Metabolism and function of β-1,3-glucan in marine diatoms

β-1,3-Glucan (chrysolaminaran) is the principal storage polysaccharide in diatoms (Bacillariophyceae), the major primary producers in the sea. The glucan generally contributes a substantial fraction of the algal biomass, but its level varies markedly in response to growth conditions. The scope of this work was to study the metabolism and function of the polysaccharide in marine diatoms. Axenic cultures of the marine planktonic diatom Skeletonema costatum (Grev.) Cleve were used in the experiments. Glucan metabolism was studied by growing the alga in batch culture, and measuring metabolite fluxes by chemical analyses as well as by 14C tracer technique using labeled bicarbonate. A photobioreactor was developed for strictly controlled growth of microalgae. Fine pH regulation was obtained by relay-activated titration with dilute acid (HCl) and base (NaOH). Irradiance and temperature were also carefully controlled. Batch cultures were grown with a 14:10 h light:dark cycle, and pH curves were recorded during different growth phases. A new method was developed for the combined determination of β-1,3-glucan and cell wall polysaccharides in diatoms, representing total cellular carbohydrate. The glucan is rapidly extracted by hot dilute H2SO4, and the cell wall polysaccharides are subsequently hydrolyzed by cold 80% H2SO4overnight. Each carbohydrate fraction is finally determined by the phenol-sulphuric acid method. This procedure is simple and rapid compared to previous methods, and applies well to laboratory cultures as well as natural phytoplankton populations dominated by diatoms. Synthesis and mobilization of β-1,3-glucan in N-limited S. costatum were studied by combined 14C tracer technique and chemical analyses. Radiolabeled bicarbonate was added to the cultures, and 14C incorporation in different metabolites was determined using biochemical fractionation. In a pulse phase, 14C label was mainly incorporated in the glucan fraction (85%) during photosynthesis under nitrogen limitation. Subsequently, a 14C chase was carried out by adding NH4+ and incubating the cells under different light conditions. Radiolabeled glucan decreased significantly (by 26% in the dark, and by 19% in low light) whereas radiolabeled amino acids, proteins and other polysaccharides increased significantly during NH4+ assimilation. Chemical analyses of β-1,3-glucan and cellular free amino acids supported the 14C measurements. Changes in amino acid composition strongly indicated that de novo biosynthesis took place, with a Gln/Glu ratio increasing from 0.4 to 10. This study provides new evidence of β-1,3-glucan supplying carbon skeletons for synthesis of amino acids and protein in diatoms. Mobilization of glucan yields glucose, which is further metabolized by the respiratory pathways to provide precursors as well as energy. The results from the 14C chase also indicated significant synthesis of other polysaccharides or possibly RNA from glucan. In a different study, dark carbon fixation in N-limited S. costatum was measured using 14C-bicarbonate. Addition of NH4+ resulted in 4-fold increase in carboxylation rate, and biochemical fractionation showed that mainly amino acids were radiolabeled. Chemical analyses confirmed that cellular free amino acids increased rapidly (with increasing Gln/Glu), and showed that cellular glucan decreased significantly (by 28%) during NH4+ assimilation. The results strongly indicate that β-carboxylation provides C4 precursors for amino acid synthesis, and β-1,3-glucan is likely to be the ultimate substrate for β-carboxylation. Moreover, a C/N uptake ratio of 0.33 indicated that β-carboxylation was related to protein synthesis. A detailed study was made of the production of carbohydrates and amino acids by S. costatum during different growth phases. During exponential growth under diel light conditions, the glucan level oscillated between 17% (end of scotophase) and 42% (end of photophase) of cellular organic carbon, and the corresponding protein/glucan ratio alternated between 2.3 and 0.7. Concurrently, the cellular free amino acid pool oscillated between 8% (end of scotophase) and 22% (end of photophase) of cellular organic nitrogen, and the corresponding Gln/Glu ratio alternated between 0.05 and 2. Depletion of nitrogen from the medium resulted in rapid accumulation of glucan, reaching 75-80% of cellular organic carbon, whereas the cellular nitrogenous components decreased significantly. Consequently, the protein/glucan ratio decreased to <0.1. This study indicates that β-1,3-glucan functions both as a short-term diurnal reserve and a long-term stockpile reserve. Field investigations by other workers suggest that glucan plays a very active role in the dynamics of natural diatom populations, and the protein/glucan ratio has been used as a sensitive parameter for nutrient status. The glucan dynamics may be involved in physiological control of buoyancy. Glucan accumulation by nutrient-deplete cells causes increased cellular density and sinking below the nutricline. Upon nutrient replenishment and mobilization of glucan, the cells rise toward the surface of the water column, thereby transporting deep nutrients to the euphotic zone. β-1,3-Glucan also seems to play an important role in the development of resting stages in diatoms

