Plant U-Box 4 regulates chloroplast stress signaling and programmed cell death via Salicylic acid modulation.

In response to environmental stress, chloroplasts generate reactive oxygen species, including singlet oxygen (1O2), which regulates nuclear gene expression (retrograde signaling), chloroplast turnover, and programmed cell death (PCD). Yet, the central signaling mechanisms and downstream responses remain poorly understood. The Arabidopsis thaliana plastid ferrochelatase two (fc2) mutant conditionally accumulates 1O2 and involves Plant U-Box 4 (PUB4), a cytoplasmic E3 ubiquitin ligase, in propagating these signals. To gain insights into 1O2 signaling pathways, we compared transcriptomes of fc2 and fc2 pub4 mutants. The accumulation of 1O2 in fc2 plants broadly repressed genes involved in chloroplast function and photosynthesis, while 1O2 induced genes and transcription factors involved in abiotic and biotic stress, the biosynthesis of jasmonic acid (JA), and Salicylic acid (SA). Elevated JA and SA levels were observed in stressed fc2 plants, but were not responsible for PCD. pub4 reversed the majority of 1O2-induced gene expression in fc2 and reduced the JA content, but maintained elevated levels of SA even in the absence of 1O2 stress. Reducing SA levels in fc2 pub4 restored 1O2 signaling and light sensitivity. Together, this work demonstrates that SA plays a protective role during photo-oxidative stress and that PUB4 mediates 1O2 signaling by modulating its levels.

Immature Teratoma in Pregnancy: A Case Report.

Immature teratoma is one of the rare malignant germ cell tumours presented in pregnancy. Here, we present 26-year-old pregnant women who had an incidental finding of left adnexal mass in an anomaly scan at 19 weeks of pregnancy. Laparotomy with peritoneal fluid cytology, left salpingo-oophorectomy and omental biopsy at 20 weeks of pregnancy revealed immature teratoma stage 1A, grade 2 in the histopathology report. However, she followed up with the metastatic mass in the pouch of Douglas at 30 weeks of pregnancy in magnetic resonance imaging despite being counselled for possible chemotherapy and surveillance. A baby with a good Apgar score and grade 3 immature teratoma in the metastatic mass was revealed following the exploratory laparotomy and cesarean section at 36 weeks of pregnancy. Fertility-sparing surgery with chemotherapy during pregnancy for high-grade tumours may result in a good prognosis. Keywords: case reports; chemotherapy; immature teratoma; pregnancy; surgery.

Chloroplast quality control pathways are dependent on plastid DNA synthesis and nucleotides provided by cytidine triphosphate synthase two.

Reactive oxygen species (ROS) produced in chloroplasts cause oxidative damage, but also signal to initiate chloroplast quality control pathways, cell death, and gene expression. The Arabidopsis thaliana plastid ferrochelatase two (fc2) mutant produces the ROS singlet oxygen in chloroplasts that activates such signaling pathways, but the mechanisms are largely unknown. Here we characterize one fc2 suppressor mutation and map it to CYTIDINE TRIPHOSPHATE SYNTHASE TWO (CTPS2), which encodes one of five enzymes in Arabidopsis necessary for de novo cytoplasmic CTP (and dCTP) synthesis. The ctps2 mutation reduces chloroplast transcripts and DNA content without similarly affecting mitochondria. Chloroplast nucleic acid content and singlet oxygen signaling are restored by exogenous feeding of the dCTP precursor deoxycytidine, suggesting ctps2 blocks signaling by limiting nucleotides for chloroplast genome maintenance. An investigation of CTPS orthologs in Brassicaceae showed CTPS2 is a member of an ancient lineage distinct from CTPS3. Complementation studies confirmed this analysis; CTPS3 was unable to compensate for CTPS2 function in providing nucleotides for chloroplast DNA and signaling. Our studies link cytoplasmic nucleotide metabolism with chloroplast quality control pathways. Such a connection is achieved by a conserved clade of CTPS enzymes that provide nucleotides for chloroplast function, thereby allowing stress signaling to occur.

The Synechocystis sp. PCC 6803 Genome Encodes Up to Four 2-Phosphoglycolate Phosphatases.

