Kinetic modeling identifies targets for engineering improved photosynthetic efficiency in potato (Solanum tuberosum cv. Solara).

Potato (Solanum tuberosum) is a significant non-grain food crop in terms of global production. However, its yield potential might be raised by identifying means to release bottlenecks within photosynthetic metabolism, from the capture of solar energy to the synthesis of carbohydrates. Recently, engineered increases in photosynthetic rates in other crops have been directly related to increased yield - how might such increases be achieved in potato? To answer this question, we derived the photosynthetic parameters Vcmax and Jmax to calibrate a kinetic model of leaf metabolism (e-Photosynthesis) for potato. This model was then used to simulate the impact of manipulating the expression of genes and their protein products on carbon assimilation rates in silico through optimizing resource investment among 23 photosynthetic enzymes, predicting increases in photosynthetic CO2 uptake of up to 67%. However, this number of manipulations would not be practical with current technologies. Given a limited practical number of manipulations, the optimization indicated that an increase in amounts of three enzymes - Rubisco, FBP aldolase, and SBPase - would increase net assimilation. Increasing these alone to the levels predicted necessary for optimization increased photosynthetic rate by 28% in potato.

Removal of plant endogenous proteins from tobacco leaf extract by freeze-thaw treatment for purification of recombinant proteins.

Plants are useful as a low-cost source for producing biopharmaceutical proteins. A significant hurdle in the production of recombinant proteins in plants, however, is the complicated process of removing plant-derived components. Removing endogenous plant proteins, including ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO), a major photosynthetic plant enzyme that catalyzes photosynthesis through carboxylation and oxygenation, is important for the purification of recombinant plant proteins. In particular, RuBisCO accounts for 50% of the soluble leaf protein; thus, the removal of RuBisCO is critical for the purification of recombinant proteins from plant materials. An effective conventional method, known as freeze-thaw treatment, was developed for the removal of RuBisCO from Nicotiana benthamiana, which expresses recombinant green fluorescent protein (GFP). Crude extracts or supernatants were frozen at - 30 °C. Upon thawing, most of the RuBisCO was precipitated by centrifugation without significant inactivation and/or yield reduction of GFP. Based on the proteomics analysis, using this method, RuBisCO large and small subunits were reduced to approximately 10% and 20% of those of the unfrozen supernatant solutions, respectively, without the need for specific reagents or equipment. The proteomic analysis also revealed that many ribosomal proteins were removed from the extracts. This method improves the purification process of recombinant proteins from plant materials. Prolonged freezing damaged recombinant ß-glucuronidase (GUS), suggesting that the applicability of this treatment should be carefully considered for each recombinant protein.

Alternative methods for RuBisCO extraction from sugar beet waste: A comparative approach of ultrasound and high voltage electrical discharge.

Ultrasound (US) and high voltage electric discharge (HVED) with water as a green solvent represent promising novel non-thermal techniques for protein extraction from sugar beet (Beta vulgaris subsp. vulgaris var. altissima) leaves. Compared to HVED, US proved to be a better alternative method for total soluble protein extraction with the aim of obtaining high yield of ribulose-1,5-bisphosphate carboxylase-oxygenase enzyme (RuBisCO). Regardless of the solvent temperature, the highest protein yields were observed at 100% amplitude and 9 min treatment time (84.60 ± 3.98 mg/gd.m. with cold and 96.75 ± 4.30 mg/gd.m. with room temperature deionized water). US treatments at 75% amplitude and 9 min treatment time showed the highest abundance of RuBisCO obtained by immunoblotting assay. The highest protein yields recorded among HVED-treated samples were observed at a voltage of 20 kV and a treatment time of 3 min, disregarding the used gas (33.33 ± 1.06 mg/gd.m. with argon and 34.89 ± 1.59 mg/gd.m. with nitrogen as injected gas), while the highest abundance of the RuBisCO among HVED-treated samples was noticed at 25 kV voltage and 3 min treatment time. By optimizing the US and HVED parameters, it is possible to affect the solubility and improve the isolation of RuBisCO, which could then be purified and implemented into new or already existing functional products.

The impact of wounding and postharvest storage conditions on retention of soluble protein in sugar beet leaves.