Metabolism and function of β-1,3-glucan in marine diatoms

β-1,3-Glucan (chrysolaminaran) is the principal storage polysaccharide in diatoms (Bacillariophyceae), the major primary producers in the sea. The glucan generally contributes a substantial fraction of the algal biomass, but its level varies markedly in response to growth conditions. The scope of this work was to study the metabolism and function of the polysaccharide in marine diatoms. Axenic cultures of the marine planktonic diatom Skeletonema costatum (Grev.) Cleve were used in the experiments. Glucan metabolism was studied by growing the alga in batch culture, and measuring metabolite fluxes by chemical analyses as well as by 14C tracer technique using labeled bicarbonate. A photobioreactor was developed for strictly controlled growth of microalgae. Fine pH regulation was obtained by relay-activated titration with dilute acid (HCl) and base (NaOH). Irradiance and temperature were also carefully controlled. Batch cultures were grown with a 14:10 h light:dark cycle, and pH curves were recorded during different growth phases. A new method was developed for the combined determination of β-1,3-glucan and cell wall polysaccharides in diatoms, representing total cellular carbohydrate. The glucan is rapidly extracted by hot dilute H2SO4, and the cell wall polysaccharides are subsequently hydrolyzed by cold 80% H2SO4overnight. Each carbohydrate fraction is finally determined by the phenol-sulphuric acid method. This procedure is simple and rapid compared to previous methods, and applies well to laboratory cultures as well as natural phytoplankton populations dominated by diatoms. Synthesis and mobilization of β-1,3-glucan in N-limited S. costatum were studied by combined 14C tracer technique and chemical analyses. Radiolabeled bicarbonate was added to the cultures, and 14C incorporation in different metabolites was determined using biochemical fractionation. In a pulse phase, 14C label was mainly incorporated in the glucan fraction (85%) during photosynthesis under nitrogen limitation. Subsequently, a 14C chase was carried out by adding NH4+ and incubating the cells under different light conditions. Radiolabeled glucan decreased significantly (by 26% in the dark, and by 19% in low light) whereas radiolabeled amino acids, proteins and other polysaccharides increased significantly during NH4+ assimilation. Chemical analyses of β-1,3-glucan and cellular free amino acids supported the 14C measurements. Changes in amino acid composition strongly indicated that de novo biosynthesis took place, with a Gln/Glu ratio increasing from 0.4 to 10. This study provides new evidence of β-1,3-glucan supplying carbon skeletons for synthesis of amino acids and protein in diatoms. Mobilization of glucan yields glucose, which is further metabolized by the respiratory pathways to provide precursors as well as energy. The results from the 14C chase also indicated significant synthesis of other polysaccharides or possibly RNA from glucan. In a different study, dark carbon fixation in N-limited S. costatum was measured using 14C-bicarbonate. Addition of NH4+ resulted in 4-fold increase in carboxylation rate, and biochemical fractionation showed that mainly amino acids were radiolabeled. Chemical analyses confirmed that cellular free amino acids increased rapidly (with increasing Gln/Glu), and showed that cellular glucan decreased significantly (by 28%) during NH4+ assimilation. The results strongly indicate that β-carboxylation provides C4 precursors for amino acid synthesis, and β-1,3-glucan is likely to be the ultimate substrate for β-carboxylation. Moreover, a C/N uptake ratio of 0.33 indicated that β-carboxylation was related to protein synthesis. A detailed study was made of the production of carbohydrates and amino acids by S. costatum during different growth phases. During exponential growth under diel light conditions, the glucan level oscillated between 17% (end of scotophase) and 42% (end of photophase) of cellular organic carbon, and the corresponding protein/glucan ratio alternated between 2.3 and 0.7. Concurrently, the cellular free amino acid pool oscillated between 8% (end of scotophase) and 22% (end of photophase) of cellular organic nitrogen, and the corresponding Gln/Glu ratio alternated between 0.05 and 2. Depletion of nitrogen from the medium resulted in rapid accumulation of glucan, reaching 75-80% of cellular organic carbon, whereas the cellular nitrogenous components decreased significantly. Consequently, the protein/glucan ratio decreased to <0.1. This study indicates that β-1,3-glucan functions both as a short-term diurnal reserve and a long-term stockpile reserve. Field investigations by other workers suggest that glucan plays a very active role in the dynamics of natural diatom populations, and the protein/glucan ratio has been used as a sensitive parameter for nutrient status. The glucan dynamics may be involved in physiological control of buoyancy. Glucan accumulation by nutrient-deplete cells causes increased cellular density and sinking below the nutricline. Upon nutrient replenishment and mobilization of glucan, the cells rise toward the surface of the water column, thereby transporting deep nutrients to the euphotic zone. β-1,3-Glucan also seems to play an important role in the development of resting stages in diatoms.dr.ing.dr.ing