Photorespiratory phosphoglycolate (2PG) metabolism is essential for cyanobacteria, algae, and plants. The first enzyme of the pathway, 2PG phosphatase (PGPase), is known from plants and algae but was scarcely investigated in cyanobacteria. In silico analysis revealed four candidate genes (slr0458, slr0586, sll1349, and slr1762) in the genome of the model cyanobacterium Synechocystis sp. PCC 6803 that all belong to the 2-haloacid dehalogenase (HAD) superfamily and could possibly encode PGPase proteins. However, in contrast to known algal and plant PGPases, the putative cyanobacterial PGPases belong to another HAD subfamily implying that PGPases in eukaryotic phototrophs did not originate from cyanobacterial PGPases. To verify their function, these four genes were inactivated both individually and in combination. A mild high-CO2-requiring (HCR) growth phenotype typical for photorespiratory mutants was observed only in Δsll1349. Combinatorial inactivation enhanced the HCR phenotype in specific double and triple mutants. Heterologous expression of the putative cyanobacterial PGPases in E. coli led to higher PGPase activities in crude cell extracts, but only the purified Slr0458 protein showed PGPase activity. Hence, we propose that a consortium of up to four photorespiratory PGPases may initiate photorespiratory 2PG metabolism in Synechocystis. We suggest that redundancy of this essential enzyme activity could be related to the highly adaptive lifestyle of cyanobacteria such as Synechocystis sp. PCC 6803, which allows them to grow under very diverse conditions.

Improved charge transportation at PbS QDs/TiO2 interface for efficient PEC hydrogen generation.

The effect of lead sulfide (PbS) quantum dots (QDs) on the photoelectrochemical properties of TiO2 with a varied number of SILAR cycles has been investigated. The study has also highlighted physical processes including band alignment, charge recombination and transportation for a PbS QDs/TiO2 interface. The inclusion of PbS QDs underneath TiO2 thin film has significantly enhanced the PEC response due to a higher number of photogenerated charge carriers along with the efficient separation and facilitation of these carriers towards their respective electrodes. The uniqueness of the work lies in the high stability of the system as PbS QDs lie beneath the TiO2 thin film, compared to the commonly used QDs sensitization over metal oxide, along with a good photoresponse.

Comparative proteomics of wild type, An+ahpC and An∆ahpC strains of Anabaena sp. PCC7120 demonstrates AhpC mediated augmentation of photosynthesis, N2-fixation and modulation of regulatory network of antioxidative proteins.

UNLABELLED: Alkylhydroperoxide reductase (AhpC), a 1-Cys peroxiredoxin is well known for maintaining the cellular homeostasis. Present study employs proteome approach to analyze and compare alterations in proteome of Anabaena PCC7120 in overexpressing (An+ahpC), deletion (An∆ahpC) and its wild type. 2-DE based analysis revealed that the major portion of identified protein belongs to energy metabolism, protein folding, modification and stress related proteins and carbohydrate metabolism. The two major traits discernible from An+ahpC were (i) augmentation of photosynthesis and nitrogen fixation (ii) modulation of regulatory network of antioxidative proteins. Increased accumulation of proteins of light reaction, dark reaction, pentose phosphate pathway and electron transfer agent FDX for nitrogenase in An+ahpC and their simultaneous downregulation in AnΔahpC demonstrates its role in augmenting photosynthesis and nitrogen fixation. Proteomic data was nicely corroborated with physiological, biochemical parameters displaying upregulation of nitrogenase (1.6 fold) PSI (1.08) and PSII (2.137) in An+ahpC. Furthermore, in silico analysis not only attested association of AhpC with peroxiredoxins but also with other players of antioxidative defense system viz. thioredoxin and thioredoxin reductase. Above mentioned findings are in agreement with 33-40% and 40-60% better growth performance of An+ahpC over wild type and An∆ahpC respectively under abiotic stresses, suggesting its role in maintenance of metabolic machinery under stress. SIGNIFICANCE: Present work explores key role of AhpC in mitigating stress in Anabaena PCC7120 through combined proteomic, biochemical and in silico investigations. This study is the first attempt to analyze and compare alterations in proteome of Anabaena PCC7120 following addition (overexpressing strain An+ahpC) and deletion (mutant An∆ahpC) of AhpC against its wild type. The effort resulted in two major traits in An+ahpC as (i) augmentation of photosynthesis and nitrogen fixation (ii) modulation of regulatory network of antioxidative proteins.

Cadmium toxicity in diazotrophic Anabaena spp. adjudged by hasty up-accumulation of transporter and signaling and severe down-accumulation of nitrogen metabolism proteins.