Sugar beet leaves can be a viable and economically interesting source of high-quality protein for the food industry. We investigated how storage conditions and leaf wounding at harvest affect the content and quality of the soluble protein. After collection, leaves were either stored intact or shredded to mimic wounding induced by commercial leaf harvesters. Leaf material was stored in small volumes at different temperatures to assess leaf physiology or in larger volumes to assess temperature development at different locations in the bins. Protein degradation was more pronounced at higher storage temperatures. Wounding accelerated the degradation of soluble protein at all temperatures. Both wounding and storage at higher temperatures greatly stimulated respiration activity and heat production. At temperatures below 5°C, ribulose-1,5-biphosphate carboxylase oxygenase (RuBisCO) in intact leaves was preserved for up to 3 weeks. At temperatures of 30-40°C, RuBisCO degradation occurred within 48 h. Degradation was more pronounced in shredded leaves. In 0.8-m3 storage bins at ambient temperature, core temperatures rapidly increased, up to 25°C in intact leaves and up to 45°C in shredded leaves within 2-3 days. Immediate storage at 5°C greatly suppressed the temperature increase in intact but not in shredded leaves. The indirect effect of excessive wounding, that is, heat production, is discussed as the pivotal factor responsible for increased degradation of protein. For optimal retention of soluble protein levels and quality in harvested sugar beet leaves, it is advised to minimize wounding and to store the material at temperatures around -5°C. PRACTICAL APPLICATION: To preserve the soluble protein content and quality for at least 3 weeks, sugar beet leaves should be harvested with minimal wounding and stored at temperatures between 1 and 5°C. When aiming to store minimally wounded leaves in larger volumes, it must be ensured that the product temperature in the core of the biomass meets the temperature criterium or the cooling strategy must be adjusted. The principles of minimal wounding and low temperature storage are transferable to other leafy crops that are harvested for food protein.

Specialized metabolism by trichome-enriched Rubisco and fatty acid synthase components.

Acylsugars, specialized metabolites with defense activities, are secreted by trichomes of many solanaceous plants. Several acylsugar metabolic genes (AMGs) remain unknown. We previously reported multiple candidate AMGs. Here, using multiple approaches, we characterized additional AMGs. First, we identified differentially expressed genes between high- and low-acylsugar-producing F2 plants derived from a cross between cultivated tomato (Solanum lycopersicum) and a wild relative (Solanum pennellii), which produce acylsugars that are ∼1% and ∼20% of leaf dry weight, respectively. Expression levels of many known and candidate AMGs positively correlated with acylsugar amounts in F2 individuals. Next, we identified lycopersicum-pennellii putative orthologs with higher nonsynonymous to synonymous substitutions. These analyses identified four candidate genes, three of which showed enriched expression in stem trichomes compared to underlying tissues (shaved stems). Virus-induced gene silencing confirmed two candidates, Sopen05g009610 [beta-ketoacyl-(acyl-carrier-protein) reductase; fatty acid synthase component] and Sopen07g006810 (Rubisco small subunit), as AMGs. Phylogenetic analysis indicated that Sopen05g009610 is distinct from specialized metabolic cytosolic reductases but closely related to two capsaicinoid biosynthetic reductases, suggesting evolutionary relationship between acylsugar and capsaicinoid biosynthesis. Analysis of publicly available datasets revealed enriched expression of Sopen05g009610 orthologs in trichomes of several acylsugar-producing species. Similarly, orthologs of Sopen07g006810 were identified as solanaceous trichome-enriched members, which form a phylogenetic clade distinct from those of mesophyll-expressed "regular" Rubisco small subunits. Furthermore, δ13C analyses indicated recycling of metabolic CO2 into acylsugars by Sopen07g006810 and showed how trichomes support high levels of specialized metabolite production. These findings have implications for genetic manipulation of trichome-specialized metabolism in solanaceous crops.

Sufficient potassium improves inorganic phosphate-limited photosynthesis in Brassica napus by enhancing metabolic phosphorus fractions and Rubisco activity.