C3 and C4 Pathways of Photosynthetic Carbon Assimilation in Marine Diatoms Are under Genetic, Not Environmental, Control1[W][OA]

Marine diatoms are responsible for up to 20% of global CO2 fixation. Their photosynthetic efficiency is enhanced by concentrating CO2 around Rubisco, diminishing photorespiration, but the mechanism is yet to be resolved. Diatoms have been regarded as C3 photosynthesizers, but recent metabolic labeling and genome sequencing data suggest that they perform C4 photosynthesis. We studied the pathways of photosynthetic carbon assimilation in two diatoms by short-term metabolic 14C labeling. In Thalassiosira weissflogii, both C3 (glycerate-P and triose-P) and C4 (mainly malate) compounds were major initial (2–5 s) products, whereas Thalassiosira pseudonana produced mainly C3 and C6 (hexose-P) compounds. The data provide evidence of C3-C4 intermediate photosynthesis in T. weissflogii, but exclusively C3 photosynthesis in T. pseudonana. The labeling patterns were the same for cells grown at near-ambient (380 μL L−1) and low (100 μL L−1) CO2 concentrations. The lack of environmental modulation of carbon assimilatory pathways was supported in T. pseudonana by measurements of gene transcript and protein abundances of C4-metabolic enzymes (phosphoenolpyruvate carboxylase and phosphoenolpyruvate carboxykinase) and Rubisco. This study suggests that the photosynthetic pathways of diatoms are diverse, and may involve combined CO2-concentrating mechanisms. Furthermore, it emphasizes the requirement for metabolic and functional genetic and enzymic analyses before accepting the presence of C4-metabolic enzymes as evidence for C4 photosynthesis

Metabolic responses of avocado plants to stress induced by Rosellinia necatrix analysed by fluorescence and thermal imaging

