Charles H. Hocart

Fatty acids and β-carotene in Australian purslane (Portulaca oleracea) varieties.

Abstract The fatty acid profile and β-carotene content of a number of Australian varieties of purslane ( Portulaca oleracea ) were determined by GC and HPLC. The total fatty acid content ranged from 1.5 to 2.5 mg/g of fresh mass in leaves, 0.6 to 0.9 mg/g in stems and 80 to 170 mg/g in seeds. α-Linolenic acid (C 18:3ω3 ) accounted for around 60% and 40% of the total fatty acid content in leaves and seeds, respectively. Longer-chain omega-3 fatty acids were not detected. The β-carotene content ranged from 22 to 30 mg/g fresh mass in leaves. These results indicate that Australian purslane varieties are a rich source of α-linolenic acid and β-carotene.

Fatty acid profiling of Chlamydomonas reinhardtii under nitrogen deprivation

The Chlamydomonas reinhardtii starch-less mutant, BAF-J5, was found to store lipids up to 65% of dry cell weight when grown photoheterotrophically and subjected to nitrogen starvation. Fourier transform infrared spectroscopy was used as a high-throughput method for semi-quantitative measurements of protein, carbohydrate and lipid content. The fatty acids of wild-type and starch mutants were identified and quantified by gas chromatography mass spectrometry. C. reinhardtii starch mutants, BAF-J5 and I7, produce significantly elevated levels of 16:0, 18:1(Δ9), 18:2(Δ9,12) and 18:3(Δ9,12,15) fatty acids. Long-chain saturated, mono- and polyunsaturated fatty acids were found under nitrogen starvation. Oleosin-like and caleosin-like genes were identified in the C. reinhardtii genome. However, proteomic analysis of isolated lipid bodies only identified a key lipid droplet associated protein. This study shows it is possible to manipulate algal biosynthetic pathways to produce high levels of lipid that may be suitable for conversion to liquid fuels.

Fractionation of carbohydrates in Arabidopsis root cell walls shows that three radial swelling loci are specifically involved in cellulose production

Abstract. Three non-allelic radial swelling mutants (rsw1, rsw2 and rsw3) of Arabidopsisthaliana L. Heynh. were shown to be specifically impaired in cellulose production. Fractionation methods that identify, characterise and quantify some of the major cell wall polysaccharides in small quantities of seedlings demonstrated that changes in the production of cellulose are much more pronounced than changes in the production of non-cellulosic polysaccharides. A crude cell wall pellet was sequentially extracted with chloroform methanol (to recover lipids), dimethyl sulphoxide (starch), ammonium oxalate (pectins) and alkali (hemicelluloses). Crystalline cellulose remained insoluble through subsequent treatments with an acetic/nitric acid mixture and with trifluoroacetic acid. Cetyltrimethylammonium bromide precipitation resolved neutral and acidic polymers in the fractions, and precipitation behaviour, monosaccharide composition and glycosidic linkage patterns identified the major polysaccharides. The deduced composition of the walls of wild-type seedlings and the structure and solubility properties of the major polymers were broadly typical of other dicots. The three temperature-sensitive, radial swelling mutants produced less cellulose in their roots than the wild type when grown at their restrictive temperature (31 °C). There were no significant differences at 21 °C where no radial swelling occurs. The limited changes seen in the monosaccharide compositions, glycosidic linkage patterns and quantities of non-cellulosic polysaccharides support the view that the RSW1, RSW2 and RSW3 genes are specifically involved in cellulose synthesis. Reduced deposition of cellulose was accompanied by increased accumulation of starch.

Functional analysis of the cellulose synthase genes CesA1, CesA2, and CesA3 in Arabidopsis.