Present study demonstrates interspecies variation in proteome and survival strategy of three Anabaena species i.e., Anabaena L31, Anabaena sp. PCC 7120 and Anabaena doliolum subjected to respective LC50 doses of Cd at 0, 1, 3, 5 and 7day intervals. The proteome coverage with 452 differentially accumulated proteins unveiled species and time specific expression and interaction network of proteins involved in important cellular functions. Statistical analysis of protein abundance across Cd-treated proteomes clustered their co-expression pattern into four groups viz., (i) early (days 1 and 3) accumulated proteins, (ii) proteins up-accumulated for longer duration, (iii) late (days 5 and 7) accumulated proteins, and (iv) mostly down-accumulated proteins. Appreciable growth of Cd treated A L31 over other two species may be ascribed to proteins contained in the first and second groups (belonging to energy and carbohydrate metabolism (TK, G6-PI, PGD, FBA, PPA, ATP synthase)), sulfur metabolism (GR, GST, PGDH, PAPS reductase, GDC-P, and SAM synthetase), fatty acid metabolism (AspD, PspA, SQD-1), phosphorous metabolism (PhoD, PstB and SQD1), molecular chaperones (Gro-EL, FKBP-type peptidylprolyl isomerase), and antioxidative defense enzymes (SOD-A, catalase). Anabaena sp. PCC 7120 harboring proteins largely from the third group qualified as a late accumulator and A. doliolum housing majority of proteins from the fourth group emerged as the most sensitive species. Thus early up-accumulation of transporter and signaling category proteins and drastic reduction of nitrogen assimilation proteins could be taken as a vital indicator of cadmium toxicity in Anabaena spp. This article is part of a Special Issue entitled: Proteomics in India.

Comparative proteomics unveils cross species variations in Anabaena under salt stress.

The present study compares protein diversity within three Anabaena species (Anabaena doliolum, Anabaena sp.PCC 7120 and Anabaena L31). 2-DE based analysis of 256 protein spots in control and 1, 3, 5, and 7days of salt treatment resulted into 96 proteins arching across fourteen functional categories were assigned to biochemical pathways using KOBAS 2.0. While 52.34% of the evaluated protein spots were common across three species, the remaining 47.66% fraction mainly comprised of the hypothetical and unknown proteins. PSORTb, CDD, Motifscan and Pfam revealed function and subcellular localization for 27 of the 31 hypothetical and unknown proteins. The differences in high salt tolerance (LC50) of A. doliolum over A. L31 was reflected by (i) many fold accumulation (as spot volumes) of Alr3090, Alr0803, peptidyl prolyl cis-trans isomerase and modulator of DNA gyrase proteins, and (ii) a better photosynthesis and energy homeostasis as indicated through photosystem activity, respiration, ATP and NADPH contents. Some common noteworthy salt effects include (i) photosystem damage, (ii) DNA damage repair, (iii) upregulated protein synthesis, (iv) enhanced sulphur metabolism, and (v) upregulated pentose phosphate pathway. 34 of the identified protein spots are novel entries to the Anabaena salt proteome. This study reveals the existence of separate strategies even within species to combat stress. BIOLOGICAL SIGNIFICANCE: This study for the first time enumerates protein diversity in three Anabaena species employing their presence/absence and relative abundance. Proteomics integrated with physiology and bioinformatics deciphers differential salt tolerance among the studied species and is the first of its kind to predict the function of hypothetical and unknown proteins. Salt-induced proteomic alterations clearly demonstrate significant metabolic shifts and existence of separate molecular phenome among the species investigated. This may be responsible for niche specificity limiting their application as biofertilizer. Of the 96 identified proteins, a large chunk are new entries to the Anabaena salt proteome while some protein genes may be used as potential candidates for engineering salt tolerant cyanobacteria.

Comparative proteomics reveals association of early accumulated proteins in conferring butachlor tolerance in three N(2)-fixing Anabaena spp.