One of the most important soilborne diseases affecting avocado (Persea americana Mill.) crops is white root rot, caused by the fungus Rosellinia necatrix. In this study we investigated the metabolic responses elicited by white root rot in the aerial part of the plant with special focus on the potential applications of imaging technique (including chlorophyll fluorescence (Chl-F), blue-green fluorescence and thermography) in early detection of the disease on leaves. The results show that leaf metabolism was significantly affected by the infection only when symptoms started to appear, which was probably related to the loss of root functionality. However, changes in some Chl-F parameters provided early indications of stress even prior to the development of symptoms. We suggest that the combinatorial analysis of several Chl-F parameters could be used as a method for early detection of stress related to white root rot, and might prove useful as a general indicator of biotic and abiotic stress in avocado plants.CICE-Junta de Andalucía (Proyectos de Excelencia P08-CVI-03475, P10-AGR-5797 and P12-AGR-0370), Plan Nacional de I+D+I del Ministerio de Ciencia e Innovación, Spain (AGL2011-30354C0201) cofinanced by FEDER, EU and RECUPERA 2020/20134R060 (Ministerio de Economía y Competitividad-CSIC, Feder funds).Peer Reviewe

Phenotyping of plant mutants using fluorescence and thermal imaging [Comunicación oral]

Congreso PhenoDays 201

Phenotyping of plant mutants using fluorescence and thermal imaging [Poster]

Peer Reviewe

Na+ transporter HKT1;2 reduces flower Na+ content and considerably mitigates the decline in tomato fruit yields under saline conditions

[EN] Genes encoding HKT1-like Na+ transporters play a key role in the salinity tolerance mechanism in Arabidopsis and other plant species by retrieving Na+ from the xylem of different organs and tissues. In this study, we investigated the role of two HKT1;2 allelic variants in tomato salt tolerance in relation to vegetative growth and fruit yield in plants subjected to salt treatment in a commercial greenhouse under real production conditions. We used two near-isogenic lines (NILs), homozygous for either the Solanum lycopersicum (NIL17) or S. cheesmaniae (NIL14) allele, at HKT1;2 loci and their respective RNAi-Sl/ScHKT1;2 lines. The results obtained show that both ScHKT1;2- and SIHKTI;2-silenced lines display hypersensitivity to salinity associated with an altered leaf Na+/K+ ratio, thus confirming that HKT1;2 plays an important role in Na+ homeostasis and salinity tolerance in tomato. Both silenced lines also showed Na+ over-accumulation and a slight, but significant, reduction in K+ content in the flower tissues of salt-treated plants and consequently a higher Na+/K+ ratio as compared to the respective unsilenced lines. This altered Na+/K+ ratio in flower tissues is associated with a sharp reduction in fruit yield, measured as total fresh weight and number of fruits, in both silenced lines under salinity conditions. Our findings demonstrate that Na+ transporter HKT1;2 protects the flower against Na+ toxicity and mitigates the reduction in tomato fruit yield under salinity conditionsWe wish to thank Emilio Jaime Fernandez (La Mayora, IHMS-CSIC) and Elena Sanchez Romero (EEZ-CSIC) for their technical assistance, the Scientific Instrumentation Service at EEZ-CSIC for their ICP-OES mineral analysis and Michael O'Shea for proofreading the text. The study was funded by EU-cofinanced grants from Agencia Estatal de Investigacion, Spanish Ministry of Economy, Industry and Competition (AGL2013-41733-R and AGL2017-82452-C2-1R to A.B., AGL2017-82452-C2-2R to M.J.A.) and the University of Granada (ACCESP2018-4 to JAT).Romero-Aranda, MR.; Gonzalez-Fernandez, P.; Pérez-Tienda, JR.; López-Díaz, MR.; Espinosa, J.; Granum, E.; Traverso, JÁ.... (2020). Na+ transporter HKT1;2 reduces flower Na+ content and considerably mitigates the decline in tomato fruit yields under saline conditions. Plant Physiology and Biochemistry. 154:341-352. https://doi.org/10.1016/j.plaphy.2020.05.012S34135215