SPL7 locally regulates copper-homeostasis-related genes in Arabidopsis.

In Arabidopsis, a central regulator of copper (Cu) homeostasis is the transcription factor SQUAMOSA promoter binding protein-like7 (SPL7). Under Cu deficiency, SPL7 induces the expression of miR398, which suppresses the expression of the genes CSD1 and CSD2, which encode cytosolic and chloroplastic isoforms of Cu/Zn superoxide dismutase, respectively. Consequently, the limited Cu is preferentially assigned to plastocyanin, which is essential for photosynthetic electron transport. Consistent with this function of miR398 related to photosynthesis, its expression is strongly induced in leaves. In this study, however, we showed that SPL7 was transcribed mainly around the vasculature in roots, where Cu levels were likely sensed. To test the possible long-distance signaling of Cu availability from roots to shoots, we conducted a series of grafting experiments using spl7 mutant and wild-type (WT) plants. Expression of Cu-responsive microRNAs and the resulting suppression of CSD1 and CSD2 mRNAs were observed in leaves only when the aerial part was from WT plants, in which a low level of SPL7 was transcribed also in the vascular tissues. Although local sensing of Cu was disturbed in the spl7 mutant, the Cu level was not affected in the shoots. SPL7 is expressed in specific cell layers in both roots and shoots and locally senses Cu availability, transmitting the information to surrounding cells.

Dynamic metabolic profiling together with transcription analysis reveals salinity-induced starch-to-lipid biosynthesis in alga Chlamydomonas sp. JSC4.

Biodiesel production using microalgae would play a pivotal role in satisfying future global energy demands. Understanding of lipid metabolism in microalgae is important to isolate oleaginous strain capable of overproducing lipids. It has been reported that reducing starch biosynthesis can enhance lipid accumulation. However, the metabolic mechanism controlling carbon partitioning from starch to lipids in microalgae remains unclear, thus complicating the genetic engineering of algal strains. We here used "dynamic" metabolic profiling and essential transcription analysis of the oleaginous green alga Chlamydomonas sp. JSC4 for the first time to demonstrate the switching mechanisms from starch to lipid synthesis using salinity as a regulator, and identified the metabolic rate-limiting step for enhancing lipid accumulation (e.g., pyruvate-to-acetyl-CoA). These results, showing salinity-induced starch-to-lipid biosynthesis, will help increase our understanding of dynamic carbon partitioning in oleaginous microalgae. Moreover, we successfully determined the changes of several key lipid-synthesis-related genes (e.g., acetyl-CoA carboxylase, pyruvate decarboxylase, acetaldehyde dehydrogenase, acetyl-CoA synthetase and pyruvate ferredoxin oxidoreductase) and starch-degradation related genes (e.g., starch phosphorylases), which could provide a breakthrough in the marine microalgal production of biodiesel.

The proteome of copper, iron, zinc, and manganese micronutrient deficiency in Chlamydomonas reinhardtii.

Trace metals such as copper, iron, zinc, and manganese play important roles in several biochemical processes, including respiration and photosynthesis. Using a label-free, quantitative proteomics strategy (MS(E)), we examined the effect of deficiencies in these micronutrients on the soluble proteome of Chlamydomonas reinhardtii. We quantified >10(3) proteins with abundances within a dynamic range of 3 to 4 orders of magnitude and demonstrated statistically significant changes in ~200 proteins in each metal-deficient growth condition relative to nutrient-replete media. Through analysis of Pearson's coefficient, we also examined the correlation between protein abundance and transcript abundance (as determined via RNA-Seq analysis) and found moderate correlations under all nutritional states. Interestingly, in a subset of transcripts known to significantly change in abundance in metal-replete and metal-deficient conditions, the correlation to protein abundance is much stronger. Examples of new discoveries highlighted in this work include the accumulation of O(2) labile, anaerobiosis-related enzymes (Hyd1, Pfr1, and Hcp2) in copper-deficient cells; co-variation of Cgl78/Ycf54 and coprogen oxidase; the loss of various stromal and lumenal photosynthesis-related proteins, including plastocyanin, in iron-limited cells; a large accumulation (from undetectable amounts to over 1,000 zmol/cell) of two COG0523 domain-containing proteins in zinc-deficient cells; and the preservation of photosynthesis proteins in manganese-deficient cells despite known losses in photosynthetic function in this condition.

Systems and trans-system level analysis identifies conserved iron deficiency responses in the plant lineage.

