Further characterisation of the PHA revealed peroxisomal biosynthesis of both 3-hydroxybutyrate (3HB) and 3-hydroxyvalyrate (3HV) monomers, forming P(HB-co-HV) copolymer. 3HB and 3HV accumulated at levels approximately 95% and 5% of total polymer, respectively. Transgenic Arabidopsis thaliana plants expressing peroxisomal-targeted PhaARe, PhaBRe and PhaCRe were also generated as a model system. Analysis of several independent A. thaliana peroxisomal PHA producing lines revealed PHA accumulation up to 1.8% dry weight in dark grown etiolated seedlings, between 0.1% and 0.16% dry weight in 10-day-old seedlings, and between 0.06% and 0.1% dry weight in 21 day old plants. P(3HB-co-3HV) copolymer was also found in these A. thaliana lines, with 3HB and 3HV monomers present in three-week-old plants in a similar ratio as measured in sugarcane. Peroxisomal PHA biosynthesis in A. thaliana had only a minor effect on plant growth as seen in etiolated hypocotyl elongation where tissue expansion was artificially dependent on peroxisomal fatty acid β-oxidation. Evidence in the literature suggests that substrate availability limits peroxisomal PHA biosynthesis in plants. Therefore several metabolic engineering strategies to overcome substrate limitation and increase peroxisomal PHA production were designed. These strategies focused on either reducing competition for peroxisomal PHA substrates by other metabolic pathways or boosting PHA substrate pools contained in the peroxisomes. Since acetyl-CoA exits peroxisomes via the formation of citrate, endogenous expression of A. thaliana peroxisomal citrate synthase genes were knocked down using artificial microRNA interference technology. A reduction in peroxisomal citrate synthase activity in conjunction with peroxisomal PHA biosynthesis increased total PHB accumulation. Higher level PHB accumulation was observed in A. thaliana peroxisomal PHA producing plants grown in liquid culture supplemented with Tween-20, a source of lauric acid (12:0).This demonstrated that peroxisomal PHA biosynthesis is limited in part by fatty acid flux through the β-oxidation cycle. These same A. thaliana plants were also fed an exogenous supply of cis-10-heptadecanoic acid which dramatically increased the proportion of total polymer comprised of HV monomer. This demonstrated that the biosynthesis of HV monomer is dependent on the presence of suitable substrate molecules, such as those arising from the β-oxidation of odd-chain-length fatty acids. A stable increase in fatty acid flux through the peroxisomal β-oxidation cycle of sugarcane and A. thaliana peroxisomal PHA producing plants was attempted with co-expression of the heterologous thioesterase FatB3 from Cuphea lanceolata. However, no increase in peroxisomal PHA biosynthesis was observed. Overall, this study reveals the potential of peroxisomal PHA biosynthesis in plants. A positive evaluation of select metabolic engineering strategies in A. thaliana warrants further application of similar strategies in sugarcane and other high biomass crops. The continuation of this work may result in peroxisomal PHA biosynthesis reaching commercially viable levels in crop plants, in either isolation or conjunction with PHA biosynthesis in other organelles of the same plant

The UVR8 UV-B Photoreceptor: Perception, Signaling and Response

Ultraviolet-B radiation (UV-B) is an intrinsic part of sunlight that is accompanied by significant biological effects. Plants are able to perceive UV-B using the UV-B photoreceptor UVR8 which is linked to a specific molecular signaling pathway and leads to UV-B acclimation. Herein we review the biological process in plants from initial UV-B perception and signal transduction through to the known UV-B responses that promote survival in sunlight. The UVR8 UV-B photoreceptor exists as a homodimer that instantly monomerises upon UV-B absorption via specific intrinsic tryptophans which act as UV-B chromophores. The UVR8 monomer interacts with COP1, an E3 ubiquitin ligase, initiating a molecular signaling pathway that leads to gene expression changes. This signaling output leads to UVR8-dependent responses including UV-B-induced photomorphogenesis and the accumulation of UV-B-absorbing flavonols. Negative feedback regulation of the pathway is provided by the WD40-repeat proteins RUP1 and RUP2, which facilitate UVR8 redimerization, disrupting the UVR8-COP1 interaction. Despite rapid advancements in the field of recent years, further components of UVR8 UV-B signaling are constantly emerging, and the precise interplay of these and the established players UVR8, COP1, RUP1, RUP2 and HY5 needs to be defined. UVR8 UV-B signaling represents our further understanding of how plants are able to sense their light environment and adjust their growth accordingly

Photoreceptor-Mediated Bending towards UV-B in Arabidopsis

Plants reorient their growth towards light to optimize photosynthetic light capture-a process known as phototropism. Phototropins are the photoreceptors essential for phototropic growth towards blue and ultraviolet-A (UV-A) light. Here we detail a phototropic response towards UV-B in etiolated Arabidopsis seedlings. We report that early differential growth is mediated by phototropins but clear phototropic bending to UV-B is maintained in phot1 phot2 double mutants. We further show that this phototropin-independent phototropic response to UV-B requires the UV-B photoreceptor UVR8. Broad UV-B-mediated repression of auxin-responsive genes suggests that UVR8 regulates directional bending by affecting auxin signaling. Kinetic analysis shows that UVR8-dependent directional bending occurs later than the phototropin response. We conclude that plants may use the full short-wavelength spectrum of sunlight to efficiently reorient photosynthetic tissue with incoming light