Crop photosynthesis (A) and productivity are often limited by a combination of nutrient stresses, such that changes in the availability of one nutrient may affect the availability of another nutrient, in turn influencing A. In this study, we examined the synergistic effects of phosphorus (P) and potassium (K) on leaf A in a nutrient amendment experiment, in which P and K were added individually or in combination to Brassica napus grown under P and K co-limitation. The data revealed that the addition of P gradually removed the dominant limiting factor (i.e. the limited availability of P) and improved leaf A. Strikingly, the addition of K synergistically improved the overall uptake of P, mainly by boosting plant growth, and compensated for the physiological demand for P by prioritizing investment in metabolic pools of P (P-containing metabolites and inorganic phosphate, Pi). The enlarged pool of metabolically active P was partially associated with the upregulation of Pi regeneration through release from triose phosphates rather than replacement of P-containing lipids. This process mitigated P restrictions on A by maintaining the ATP/NADPH and NADPH/NADP+ ratios and increasing the content and activity of Rubisco. Our findings demonstrate that sufficient K increased Pi-limited A by enhancing metabolic P fractions and Rubisco activity. Thus, ionic synergism may be exploited to mitigate nutrient-limiting factors to improve crop productivity.

Interspecies Comparison of Interaction Energies between Photosynthetic Protein RuBisCO and 2CABP Ligand.

Ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCO) functions as the initial enzyme in the dark reactions of photosynthesis, catalyzing reactions that extract CO2 from the atmosphere and fix CO2 into organic compounds. RuBisCO is classified into four types (isoforms I-IV) according to sequence-based phylogenetic trees. Given its size, the computational cost of accurate quantum-chemical calculations for functional analysis of RuBisCO is high; however, recent advances in hardware performance and the use of the fragment molecular orbital (FMO) method have enabled the ab initio analyses of RuBisCO. Here, we performed FMO calculations on multiple structural datasets for various complexes with the 2'-carboxylarabinitol 1,5-bisphosphate (2CABP) ligand as a substrate analog and investigated whether phylogenetic relationships based on sequence information are physicochemically relevant as well as whether novel information unobtainable from sequence information can be revealed. We extracted features similar to the phylogenetic relationships found in sequence analysis, and in terms of singular value decomposition, we identified residues that strongly interacted with the ligand and the characteristics of the isoforms for each principal component. These results identified a strong correlation between phylogenetic relationships obtained by sequence analysis and residue interaction energies with the ligand. Notably, some important residues were located far from the ligand, making comparisons among species using only residues proximal to the ligand insufficient.

Photosynthetic carbon and nitrogen metabolism of Camellia oleifera Abel during acclimation to low light conditions.

Tea-oil tree (Camellia oleifera Abel) is an important woody oil crop with high economic value. However, it has low photosynthetic production considering the low light intensity of its growth environment. To understand the acclimation mechanism of tea-oil trees to low light conditions, three light intensity treatments were conducted: high light (450-500 µmol. m-2. s-1), medium light (180-200 µmol. m-2. s-1), and low light (45-50 µmol. m-2. s-1). The carbon (C) and nitrogen (N) metabolism network were constructed by investigating the leaf anatomy, photosynthetic characteristics, N partitioning, transcriptome and metabolome. Results demonstrated that a larger proportion light energy was used for photochemical reactions in an environment with lower light intensity, which resulted in an increase in photosystem II photochemical efficiency and instantaneous light use efficiency (LUE) at the leaf level. As the light intensity increased, decreased electron transfer and carboxylation efficiencies, photorespiration and dark respiration rates, LUE at plant level, and N use efficiency (PNUE) were observed. Leaves trended to harvest more light using higher expression levels of light-harvesting protein genes, higher chlorophyll content, more granum and more tightly stacked granum lamella under lower light intensity. At transcriptional and metabolic levels, the TCA cycle, and the synthesis of starch and saccharides were weakened as light intensity decreased, while the Calvin cycle did not show the regularity between different treatments. Less N was distributed in Rubisco, respiration, and cell wall proteins as light decreased. Storage N was prominently accumulated in forms of amino acids (especially L-arginine) and amino acid derivatives as under medium and low light environments, to make up for C deficiency. Therefore, tea-oil trees actively improve light-harvesting capacity and enlarges the storage N pool to adapt to a low light environment, at the cost of a decrease of photosynthetic C assimilation and PNUE.