Polysaccharide analyses of mutants link several of the glycosyltransferases encoded by the 10 CesA genes of Arabidopsis to cellulose synthesis. Features of those mutant phenotypes point to particular genes depositing cellulose predominantly in either primary or secondary walls. We used transformation with antisense constructs to investigate the functions of CesA2(AthA) and CesA3 (AthB), genes for which reduced synthesis mutants are not yet available. Plants expressing antisense CesA1 (RSW1) provided a comparison with a gene whose mutant phenotype (Rsw1−) points mainly to a primary wall role. The antisense phenotypes of CesA1 and CesA3were closely similar and correlated with reduced expression of the target gene. Reductions in cell length rather than cell number underlay the shorter bolts and stamen filaments. Surprisingly, seedling roots were unaffected in both CesA1 and CesA3antisense plants. In keeping with the mild phenotype compared with Rsw1−, reductions in total cellulose levels in antisenseCesA1 and CesA3 plants were at the borderline of significance. We conclude that CesA3, likeCesA1, is required for deposition of primary wall cellulose. To test whether there were important functional differences between the two, we overexpressed CesA3 inrsw1 but were unable to complement that mutants defect in CesA1. The function of CesA2 was less obvious, but, consistent with a role in primary wall deposition, the rate of stem elongation was reduced in antisense plants growing rapidly at 31°C.

Increased endogenous cytokinin in the Arabidopsis amp1 mutant corresponds with de-etiolation responses

The high-cytokinin Arabidopsis thaliana (L.) Heynh. mutant amp1 of has been further characterised. We extend our previous work on the cytokinin level in the amp1 mutant and show that it contains high levels of endogenous cytokinins in both light- and dark-grown plants, and that amp1 can be phenocopied in the dark by growing wild-type plants on exogenous cytokinin. Previously we showed that dark-grown amp1 plants display de-etiolated characteristics such as short unhooked hypocotyls, opened cotyledons and formation of leaves. We further show that amp1 mutants display de-etiolated plastid morphology and increased levels of transcripts of light-regulated genes, indicating that in this mutant light-mediated processes are partially induced in the dark. The amp1 mutant also shows a reduced level of expression of several light-regulated genes compared with the wild type when grown in light, and has an altered dark-adaptation response when compared with the wild type. These results demonstrate an association between high cytokinin levels and de-etiolation, and we infer that cytokinin itself or a cytokinin-mediated process is involved in regulation of etiolation. The map location, phenotypes and de-etiolation responses in the amp1 mutant are different from those of previously described de-etiolated mutants such as det1, det2, cop1 and cop9. We propose a model in which cytokinin acts as a component of the induction of photomorphogenic processes via a signal transduction pathway which is independent of light.

Cortical microtubules optimize cell-wall crystallinity to drive unidirectional growth in Arabidopsis.

The shape of plants depends on cellulose, a biopolymer that self-assembles into crystalline, inextensible microfibrils (CMFs) upon synthesis at the plasma membrane by multi-enzyme cellulose synthase complexes (CSCs). CSCs are displaced in directions predicted by underlying parallel arrays of cortical microtubules, but CMFs remain transverse in cells that have lost the ability to expand unidirectionally as a result of disrupted microtubules. These conflicting findings suggest that microtubules are important for some physico-chemical property of cellulose that maintains wall integrity. Using X-ray diffraction, we demonstrate that abundant microtubules enable a decrease in the degree of wall crystallinity during rapid growth at high temperatures. Reduced microtubule polymer mass in the mor1-1 mutant at high temperatures is associated with failure of crystallinity to decrease and a loss of unidirectional expansion. Promotion of microtubule bundling by over-expressing the RIC1 microtubule-associated protein reduced the degree of crystallinity. Using live-cell imaging, we detected an increase in the proportion of CSCs that track in microtubule-free domains in mor1-1, and an increase in the CSC velocity. These results suggest that microtubule domains affect glucan chain crystallization during unidirectional cell expansion. Microtubule disruption had no obvious effect on the orientation of CMFs in dark-grown hypocotyl cells. CMFs at the outer face of the hypocotyl epidermal cells had highly variable orientation, in contrast to the transverse CMFs on the radial and inner periclinal walls. This suggests that the outer epidermal mechanical properties are relatively isotropic, and that axial expansion is largely dependent on the inner tissue layers.

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.

