Comparative proteomic analysis of salt response proteins in seedling roots of two wheat varieties

A comparative proteomic analysis was made of salt response in seedling roots of wheat cultivars Jing-411 (salt tolerant) and Chinese Spring (salt sensitive) subjected to a range of salt stress concentrations (0.5%, 1.5% and 2.5%) for 2 days. One hundred and ninety eight differentially expressed protein spots (DEPs) were located with at least two-fold differences in abundance on 2-DE maps, of which 144 were identified by MALDI-TOF-TOF MS. These proteins were involved primarily in carbon metabolism (31.9%), detoxification and defense (12.5%), chaperones (5.6%) and signal transduction (4.9%). Comparative analysis showed that 41 DEPs were salt responsive with significant expression changes in both varieties under salt stress, and 99 (52 in Jing-411 and 47 in Chinese Spring) were variety specific. Only 15 and 9 DEPs in Jing-411 and Chinese Spring, respectively, were up-regulated in abundance under all three salt concentrations. All dynamics of the DEPs were analyzed across all treatments. Some salt responsive DEPs, such as guanine nucleotide-binding protein subunit beta-like protein, RuBisCO large subunit-binding protein subunit alpha and pathogenesis related protein 10, were up-regulated significantly in Jing-411 under all salt concentrations, whereas they were down-regulated in salinity-stressed Chinese Spring.     

Characterization of the pyrenoid isolated from unicellular green algaChlamydomonas reinhardtii: Particulate form of RuBisCO protein

Summary The pyrenoid is a protein complex in the chloroplast stroma of eukaryotic algae. After the treatment with mercury chloride, pyrenoids were isolated by sucrose density gradient centrifugation from cell-wall less mutant cells, CW-15, as well as wild type cells, C-9, of unicellular green algaChlamydomonas reinhardtii. Pyrenoids were characterized as a fraction whose protein/chlorophyll ratio was very high, and also examined by Nomarski differential interference microscopy. Most of the components consisted of 55 kDa and 16 kDa polypeptides (11) which were immunologically identified as the large and small subunit of RuBisCO (ribulose-1,5-bisphosphate carboxylase/oxygenase) protein, respectively. Some minor polypeptides were also detected. Substantial amount of RuBisCO protein is present as a particulate form in the pyrenoid in addition to the soluble form in algal chloroplast stroma.Abbreviations BPB bromophenol blue - DAB 3,3-diaminobenzidine - DTT dithiothreitol - ELISA enzyme-linked immunosorbent assay - High-CO₂ cells cells grown under air enriched with 4% CO₂ - Low-CO₂ cells cells grown under ordinary air (containing 0.04% CO₂) - NP-40 nonionic detergent (Nonidet) P-40 - PAGE polyacrylamide gel electrophoresis - PAP peroxidase-antiperoxidase conjugate - RuBisCO ribulose-1,5-bisphosphate carboxylase/oxygenase - RuBP ribulose-1,5-bisphosphate - SDS sodium dodecylsulfate     

How do vascular plants perform photosynthesis in extreme environments? An integrative ecophysiological and biochemical story

In this work, we review the physiological and molecular mechanisms that allow vascular plants to perform photosynthesis in extreme environments, such as deserts, polar and alpine ecosystems. Specifically, we discuss the morpho/anatomical, photochemical and metabolic adaptive processes that enable a positive carbon balance in photosynthetic tissues under extreme temperatures and/or severe water‐limiting conditions in C₃ species. Nevertheless, only a few studies have described the in situ functioning of photoprotection in plants from extreme environments, given the intrinsic difficulties of fieldwork in remote places. However, they cover a substantial geographical and functional range, which allowed us to describe some general trends. In general, photoprotection relies on the same mechanisms as those operating in the remaining plant species, ranging from enhanced morphological photoprotection to increased scavenging of oxidative products such as reactive oxygen species. Much less information is available about the main physiological and biochemical drivers of photosynthesis: stomatal conductance (g_s), mesophyll conductance (g_m) and carbon fixation, mostly driven by RuBisCO carboxylation. Extreme environments shape adaptations in structures, such as cell wall and membrane composition, the concentration and activation state of Calvin–Benson cycle enzymes, and RuBisCO evolution, optimizing kinetic traits to ensure functionality. Altogether, these species display a combination of rearrangements, from the whole‐plant level to the molecular scale, to sustain a positive carbon balance in some of the most hostile environments on Earth.     

Exploration of green integrated approach for effluent treatment through mass culture and biofuel production from unicellular alga,Acutodesmus obliquus RDS01

Abstract

This study deals with the open pond (OP) pilot scale treatment of cassava effluent and enhancement of Ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) enzyme through CO₂ utilization by the microalga, Acutodesmus obliquus RDS01. The cassava effluent treatment (ET) revealed maximum reduction of ammonia (96.8%), calcium (94.6%), chloride (95.2%), chlorine (98.5%), inorganic phosphate (94.6%), magnesium (96.8%), nitrate (96.89%), organic carbon (95.9%), organic phosphorus (96.3%), potassium (97.9%), sodium (97.1%), and sulfate (95.4%) on 15^th day using A. obliquus. The microalga produced highest RuBisCO enzyme activity (90%), CO₂ utilization efficiency (95%), biomass (8.9 gL^?1), lipid (176.65?mg mL^?1), carbohydrate (96.78?mg mL^?1), biodiesel (4.1?mL g^?1), and bioethanol (3.7?mL g^?1) during OP treatment. The isolated RuBisCO gene (rbcL) was used to construct the protein model by homology modeling. The microalgal-lipid content was analyzed through thin layer chromatography, the biodiesel produced was analyzed using Fourier-transform infrared spectroscopy and gas chromatography mass spectrometry (GCMS). The bioethanol production was confirmed by high performance liquid chromatography and GCMS analyses. A. obliquus produced of 98.75% biodiesel and 96.83% bioethanol in the OP pilot scale treatment A. obliquus. Overall, the microalga A. obliquus could act as an effective CO₂ capturing and bioremediation agent in the cassava ET, and also for the biodiesel and bioethanol can be produced.     