An orally active plant Rubisco-derived peptide increases neuronal leptin responsiveness.

Nutrient excess, such as the intake of a high-fat diet, reduces hypothalamic responses to exogenously administered leptin and induces dietary obesity; however, orally active components that attenuate neural leptin dysregulation have yet to be identified. We herein demonstrated that YHIEPV, derived from the pepsin-pancreatin digestion of the green leaf protein Rubisco, increased the leptin-induced phosphorylation of STAT3 in ex vivo hypothalamic slice cultures. We also showed that YHIEPV mitigated palmitic acid-induced decreases in leptin responsiveness. Furthermore, orally administered YHIEPV promoted leptin-induced reductions in body weight and food intake in obese mice. In addition, dietary-induced body weight gain was significantly less in mice orally or centrally administered YHIEPV daily than in saline-control mice. Cellular leptin sensitivity and the levels of proinflammatory-related factors, such as IL1ß and Socs-3, in the hypothalamus of obese mice were also restored by YHIEPV. YHIEPV blocked cellular leptin resistance induced by forskolin, which activates Epac-Rap1 signaling, and reduced the level of the GTP-bound active form of Rap1 in the brains of obese mice. Collectively, the present results demonstrated that the orally active peptide YHIEPV derived from a major green leaf protein increased neural leptin responsiveness and reduced body weight gain in mice with dietary obesity.

Global variation in the fraction of leaf nitrogen allocated to photosynthesis.

Plants invest a considerable amount of leaf nitrogen in the photosynthetic enzyme ribulose-1,5-bisphosphate carboxylase-oxygenase (RuBisCO), forming a strong coupling of nitrogen and photosynthetic capacity. Variability in the nitrogen-photosynthesis relationship indicates different nitrogen use strategies of plants (i.e., the fraction nitrogen allocated to RuBisCO; fLNR), however, the reason for this remains unclear as widely different nitrogen use strategies are adopted in photosynthesis models. Here, we use a comprehensive database of in situ observations, a remote sensing product of leaf chlorophyll and ancillary climate and soil data, to examine the global distribution in fLNR using a random forest model. We find global fLNR is 18.2 ± 6.2%, with its variation largely driven by negative dependence on leaf mass per area and positive dependence on leaf phosphorus. Some climate and soil factors (i.e., light, atmospheric dryness, soil pH, and sand) have considerable positive influences on fLNR regionally. This study provides insight into the nitrogen-photosynthesis relationship of plants globally and an improved understanding of the global distribution of photosynthetic potential.

Sodium lignosulfonate improves shoot growth of Oryza sativa via enhancement of photosynthetic activity and reduced accumulation of reactive oxygen species.

Lignosulfonate (LS) is a by-product obtained during sulfite pulping process and is commonly used as a growth enhancer in plant growth. However, the underlying growth promoting mechanism of LS on shoot growth remains largely unknown. Hence, this study was undertaken to determine the potential application of eco-friendly ion-chelated LS complex [sodium LS (NaLS) and calcium LS (CaLS)] to enhance recalcitrant indica rice MR 219 shoot growth and to elucidate its underlying growth promoting mechanisms. In this study, the shoot apex of MR 219 rice was grown on Murashige and Skoog medium supplemented with different ion chelated LS complex (NaLS and CaLS) at 100, 200, 300 and 400 mg/L The NaLS was shown to be a better shoot growth enhancer as compared to CaLS, with optimum concentration of 300 mg/L. Subsequent comparative proteomic analysis revealed an increase of photosynthesis-related proteins [photosystem II (PSII) CP43 reaction center protein, photosystem I (PSI) iron-sulfur center, PSII CP47 reaction center protein, PSII protein D1], ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), carbohydrate metabolism-related proteins (glyceraldehyde-3-phosphate dehydrogenase 3, fructose-bisphosphate aldolase) and stress regulator proteins (peptide methionine sulfoxide reductase A4, delta-1-pyrroline-5-carboxylate synthase 1) abundance in NaLS-treated rice as compared to the control (MSO). Consistent with proteins detected, a significant increase in biochemical analyses involved in photosynthetic activities, carbohydrate metabolism and protein biosynthesis such as total chlorophyll, rubisco activity, total sugar and total protein contents were observed in NaLS-treated rice. This implies that NaLS plays a role in empowering photosynthesis activities that led to plant growth enhancement. In addition, the increased in abundance of stress regulator proteins were consistent with low levels of peroxidase activity, malondialdehyde content and phenylalanine ammonia lyase activity observed in NaLS-treated rice. These results suggest that NaLS plays a role in modulating cellular homeostasis to provide a conducive cellular environment for plant growth. Taken together, NaLS improved shoot growth of recalcitrant MR 219 rice by upregulation of photosynthetic activities and reduction of ROS accumulation leading to better plant growth.

