When the unicellular green soil alga Chlamydomonas reinhardtii is deprived of nitrogen after entering stationary phase in liquid culture, the cells produce abundant cytoplasmic lipid bodies (LBs), as well as abundant starch, via a pathway that accompanies a regulated autophagy program. After 48 h of N starvation in the presence of acetate, the wild-type LB content has increased 15-fold. When starch biosynthesis is blocked in the sta6 mutant, the LB content increases 30-fold, demonstrating that genetic manipulation can enhance LB production. The use of cell wall-less strains permitted development of a rapid "popped-cell" microscopic assay to quantitate the LB content per cell and permitted gentle cell breakage and LB isolation. The highly purified LBs contain 90% triacylglycerol (TAG) and 10% free fatty acids (FFA). The fatty acids associated with the TAGs are ϳ50% saturated (C 16 and C 18 ) fatty acids and ϳ50% unsaturated fatty acids, half of which are in the form of oleic acid (C 18:1 ). The FFA are ϳ50% C 16 and ϳ50% C 18 . The LB-derived TAG yield from a liter of sta6 cells at 10 7 cells/ml after starvation for 48 h is calculated to approach 400 mg. The LB fraction also contains low levels of charged glycerolipids, with the same profile as whole-cell charged glycerolipids, that presumably form LB membranes; chloroplast-specific neutral glycerolipids (galactolipids) are absent. Very low levels of protein are also present, but all matrix-assisted laser desorption ionization-identified species are apparent contaminants. Nitrogen stress-induced LB production in C. reinhardtii has the hallmarks of a discrete pathway that should be amenable to additional genetic and culture condition manipulation.
SummaryThe Arabidopsis thaliana chlorophyll a/b binding protein underexpressed (cue1) mutant, which has been isolated in a screen for chlorophyll a/b binding protein (CAB) underexpressors, exhibits a reticulate leaf phenotype combined with delayed chloroplast development and aberrant shape of the palisade parenchyma cells. The affected gene in cue1 is a phosphoenolpyruvate (PEP)/phosphate translocator (PPT) of the plastid inner envelope membrane. The proposed function of the PPT in C 3 -plants is the import of PEP into the stroma as one of the substrates for the shikimate pathway, from which aromatic amino acids and a variety of secondary plant products derive. The mutant phenotype could be: (i) complemented by constitutive overexpression of a heterologous PPT from cauli¯ower; and (ii) rescued by overexpression of a C 4 -type pyruvate,orthophosphate dikinase (PPDK). The latter approach indicates that PEP de®ciency within plastids triggers developmental constraints in cue1. The impact of the mutation on aspects of primary and secondary metabolism was assessed in cue1 as well as in the individual transformant lines. The majority of the data obtained in this and an accompanying paper suggest that the mutant phenotype is not simply caused by a general restriction of the shikimate pathway because of a defect in a PPT.Keywords: phenylpropanoids, secondary metabolism, phosphoenolpyruvate transport, aromatic amino acids, cue1 mutant, plastids. IntroductionThe Arabidopsis chlorophyll a/b binding protein underexpressed (cue1) mutant was identi®ed in a screen for genes that confer the phytochrome-dependent activation of nuclear promotors by monitoring the de-repression of the chlorophyll a/b binding protein 3 (CAB3) promotor during de-etiolation (Li et al., 1995). Cue1 exhibits a reticulate leaf phenotype with dark green paraveinal and light green interveinal regions. Moreover, cue1 is impaired in chloroplast and mesophyll development, e.g. the palisade parenchyma cells show a spheric rather than a columellar shape with larger airspaces and smaller chloroplasts, whereas cells (and chloroplasts therein) close to the vascular bundles are not affected (Li et al., 1995;Streat®eld et al., 1999). To date, the cue1 mutant is unique in that it combines lower responsiveness to phytochrome signals with delayed chloroplast development.The gene affected in cue1 codes for a phosphoenolpyruvate (PEP)/phosphate translocator (PPT) of the inner plastid envelope membrane (Fischer et al., 1997;Streat®eld et al., 1999). The proposed physiological role of the PPT in leaves of C 3 -plants is the supply of PEP to the stroma as a precursor for the shikimate pathway. The import of PEP appears to be essential as chloroplasts, and most nongreen plastids have been reported to be incapable of producing PEP from 3-phosphoglycerate (3-PGA) via glycolysis because of a de®ciency in phosphoglycerate mutase and/or enolase (Bagge and Larsson, 1986;Journet and Douce, 1985;Stitt and ap Rees, 1979). In plastids, PEP can also doi: 10.1046/j.1365-313X.2003.01889...
Hydroxyproline-rich glycoproteins (HRGPs) are the major proteinaceous components of higher plant walls and the predominant components of the cell wall of the green alga Chlamydomonas reinhardtii. The GP1 protein, an HRGP of the C. reinhardtii wall, is shown to adopt a polyproline II helical configuration and to carry a complex array of arabinogalactoside residues, many branched, which are necessary to stabilize the helical conformation. The deduced GP1 amino acid sequence displays two Ser-Pro-rich domains, one with a repeating (SP)(x)() motif and the other with a repeating (PPSPX)(x)() motif. A second cloned gene a2 also carries the PPSPX repeat, defining a novel gene family in this lineage. The SP-repeat domains of GP1 form a 100-nm shaft with a flexible kink 28 nm from the head. The gp1 gene encodes a PPPPPRPPFPANTPM sequence at the calculated kink position, generating the proposal that this insert interrupts the PPII helix, with the resultant kink exposing amino acids necessary for GP1 to bind to partner molecules. It is proposed that similar kinks in the higher plant HRGPs called extensins may play a comparable role in wall assembly.
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