An evolutionally conserved Lys122 is essential for function in Rhodospirillum rubrum bona fide RuBisCO and Bacillus subtilis RuBisCO-like protein

Ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) and RuBisCO-like protein (RLP) catalyze similar enolase-type reactions. Both enzymes have a conserved non-catalytic Lys122 or Arg122 on the β-strand E lying in the interface between the N- and C-terminal domains. We used site-directed mutagenesis to analyze the function of Lys122 in the form II Rhodospirillum rubrum RuBisCO (RrRuBisCO) and Bacillus subtilis RLP (BsRLP). The K122R mutant of RrRuBisCO had a 40% decrease in k_cat for carboxylase activity, a 2-fold increase in K_m for CO₂, and a 1.9-fold increase in K_m for ribulose-1,5-bisphosphate. K122M and K122E mutants of RrRuBisCO were almost inactive. None of the substitutions affected the thermal stability of RrRuBisCO. The K122R mutant of BsRLP had a 32% decrease in k_cat and lower thermal stability than the wild-type enzyme. The K122M and K122E mutants of BsRLP failed to form a catalytic dimer. Our results suggest that the lysine residue is essential for function in both enzymes, although in each case, its role is likely distinct.     

Lysine acetylome profiling uncovers novel histone deacetylase substrate proteins in Arabidopsis

Histone deacetylases have central functions in regulating stress defenses and development in plants. However, the knowledge about the deacetylase functions is largely limited to histones, although these enzymes were found in diverse subcellular compartments. In this study, we determined the proteome‐wide signatures of the RPD3/HDA1 class of histone deacetylases in Arabidopsis. Relative quantification of the changes in the lysine acetylation levels was determined on a proteome‐wide scale after treatment of Arabidopsis leaves with deacetylase inhibitors apicidin and trichostatin A. We identified 91 new acetylated candidate proteins other than histones, which are potential substrates of the RPD3/HDA1‐like histone deacetylases in Arabidopsis, of which at least 30 of these proteins function in nucleic acid binding. Furthermore, our analysis revealed that histone deacetylase 14 (HDA14) is the first organellar‐localized RPD3/HDA1 class protein found to reside in the chloroplasts and that the majority of its protein targets have functions in photosynthesis. Finally, the analysis of HDA14 loss‐of‐function mutants revealed that the activation state of RuBisCO is controlled by lysine acetylation of RuBisCO activase under low‐light conditions.     

CARBON FIXATION IN CULTURED MARINE BENTHIC DIATOMS1

The enzyme activity of ribulose 1, 5-bisphosphate carboxylase-oxygenase (RuBisCO) and phosphoenolpyruvate carboxylase (PEPC) was measured in four species of marine benthic diatoms isolated from subtidal sediments of Graveline Bayou, Mississippi. Enzyme activities were measured in cultures of Amphora micrometra Giffen, A. tenerrima Aleem and Hustedt, Nitzschia fontifuga Cholnoky, and Nitzschia vermicularis Grunow that were grown at light levels supporting μ_max and at light-limiting irradiances. All four species exhibited similar RuBisCO: PEP ratios (range = 1–1.8) at μ_max the lowest ratio (0.4) was observed in A. micrometra. Reduced light levels increased PEPC relative to that measured at μ_max in two species. Two-dimensional paper chromatography was used to determine the first products of carbon fixation in A. micrometra After a 15 s incorporation period, the first product of photosynthetic carbon fixation was 3-phosphoglycerate even though this alga had a PEPC activity that was three times higher than that of RuBisCO. After 30 s, over 50% of the recovered radioactivity was still in this compound. Stable carbon isotope analyses of a mixture of the four pennate diatoms also suggest the predominant carbon fixation pathway in these benthic diatoms was similar to C3 plants.     

The CbbQO-type rubisco activases encoded in carboxysome gene clusters can activate carboxysomal form IA rubiscos

The CO₂-fixing enzyme rubisco is responsible for almost all carbon fixation. This process frequently requires rubisco activase (Rca) machinery, which couples ATP hydrolysis to the removal of inhibitory sugar phosphates, including the rubisco substrate ribulose 1,5-bisphosphate (RuBP). Rubisco is sometimes compartmentalized in carboxysomes, bacterial microcompartments that enable a carbon dioxide concentrating mechanism (CCM). Characterized carboxysomal rubiscos, however, are not prone to inhibition, and often no activase machinery is associated with these enzymes. Here, we characterize two carboxysomal rubiscos of the form IA^C clade that are associated with CbbQO-type Rcas. These enzymes release RuBP at a much lower rate than the canonical carboxysomal rubisco from Synechococcus PCC6301. We found that CbbQO-type Rcas encoded in carboxysome gene clusters can remove RuBP and the tight-binding transition state analog carboxy-arabinitol 1,5-bisphosphate from cognate rubiscos. The Acidithiobacillus ferrooxidans genome encodes two form IA rubiscos associated with two sets of cbbQ and cbbO genes. We show that the two CbbQO activase systems display specificity for the rubisco enzyme encoded in the same gene cluster, and this property can be switched by substituting the C-terminal three residues of the large subunit. Our findings indicate that the kinetic and inhibitory properties of proteobacterial form IA rubiscos are diverse and predict that Rcas may be necessary for some α-carboxysomal CCMs. These findings will have implications for efforts aiming to introduce biophysical CCMs into plants and other hosts for improvement of carbon fixation of crops.