Haitao Ge

The group 2 σ factor for RNA polymerase SigE plays important role in regulating central carbon metabolism in cyanobacteria. However, the regulation of SigE for these pathways at a proteome level remains unknown. Using a sigE‐deficient strain (ΔsigE) of Synechocystis sp. PCC 6803 and quantitative proteomics, we found that SigE depletion induces differential protein expression for sugar catabolic pathways including glycolysis, oxidative pentose phosphate (OPP) pathway, and glycogen catabolism. Two glycogen debranching enzyme homologues Slr1857 and Slr0237 are found differentially expressed in ΔsigE. Glycogen determination indicated that Δslr0237 accumulated glycogen under photomixotrophic condition but was unable to utilize these reserves in the dark, whereas Δslr1857 accumulates and utilizes glycogen in a similar way as the WT strain does in the same condition. These results suggest that Slr0237 plays the major role as the glycogen debranching enzyme in Synechocystis.

The group 2 σ factor for RNA polymerase SigE plays important role in regulating central carbon metabolism in cyanobacteria. However, the regulation of SigE for these pathways at a proteome level remains unknown. Using a sigE-deficient strain (ΔsigE) of Synechocystis sp. PCC 6803 and quantitative proteomics, we found that SigE depletion induces differential protein expression for sugar catabolic pathways including glycolysis, oxidative pentose phosphate (OPP) pathway, and glycogen catabolism. Two glycogen debranching enzyme homologues Slr1857 and Slr0237 are found differentially expressed in ΔsigE. Glycogen determination indicated that Δslr0237 accumulated glycogen under photomixotrophic conditions but was unable to utilize these reserves in the dark, whereas Δslr1857 accumulates and utilize glycogen in a similar way as the WT strain does in the same conditions. These results suggest that Slr0237 plays the major role as the glycogen debranching enzyme in Synechocystis. To our knowledge, this is the first study to report the functional difference of two glycogen debranching enzyme in Synechocystis and the research highlights the intricate regulation of glycogen breakdown.

Spatial proteome reorganization in response to a changing environment represents a different layer of adaptation mechanism in addition to differential expression of a subset of stress responsive genes in photosynthetic organisms. Profiling such reorganization events is critically important to extend our understanding how photosynthetic organisms adapt to adverse environments. Thus, we treated a unicellular photosynthetic model cyanobacterium, Synechocystis sp. PCC 6803 (hereafter referred to as Synechocystis), with five different types of abiotic stresses including nitrogen starvation, iron deficiency, cold, heat, and darkness, and systematically identified proteins showing stress-induced differential expression and/or redistribution between the membrane and the soluble fractions using a quantitative proteomics approach. A number of proteins showing such a redistribution in response to a single or multiple types of abiotic stresses were identified. These include 12 ribosomal proteins displaying unanimous cold-induced redistribution to the membrane and the protein FurA, a master regulator of iron acquisition, displaying iron deficiency- and nitrogen starvation-induced redistribution to the membrane. Such findings shed light on a novel regulatory mechanism underlying the corresponding stress responses, and establish the results in the present study as an important resource for future studies intended to understand how photosynthetic organisms cope with adverse environments.

Photosynthesis and the biosynthesis of many important metabolites occur in chloroplasts. In these semi-autonomous organelles, the chloroplast genome encodes approximately 100 proteins. The remaining chloroplast proteins, close to 3,000, are encoded by nuclear genes whose products are translated in the cytosol and imported into chloroplasts. However, there is still no consensus on the composition of the protein import machinery including its motor proteins and on how newly imported chloroplast proteins are refolded. In this study, we have examined the function of orf2971, the largest chloroplast gene of Chlamydomonas reinhardtii. The depletion of Orf2971 causes the accumulation of protein precursors, partial proteolysis and aggregation of proteins, increased expression of chaperones and proteases, and autophagy. Orf2971 interacts with the TIC (translocon at the inner chloroplast envelope) complex, catalyzes ATP (adenosine triphosphate) hydrolysis, and associates with chaperones and chaperonins. We propose that Orf2971 is intimately connected to the protein import machinery and plays an important role in chloroplast protein quality control.

High salinity is a common environmental factor that limits productivity and growth for photosynthetic organisms. Here, we identified a mutant defected in gene sll1770, which encodes a Ser/Thr protein kinase SpkI, with a significantly low maximal quantum yield of PSII under high salt condition in Synechocystis sp. PCC 6803. Physiological characterization demonstrated that the ΔspkI mutant had normal growth and photosynthesis under control condition. And a significantly higher NPQ capacity was also observed in ΔspkI when grown under control condition. However, when grown under high salt condition, ΔspkI exhibited apparently slower growth as well as decreased net photosynthesis and PSII activity. Western blot analysis demonstrated that the amount of major photosynthetic proteins declined sharply in ΔspkI when cells grown under high salt condition. Redox kinetics measurement suggested that the activities of PSI and cytochrome b₆f complex were modified in ΔspkI under high salt condition, which resulted in a more reduced PQ pool in ΔspkI. Chlorophyll fluorescence traces suggested that the O_A⁻ reoxidation and state transition was also impaired in ΔspkI under high salt condition. Above all, we propose that Ser/Thr protein kinase SpkI plays a role in maintaining high-effective photosynthesis during high-salt acclimation process in Synechocystis.

Upon exposure of photosynthetic organisms to high light (HL), several HL acclimation responses are triggered. Herein, we identified a novel gene, slr0320, critical for HL acclimation in Synechocystis sp. PCC 6803. The growth rate of the Δslr0320 mutant was similar to wild type (WT) under normal light (NL) but severely declined under HL. Net photosynthesis of the mutant was lower under HL, but maximum photosystem II (PSII) activity was higher under NL and HL. Immunodetection revealed the accumulation and assembly of PSII were similar between WT and the mutant. Chlorophyll fluorescence traces showed the stable fluorescence of the mutant under light was much higher. Kinetics of single flash-induced chlorophyll fluorescence increase and decay revealed the slower electron transfer from Q_A to Q_B in the mutant. These data indicate that, in the Δslr0320 mutant, the number of functional PSIIs was comparable to WT even under HL but the electron transfer between Q_A and Q_B was inefficient. Quantitative proteomics and real-time PCR revealed that expression profiles of psbL, psbH and psbI were significantly altered in the Δslr0320 mutant. Thus, Slr0320 protein plays critical roles in optimizing PSII activity during HL acclimation and is essential for PSII electron transfer from Q_A to Q_B.

The histidine kinase Hik33 plays a central role in acclimation to changing environments in cyanobacteria. Deletion of hik33 induces a strong stress‐like response in Synechocystis sp. PCC 6803 ( Synechocystis ) as represented by repressed photoautotrophic growth and photosynthesis, and differential expression of stress‐responsive proteins. In contrast, the photomixotrophic growth of the hik33‐deletion mutant (Δhik33) with glucose as the exogenous carbon source is only marginally repressed. To investigate how glucose rescues the growth of Δhik33, the proteomes of the photomixotrophically growing wild‐type (WT) and the mutant strains of Synechocystis are quantitatively analyzed. It is found that glucose induces upregulation of the oxidative pentose phosphate (OPP) pathway. Depletion of glucose‐6‐phosphate dehydrogenase (G6PDH), which catalyzes the first and the rate‐limiting step of the OPP pathway, significantly inhibits the photomixotrophic growth of Δhik33 but not of the WT. The result suggests that the OPP pathway, which is usually nonfunctional in the photomixotrophically growing WT, plays a major role in the photomixotrophic growth of Δhik33.