Modifying Plant Photosynthesis and Growth via Simultaneous Chloroplast Transformation of Rubisco Large and Small Subunits.

Engineering improved Rubisco for the enhancement of photosynthesis is challenged by the alternate locations of the chloroplast rbcL gene and nuclear RbcS genes. Here we develop an RNAi-RbcS tobacco (Nicotiana tabacum) master-line, tobRrΔS, for producing homogenous plant Rubisco by rbcL-rbcS operon chloroplast transformation. Four genotypes encoding alternative rbcS genes and adjoining 5'-intergenic sequences revealed that Rubisco production was highest (50% of the wild type) in the lines incorporating a rbcS gene whose codon use and 5' untranslated-region matched rbcL Additional tobacco genotypes produced here incorporated differing potato (Solanum tuberosum) rbcL-rbcS operons that either encoded one of three mesophyll small subunits (pS1, pS2, and pS3) or the potato trichome pST-subunit. The pS3-subunit caused impairment of potato Rubisco production by ∼15% relative to the lines producing pS1, pS2, or pST However, the ßA-ßB loop Asn-55-His and Lys-57-Ser substitutions in the pS3-subunit improved carboxylation rates by 13% and carboxylation efficiency (CE) by 17%, relative to potato Rubisco incorporating pS1 or pS2-subunits. Tobacco photosynthesis and growth were most impaired in lines producing potato Rubisco incorporating the pST-subunit, which reduced CE and CO2/O2 specificity 40% and 15%, respectively. Returning the rbcS gene to the plant plastome provides an effective bioengineering chassis for introduction and evaluation of novel homogeneous Rubisco complexes in a whole plant context.

Jasmonic acid and methyl jasmonate modulate growth, photosynthetic activity and expression of photosystem II subunit genes in Brassica oleracea L.

The effects of jasmonic acid (JA) and methyl jasmonate (Me-JA) on photosynthetic efficiency and expression of some photosystem (PSII) related in different cultivars of Brassica oleracea L. (var. italica, capitata, and botrytis) were investigated. Plants raised from seeds subjected to a pre-sowing soaking treatment of varying concentrations of JA and Me-JA showed enhanced photosynthetic efficiency in terms of qP and chlorophyll fluorescence. Maximum quantum efficiency of PSII (Fv/Fm) was increased over that in the control seedlings. This enhancement was more pronounced in the Me-JA-treated seedlings compared to that in JA-treated ones. The expression of PSII genes was differentially regulated among the three varieties of B. oleracea. The gene PsbI up-upregulated in var. botrytis after treatment of JA and Me-JA, whereas PsbL up-regulated in capitata and botrytis after supplementation of JA. The gene PsbM showed many fold enhancements in these expressions in italica and botrytis after treatment with JA. However, the expression of the gene PsbM increased by both JA and Me-JA treatments. PsbTc(p) and PsbTc(n) were also found to be differentially expressed which revealed specificity with the variety chosen as well as JA or Me-JA treatments. The RuBP carboxylase activity remained unaffected by either JA or Me-JA supplementation in all three varieties of B. oleracea L. The data suggest that exogenous application of JA and Me-JA to seeds before germination could influence the assembly, stability, and repair of PS II in the three varieties of B. oleracea examined. Furthermore, this improvement in the PS II machinery enhanced the photosynthetic efficiency of the system and improved the photosynthetic productivity in terms of saccharides accumulation.

Phosphorus toxicity disrupts Rubisco activation and reactive oxygen species defence systems by phytic acid accumulation in leaves.