We surveyed the iron nutrition-responsive transcriptome of Chlamydomonas reinhardtii using RNA-Seq methodology. Presumed primary targets were identified in comparisons between visually asymptomatic iron-deficient versus iron-replete cells. This includes the known components of high-affinity iron uptake as well as candidates for distributive iron transport in C. reinhardtii. Comparison of growth-inhibited iron-limited versus iron-replete cells revealed changes in the expression of genes in chloroplastic oxidative stress response pathways, among hundreds of other genes. The output from the transcriptome was validated at multiple levels: by quantitative RT-PCR for assessing the data analysis pipeline, by quantitative proteomics for assessing the impact of changes in RNA abundance on the proteome, and by cross-species comparison for identifying conserved or universal response pathways. In addition, we assessed the functional importance of three target genes, Vitamin C 2 (VTC2), monodehydroascorbate reductase 1 (MDAR1), and conserved in the green lineage and diatoms 27 (CGLD27), by biochemistry or reverse genetics. VTC2 and MDAR1, which are key enzymes in de novo ascorbate synthesis and ascorbate recycling, respectively, are likely responsible for the 10-fold increase in ascorbate content of iron-limited cells. CGLD27/At5g67370 is a highly conserved, presumed chloroplast-localized pioneer protein and is important for growth of Arabidopsis thaliana in low iron.

SQUAMOSA Promoter Binding Protein-Like7 Is a Central Regulator for Copper Homeostasis in Arabidopsis.

Expression of miR398 is induced in response to copper deficiency and is involved in the degradation of mRNAs encoding copper/zinc superoxide dismutase in Arabidopsis thaliana. We found that SPL7 (for SQUAMOSA promoter binding protein-like7) is essential for this response of miR398. SPL7 is homologous to Copper response regulator1, the transcription factor that is required for switching between plastocyanin and cytochrome c(6) in response to copper deficiency in Chlamydomonas reinhardtii. SPL7 bound directly to GTAC motifs in the miR398 promoter in vitro, and these motifs were essential and sufficient for the response to copper deficiency in vivo. SPL7 is also required for the expression of multiple microRNAs, miR397, miR408, and miR857, involved in copper homeostasis and of genes encoding several copper transporters and a copper chaperone, indicating its central role in response to copper deficiency. Consistent with this idea, the growth of spl7 plants was severely impaired under low-copper conditions.

How do plants respond to copper deficiency?

The transition metal copper is essential for all organisms yet excess copper is toxic because of production of free radicals via its free form. Therefore, the levels of copper are precisely regulated in a cell. Under copper depleted conditions, the expression of Cu/Zn superoxide dismutase (SOD) is downregulated and its function is compensated by Fe SOD in chloroplasts of higher plants. We presented evidence that a microRNA, miR398, is involved in this downregulation of Cu/Zn SOD genes in Arabidopsis thaliana when grown at low copper levels, corresponding to less than 1 microM Cu in tissue culture media. However, a green alga, Chlamydomonas reinhardtii, adjusts to copper depletion by modifying the photosynthetic apparatus from copper containing plastocyanin to iron containing cytochrome c(6). During evolution plants modified one of the main strategies to respond to copper deficiency probably to adapt to different metal environments.

Regulation of copper homeostasis by micro-RNA in Arabidopsis.

Major copper proteins in the cytoplasm of plant cells are plastocyanin, copper/zinc superoxide dismutase, and cytochrome c oxidase. Under copper limited conditions, expression of copper/zinc superoxide dismutase is down-regulated and the protein is replaced by iron superoxide dismutase in chloroplasts. We present evidence that a micro-RNA, miR398, mediates this regulation in Arabidopsis thaliana, by directing the degradation of copper/zinc superoxide dismutase mRNA when copper is limited. Sequence analysis indicated that the transcripts encoding cytosolic copper/zinc superoxide dismutase and COX5b-1, a subunit of the mitochondrial cytochrome c oxidase, are also targeted by miR398. This regulation via miR398 takes place in response to changes in a low range of copper levels (0.2-0.5 microM), indicating that miR398 is involved in a response to copper limitation. On the other hand, another major copper protein, plastocyanin, which is involved in photosynthetic electron flow and is essential in higher plants, was not regulated via miR398. We propose that miR398 is a key factor in copper homeostasis in plants and regulates the stability of mRNAs of major copper proteins under copper-limited conditions.