Biosynthetic origin of the saw-toothed profile in δ13C and δ2Η of n-alkanes and systematic isotopic differences between n-, iso- and anteiso-alkanes in leaf waxes of land plants

The n-fatty acids containing an even number of carbons (ECN-n-FAs) in higher plants are biosynthesised by repetitive addition of a two carbon unit from malonyl-ACP. The n-alkanes containing an odd number of carbon atoms (OCN-n-alkanes) are generally formed by the decarboxylation of ECN-n-FAs, but it is unknown how the less abundant even-carbon-numbered alkanes (ECN-n-alkanes) are biosynthesised in higher plants. There is a distinctive compositional pattern of incorporation of stable carbon ((13)C) and hydrogen ((2)H) isotopes in co-existing ECN- and OCN-n-alkanes in leaves of higher plants, such that the OCN n-alkanes are relatively enriched in (13)C but relatively depleted in (2)H against the ECN-n-alkanes. This is consistent with the OCN-n-fatty acids having a propionate precursor which is derived from reduction of pyruvate. A tentative pathway is presented with propionate produced by enzymatic reduction of pyruvate which is then thio-esterified with CoSH (coenzyme A thiol) in the chloroplast to form the terminal precursor molecule propionyl-CoA. This is then repetitively extended/elongated with the 2-carbon unit from malonyl-ACP to form the long chain OCN-n-fatty acids. The anteiso- and iso-alkanes in Nicotiana tabacum leaf waxes have previously been found to be systematically enriched in (13)C compared with the n-alkanes by Grice et al. (2008). This is consistent with the isotopic composition of their putative respective precursors (pyruvate as precursor for n-alkanes, valine for iso-alkanes and isoleucine for anteiso-alkanes). The current study complements that of Grice et al. (2008) and looks at the distribution of hydrogen isotopes. The n-alkanes were found to be more enriched in deuterium ((2)H) than the iso-alkanes which in turn were more enriched than the anteiso-alkanes. We propose therefore that the depletion of (2)H in the iso-alkanes, relative to the n-alkanes is the consequence of accepting highly (2)H-depleted hydrogen atoms from NADPH during their biosynthesis. The anteiso-alkanes are further depleted again because there are three NADPH-derived hydrogen atoms in their precursor isoleucine, as compared with only one NADPH-derived hydrogen in valine, the precursor of the iso-alkanes.

Alteration of flavonoid accumulation patterns in transparent testa mutants disturbs auxin transport, gravity responses, and imparts long-term effects on root and shoot architecture

Flavonoids have broad cross-kingdom biological activity. In Arabidopsis, flavonoid accumulation in specific tissues, notably the root elongation zone and root/shoot junction modulate auxin transport, affect root gravitropism, and influence overall plant architecture. The relative contribution made by aglycones and their glycosides remains undetermined, and the longer-term phenotypic effects of altered flavonoid accumulation are not fully assessed. We tested Arabidopsis thaliana mutants that accumulate different flavonoids to determine which flavonoids were causing these affects. Tandem mass spectrometry and in situ fluorescence localisation were used to determine the in vivo levels of aglycones in specific tissues of 11 transparent testa mutants. We measured rootward and shootward auxin transport, gravitropic responses, and identified the long-term changes to root and shoot architecture. Unexpected aglycone species accumulated in vivo in several flavonoid-pathway mutants, and lower aglycone levels occurred in transcription factor mutants. Mutants accumulating more quercetin and quercetin-glycosides changed the greatest in auxin transport, gravitropism, and aerial tissue growth. Early flavonoid-pathway mutants showed aberrant lateral root initiation patterns including clustered lateral root initiations at a single site. Transcription factor mutants had multiple phenotypes including shallow root systems. These results confirm that aglycones are present at very low levels, show that lateral root initiation is perturbed in early flavonoid-pathway mutants, and indicate that altered flavonoid accumulation affects multiple aspects of plant architecture.

Temperature modulation of fatty acid profiles for biofuel production in nitrogen deprived Chlamydomonas reinhardtii

This study investigated the changes in the fatty acid content and composition in the nitrogen-starved Chlamydomonas reinhardtii starchless mutant, BAF-J5, grown at different temperatures. The optimal temperature for vegetative growth under nitrogen sufficient conditions was found to be 32 °C. Shifting temperature from 25 to 32 °C, in conjunction with nitrogen starvation, resulted in BAF-J5 storing the maximum quantity of fatty acid (76% of dry cell weight). Shifting to temperatures lower than 25 °C, reduced the total amount of stored fatty acid content and increased the level of desaturation in the fatty acids. The optimal fatty acid composition for biodiesel was at 32 °C. This study demonstrates how a critical environmental factor, such as temperature, can modulate the amount and composition of fatty acids under nitrogen deprivation and reduce the requirement for costly refining of biofuels.