Revisiting chromatin binding of the Arabidopsis UV-B photoreceptor UVR8

Plants perceive UV-B through the UV RESISTANCE LOCUS 8 (UVR8) photoreceptor and UVR8 activation leads to changes in gene expression such as those associated with UV-B acclimation and stress tolerance. Albeit functionally unrelated, UVR8 shows some homology with RCC1 (Regulator of Chromatin Condensation 1) proteins from non-plant organisms at the sequence level. These proteins act as guanine nucleotide exchange factors for Ran GTPases and bind chromatin via histones. Subsequent to the revelation of this sequence homology, evidence was presented showing that UVR8 activity involves interaction with chromatin at the loci of some target genes through histone binding. This suggested a UVR8 mode-of-action intimately and directly linked with gene transcription. However, several aspects of UVR8 chromatin association remained undefined, namely the impact of UV-B on the process and how UVR8 chromatin association related to the transcription factor ELONGATED HYPOCOTYL 5 (HY5), which is important for UV-B signalling and has overlapping chromatin targets. Therefore, we have investigated UVR8 chromatin association in further detail

UV-B Perception and Acclimation in Chlamydomonas reinhardtii

Plants perceive UV-B, an intrinsic component of sunlight, via a signaling pathway that is mediated by the photoreceptor UV RESISTANCE LOCUS8 (UVR8) and induces UV-B acclimation. To test whether similar UV-B perception mechanisms exist in the evolutionarily distant green alga Chlamydomonas reinhardtii, we identified Chlamydomonas orthologs of UVR8 and the key signaling factor CONSTITUTIVELY PHOTOMORPHOGENIC1 (COP1). Cr-UVR8 shares sequence and structural similarity to Arabidopsis thaliana UVR8, has conserved tryptophan residues for UV-B photoreception, monomerizes upon UV-B exposure, and interacts with Cr-COP1 in a UV-B-dependent manner. Moreover, Cr-UVR8 can interact with At-COP1 and complement the Arabidopsis uvr8 mutant, demonstrating that it is a functional UV-B photoreceptor. Chlamydomonas shows apparent UV-B acclimation in colony survival and photosynthetic efficiency assays. UV-B exposure, at low levels that induce acclimation, led to broad changes in the Chlamydomonas transcriptome, including in genes related to photosynthesis. Impaired UV-B-induced activation in the Cr-COP1 mutant hit1 indicates that UVR8-COP1 signaling induces transcriptome changes in response to UV-B. Also, hit1 mutants are impaired in UV-B acclimation. Chlamydomonas UV-B acclimation preserved the photosystem II core proteins D1 and D2 under UV-B stress, which mitigated UV-B-induced photoinhibition. These findings highlight the early evolution of UVR8 photoreceptor signaling in the green lineage to induce UV-B acclimation and protection

Heterotrimeric G Protein γ Subunits Provide Functional Selectivity in Gβγ Dimer Signaling in Arabidopsis[OA]

The Arabidopsis thaliana heterotrimeric G protein complex is encoded by single canonical Gα and Gβ subunit genes and two Gγ subunit genes (AGG1 and AGG2), raising the possibility that the two potential G protein complexes mediate different cellular processes. Mutants with reduced expression of one or both Gγ genes revealed specialized roles for each Gγ subunit. AGG1-deficient mutants, but not AGG2-deficient mutants, showed impaired resistance against necrotrophic pathogens, reduced induction of the plant defensin gene PDF1.2, and decreased sensitivity to methyl jasmonate. By contrast, both AGG1- and AGG2-deficient mutants were hypersensitive to auxin-mediated induction of lateral roots, suggesting that Gβγ1 and Gβγ2 synergistically inhibit auxin-dependent lateral root initiation. However, the involvement of each Gγ subunit in this root response differs, with Gβγ1 acting within the central cylinder, attenuating acropetally transported auxin signaling, while Gβγ2 affects the action of basipetal auxin and graviresponsiveness within the epidermis and/or cortex. This selectivity also operates in the hypocotyl. Selectivity in Gβγ signaling was also found in other known AGB1-mediated pathways. agg1 mutants were hypersensitive to glucose and the osmotic agent mannitol during seed germination, while agg2 mutants were only affected by glucose. We show that both Gγ subunits form functional Gβγ dimers and that each provides functional selectivity to the plant heterotrimeric G proteins, revealing a mechanism underlying the complexity of G protein–mediated signaling in plants

Additional file 3: of Revisiting chromatin binding of the Arabidopsis UV-B photoreceptor UVR8

UVR8 and HY5 do not interact in yeast. (PDF 143 kb

Additional file 5: of Revisiting chromatin binding of the Arabidopsis UV-B photoreceptor UVR8

List of primers used for ChIP-qPCR in this work. (PDF 104 kb