Phosphorus (P) is an essential mineral nutrient for plants. Nevertheless, excessive P accumulation in leaf mesophyll cells causes necrotic symptoms in land plants; this phenomenon is termed P toxicity. However, the detailed mechanisms underlying P toxicity in plants have not yet been elucidated. This study aimed to investigate the molecular mechanism of P toxicity in rice. We found that under excessive inorganic P (Pi) application, Rubisco activation decreased and photosynthesis was inhibited, leading to lipid peroxidation. Although the defence systems against reactive oxygen species accumulation were activated under excessive Pi application conditions, the Cu/Zn-type superoxide dismutase activities were inhibited. A metabolic analysis revealed that excessive Pi application led to an increase in the cytosolic sugar phosphate concentration and the activation of phytic acid synthesis. These conditions induced mRNA expression of genes that are activated under metal-deficient conditions, although metals did accumulate. These results suggest that P toxicity is triggered by the attenuation of both photosynthesis and metal availability within cells mediated by phytic acid accumulation. Here, we discuss the whole phenomenon of P toxicity, beginning from the accumulation of Pi within cells to death in land plants.

Physiological and biochemical responses to aluminum-induced oxidative stress in two cyanobacterial species.

Phycoremediation technologies significantly contribute to solving serious problems induced by heavy metals accumulation in the aquatic systems. Here we studied the mechanisms underlying Al stress tolerance in two diazotrophic cyanobacterial species, to identify suitable species for Al phycoremediation. Al uptake as well as the physiological and biochemical responses of Anabaena laxa and Nostoc muscorum to 7 days Al exposure at two different concentrations i.e., mild (100 µM) and high dose (200 µM), were investigated. Our results revealed that A. laxa accumulated more Al, and it could acclimatize to long-term exposure of Al stress. Al induced a dose-dependent decrease in photosynthesis and its related parameters e.g., chlorophyll content (Chl a), phosphoenolpyruvate carboxylase (PEPC) and Ribulose‒1,5‒bisphosphate carboxylase/oxygenase (RuBisCo) activities. The affect was less pronounced in A. laxa than N. muscorum. Moreover, Al stress significantly increased cellular membrane damage as indicated by induced H2O2, lipid peroxidation, protein oxidation, and NADPH oxidase activity. However, these increases were lower in A. laxa compared to N. muscorum. To mitigate the impact of Al stress, A. laxa induced its antioxidant defense system by increasing polyphenols, flavonoids, tocopherols and glutathione levels as well as peroxidase (POX), catalase (CAT), glutathione reductase (GR) and glutathione peroxidase (GPX) enzymes activities. On the other hand, the antioxidant increases in N. muscorum were only limited to ascorbate (ASC) cycle. Overall, high biosorption/uptake capacity and efficient antioxidant defense system of A. laxa recommend its feasibility in the treatment of Al contaminated waters/soils.

Role of Ca2+ as protectant under heat stress by regulation of photosynthesis and membrane saturation in Anabaena PCC 7120.

The present study was aimed at understanding the effects of heat stress on selected physiological and biochemical parameters of a model cyanobacterium, Anabaena PCC 7120 in addition to amelioration strategy using exogenous Ca2+. A comparison of the cells exposed to heat stress (0-24 h) in the presence or absence of Ca2+ clearly showed reduction in colony-forming ability and increase in reactive oxygen species (ROS) leading to loss in the viability of cells of Ca2+-deficient cultures. There was higher level of saturation in membrane lipids of the cells supplemented with Ca2+ along with higher accumulation of proline. Similarly, higher quantum yield (7.8-fold) in Ca2+-supplemented cultures indicated role of Ca2+ in regulation of photosynthesis. Relative electron transport rate (rETR) decreased in both the sets with the difference in the rate of decrease (slow) in Ca2+-supplemented cultures. The Ca2+-supplemented sets also maintained high levels of open reaction centers of PS II in comparison to Ca2+-deprived cells. Increase in transcripts of both subunits ((rbcL and rbcS) of RubisCO from Ca2+-supplemented Anabaena cultures pointed out the role of Ca2+ in sustenance of photosynthesis of cells via CO2 fixation, thus, playing an important role in maintaining metabolic status of the heat-stressed cyanobacterium.