Butachlor an extensively used rice field herbicide negatively affects the cyanobacterial proliferation, yet the molecular mechanism underlying its toxicity in diazotrophic cyanobacteria is largely unknown. The present study focuses on the comparative proteomics to decode the molecular basis of butachlor toxicity/tolerance in three Anabaena species e.g. Anabaena sp. PCC 7120, Anabaena doliolum and Anabaena L31. 75 differentially expressed proteins from each Anabaena sp. included those involved in photosynthesis, C, N and protein metabolism, redox homeostasis, and signal transduction. While early accumulated proteins related to photosynthesis (atpA, atpB), carbon metabolism (glpx, fba and prk), protein folding (groEL, PPIase), regulation (orrA) and other function (OR, akr) appeared crucial for tolerance of Anabaena L31, the late accumulated proteins in Anabaena 7120 presumably offer acclimation during prolonged exposure to butachlor. Contrary to the above, a multitude of down-accumulated proteins vis-a-vis metabolisms augment sensitivity of A. doliolum to butachlor. A cluster of high abundant proteins (atpA, groEL, OR, AGTase, Alr0803, Alr0806, Alr3090, Alr3199, All4050 and All4051) common across the three species may be taken as markers for butachlor tolerance and deserve exploitation for stress management and transgenic development. BIOLOGICAL SIGNIFICANCE: Cyanobacteria offer an eco-friendly alternative to chemical fertilizers for increasing productivity, especially in rice cultivation. This study is the first to compare the proteome of three diazotrophic cyanobacteria subjected to butachlor, a pre-emergent herbicide extensively used in rice paddy. Changes in protein dynamics over time along with physiological and biochemical attributes clearly provide a comprehensive overview on differential tolerance of Anabaena species to butachlor. Molecular docking further added a new dimension in identification of potential protein candidates for butachlor stress management in cyanobacteria. This study strongly recommends combined application of Anabaena spp. A. L31 and A. PCC7120 as biofertilizer in paddy fields undergoing butachlor treatment.

Salt and UV-B induced changes in Anabaena PCC 7120: physiological, proteomic and bioinformatic perspectives.

This study examines response of Anabaena sp. PCC 7120 to salt and UV-B stress by combining physiological, biochemical, proteomics and bioinformatics approaches. Sixty five significantly altered protein spots corresponding to 51 protein genes identified using MALDI-TOF MS/MS were divided into nine functional categories. Based on relative abundance, these proteins were grouped into four major sets. Of these, 27 and 5 proteins were up- and downregulated, respectively, both under salt and UV-B while 8 and 11 proteins showed accumulation in salt and UV-B applied singly. Some responses common to salt and UV-B included (i) enhanced expression of FeSOD, alr3090 and accumulation of MDA indicating oxidative stress, (ii) accumulation of PDH, G6P isomerase, FBPaldolase, TK, GAPDH and PGK suggesting enhanced glycolysis, (iii) upregulation of 6-PGD, 6PGL and NADPH levels signifying operation of pentose phosphate pathway, (iv) upregulation of Dps, NDK and alr3199 indicating DNA damage, and (v) accumulation of proteins of ribosome assembly, transcriptional and translational processing. In contrast, enhanced expression of RUBISCO, increased glycolate oxidase activity and ammonium content under salt signify the difference. Salt was found to be more damaging than UV-B probably due to a cumulative effect of ionic, osmotic and oxidative damage. A group of proteins having common expression represent decreased toxicity of salt and UV-B when applied in combination.

alr0882 encoding a hypothetical protein of Anabaena PCC7120 protects Escherichia coli from nutrient starvation and abiotic stresses.

This study is the first to demonstrate cloning of alr0882, a hypothetical protein gene of Anabaena PCC7120, its heterologous expression in Escherichia coli strain LN29MG1655 (∆uspA::Kan) and functional complementation of abiotic stress tolerance of E. coli UspA. The recombinant vector pGEX-5X-2-alr0882 was used to transform ∆uspA E. coli strain. The IPTG induced expression of a 56.6kDa GST fusion protein was visualized on SDS-PAGE and attested by immunoblotting. E. coli ∆uspA strain harboring pGEX-5X-2-alr0882 when grown under carbon, nitrogen, phosphorus and sulphur limitation and abiotic stresses e.g. nalidixic acid, cycloserine, CdCl(2), H(2)O(2), UV-B, phenazine methosulphate (PMS), dinitrophenol (DNP), NaCl, heat, carbofuron and CuCl(2) demonstrated about 22.6-51.6% increase in growth over the cells transformed with empty vector. Expression of alr0882 gene in mutant E. coli as measured by semi-quantitative RT-PCR at different time points under selected treatments reaffirmed its role in tolerance against stresses employed in this study. Thus the results of this study vividly demonstrated that the novel protein alr0882, although appreciably different from the known UspA of E. coli, offers tolerance to abiotic stresses hence holds potential for the development of transgenic cyanobacteria.