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.

Developmental mechanisms that yield multicellular diversity are proving to be well conserved within lineages, generating interest in their origins in unicellular ancestors. We report that molecular regulation of the haploid-diploid transition in Chlamydomonas, a unicellular green soil alga, shares common ancestry with differentiation pathways in land plants. Two homeoproteins, Gsp1 and Gsm1, contributed by gametes of plus and minus mating types respectively, physically interact and translocate from the cytosol to the nucleus upon gametic fusion, initiating zygote development. Their ectopic expression activates zygote development in vegetative cells and, in a diploid background, the resulting zygotes undergo a normal meiosis. Gsm1/Gsp1 dyads share sequence homology with and are functionally related to KNOX/BELL dyads regulating stem-cell (meristem) specification in land plants. We propose that combinatorial homeoprotein-based transcriptional control, a core feature of the fungal/animal radiation, may have originated in a sexual context and enabled the evolution of land-plant body plans.

SummaryEthylene is an important hormone in plant growth, development and responses to environmental stimuli. The ethylene-signaling pathway is initiated by the induction of ethylene biosynthesis, which is under tight regulation at both transcriptional and post-transcriptional levels by exogenous and endogenous cues. 1-Aminocyclopropane-1-carboxylic acid synthase (ACS) is the rate-limiting enzyme that catalyzes the committing step of ethylene biosynthesis. Recently, we found that ACS2 and ACS6, two isoforms of the Arabidopsis ACS family, are substrates of a stress-responsive mitogen-activated protein kinase (MAPK) cascade. Phosphorylation of ACS2/ACS6 by MPK6 leads to the accumulation of ACS proteins and the induction of ethylene. In this report, we demonstrate that unphosphorylated ACS6 protein is rapidly degraded by the 26S proteasome pathway. The degradation machinery targets the C-terminal non-catalytic domain of ACS6, which is sufficient to confer instability to green fluorescent protein and luciferase reporters. Phosphorylation of ACS6 introduces negative charges to the C-terminus of ACS6, which reduces the turnover of ACS6 by the degradation machinery. Consistent with this, other nearby conserved negatively charged amino acid residues are essential for ACS6 stability regulation. Protein degradation and phosphorylation are two important posttranslational modifications of proteins. This research reveals an intricate interplay between these two important processes in controlling the levels of cellular ACS activity, and thus ethylene biosynthesis. The posttranslational nature of both processes ensures a rapid response of ethylene induction, which is detectable within minutes after plants are exposed to stress.

The sexual cycle of the unicellular Chlamydomonas reinhardtii culminates in the formation of diploid zygotes that differentiate into dormant spores that eventually undergo meiosis. Mating between gametes induces rapid cell wall shedding via the enzyme g-lysin; cell fusion is followed by heterodimerization of sex-specific homeobox transcription factors, GSM1 and GSP1, and initiation of zygote-specific gene expression. To investigate the genetic underpinnings of the zygote developmental pathway, we performed comparative transcriptome analysis of both pre-and post-fertilization samples. We identified 253 transcripts specifically enriched in early zygotes, 82% of which were not up-regulated in gsp1 null zygotes. We also found that the GSM1/GSP1 heterodimer negatively regulates the vegetative wall program at the posttranscriptional level, enabling prompt transition from vegetative wall to zygotic wall assembly. Annotation of the g-lysin-induced and early zygote genes reveals distinct vegetative and zygotic wall programs, supported by concerted up-regulation of genes encoding cell wall-modifying enzymes and proteins involved in nucleotide-sugar metabolism. The haploid-to-diploid transition in Chlamydomonas is masterfully controlled by the GSM1/GSP1 heterodimer, translating fertilization and gamete coalescence into a bona fide differentiation program. The fertilization-triggered integration of genes required to make related, but structurally and functionally distinct organelles-the vegetative versus zygote cell wall-presents a likely scenario for the evolution of complex developmental gene regulatory networks.