Comparison of the functional properties of RuBisCO protein isolate extracted from sugar beet leaves with commercial whey protein and soy protein isolates.

BACKGROUND: RuBisCO was extracted from sugar beet leaves using soft and food-compatible technologies. Proximate composition, solubility, emulsifying, foaming and gelling properties of the protein isolate were determined. All these properties were systematically benchmarked against commercial whey and soy protein isolates used in food applications. RESULTS: RuBisCO protein isolate (RPI) contained 930 g kg-1 of crude protein. Protein solubility was higher than 80% at pH values lower than 4.0 or higher than 5.5. Foaming capacity of RPI was better at pH 4.0 than at pH 7.0. Interestingly, 10 g kg-1 protein foams were more stable (pH 7.0 and 4.0) than foams obtained with whey or soy protein. Moreover, 10 g kg-1 RPI emulsions at pH 4.0 or 7.0 exhibited good stability, being similar to whey protein isolate. Remarkable gelling properties were observed at pH 7.0, where 50 g kg-1 protein solutions of RPI formed self-supporting gels while more concentrated solutions were needed for whey or soy protein. CONCLUSION: RuBisCO showed comparable or superior functional properties to those of currently used whey and soy protein isolates. These results highlight the high potential of sugar beet leaf protein isolate as a nutritious and functional food ingredient to face global food security and protein supply. © 2018 Society of Chemical Industry.

Extraction of RuBisCO to Determine Catalytic Constants.

RuBisCO enables net carbon fixation through the carboxylation of RuBP during photosynthesis. Its complex biochemistry and catalytic diversity found among different plants make characterization of RuBisCO properties useful for investigations aimed at improving photosynthetic performance. This chapter reports methods for rapid extraction of soluble proteins to examine RuBisCO catalytic properties, and for large-scale purification of RuBisCO from leaves to measure the specificity of the enzyme toward its gaseous substrates.

Spectrophotometric Determination of RuBisCO Activity and Activation State in Leaf Extracts.

RuBisCO plays a central role in photosynthesis and, due to its catalytic inefficiencies, frequently limits CO2 assimilation in fully illuminated leaves at the top of unstressed crop canopies. The CO2-fixing enzyme is heavily regulated and not all the enzyme present in the leaf is active at any given moment. In this chapter, a spectrophotometric assay is described for measuring RuBisCO activity and activation state in leaf extracts. Most of the assay components are available commercially and others can be produced by established protocols, making adoption of the assay achievable by most plant biochemistry laboratories. Its relative high-throughput capacity enables large-scale experiments aimed at screening germplasm for improved RuBisCO function.

Leaf proteomics of drought-sensitive and -tolerant genotypes of fennel.

Fennel is attracted attention as a useful resource as researching medicinal plant for drought tolerance. To elucidate the response mechanism in drought-sensitive and -tolerant genotypes of fennel leaf, a gel-free/label-free proteomic technique was used. Fifty-day-old plants were subjected to drought stress for 60days. The relative water and proline contents were decreased and increased in sensitive genotypes, respectively; however, they were not a big change in tolerant genotypes. Photosynthesis was decreased in the sensitive genotypes under drought; however, it was increased in the tolerant genotype. In both drought-sensitive and -tolerant genotypes, proteins related to protein metabolism and cell organization were predominately affected under drought stress. The abundance of phosphoribulokinase and phosphoglycerate kinase enzymes were decreased and increased in drought-sensitive and -tolerant genotypes, respectively; however, the abundance of RuBisCO and glyceraldehyde-3-phosphate dehydrogenase enzymes were increased and decreased in drought-sensitive and -tolerant genotypes, respectively. Under drought stress, the abundance of glycolysis-related proteins was decreased in sensitive genotypes; however, they were increased in tolerance genotypes. Commonly changed proteins with polyethylene glycol fractionation such as cobalamin-independent methionine synthase were decreased and increased in drought-sensitive and -tolerant genotypes, respectively. These results suggest that cobalamin-independent methionine synthetase is involved in the tolerance of drought-tolerant fennel leaf under drought stress.