Down-Regulation of Phosphoenolpyruvate Carboxylase Kinase in Grapevine Cell Cultures and Leaves Is Linked to Enhanced Resveratrol Biosynthesis.

In grapevine, trans-Resveratrol (tR) is produced as a defence mechanism against stress or infection. tR is also considered to be important for human health, which increases its interest to the scientific community. Transcriptomic analysis in grapevine cell cultures treated with the defence response elicitor methyl-ß-cyclodextrin (CD) revealed that both copies of PHOSPHOENOLPYRUVATE CARBOXYLASE KINASE (PPCK) were down-regulated significantly. A role for PPCK in the defence response pathway has not been proposed previously. We therefore analysed the control of PPCK transcript levels in grapevine cell cultures and leaves elicited with CD. Moreover, phosphoenolpyruvate carboxylase (PPC), stilbene synthase (STS), and the transcription factors MYB14 and WRKY24, which are involved in the activation of STS transcription, were also analysed by RT-qPCR. The results revealed that under CD elicitation conditions PPCK down-regulation, increased stilbene production and loss of PPC activity occurs in both tissues. Moreover, STS transcripts were co-induced with MYB14 and WRKY24 in cell cultures and leaves. These genes have not previously been reported to respond to CD in grape leaves. Our findings thus support the hypothesis that PPCK is involved in diverting metabolism towards stilbene biosynthesis, both for in vitro cell culture and whole leaves. We thus provide new evidence for PEP being redirected between primary and secondary metabolism to support tR production and the stress response.

Updating the steady-state model of C4 photosynthesis.

C4 plants play a key role in world agriculture. For example, C4 crops such as maize and sorghum are major contributors to food production in both developed and developing countries, and the C4 grasses sugarcane, miscanthus, and switchgrass are major plant sources of bioenergy. In the challenge to manipulate and enhance C4 photosynthesis, steady-state models of leaf photosynthesis provide an important tool for gas exchange analysis and thought experiments that can explore photosynthetic pathway changes. Here a previous C4 photosynthetic model developed by von Caemmerer and Furbank has been updated with new kinetic parameterization and temperature dependencies added. The parameterization was derived from experiments on the C4 monocot, Setaria viridis, which for the first time provides a cohesive parameterization. Mesophyll conductance and its temperature dependence have also been included, as this is an important step in the quantitative correlation between the initial slope of the CO2 response curve of CO2 assimilation and in vitro phosphoenolpyruvate carboxylase activity. Furthermore, the equations for chloroplast electron transport have been updated to include cyclic electron transport flow, and equations have been added to calculate the electron transport rate from measured CO2 assimilation rates.

Evolutionary Convergence of C4 Photosynthesis: A Case Study in the Nyctaginaceae.

C4 photosynthesis evolved over 65 times, with around 24 origins in the eudicot order Caryophyllales. In the Caryophyllales family Nyctaginaceae, the C4 pathway is known in three genera of the tribe Nyctagineae: Allionia, Okenia and Boerhavia. Phylogenetically, Allionia and Boerhavia/Okenia are separated by three genera whose photosynthetic pathway is uncertain. To clarify the distribution of photosynthetic pathways in the Nyctaginaceae, we surveyed carbon isotope ratios of 159 species of the Nyctaginaceae, along with bundle sheath (BS) cell ultrastructure, leaf gas exchange, and C4 pathway biochemistry in five species from the two C4 clades and closely related C3 genera. All species in Allionia, Okenia and Boerhavia are C4, while no C4 species occur in any other genera of the family, including three that branch between Allionia and Boerhavia. This demonstrates that C4 photosynthesis evolved twice in Nyctaginaceae. Boerhavia species use the NADP-malic enzyme (NADP-ME) subtype of C4 photosynthesis, while Allionia species use the NAD-malic enzyme (NAD-ME) subtype. The BS cells of Allionia have many more mitochondria than the BS of Boerhavia. Bundle sheath mitochondria are closely associated with chloroplasts in Allionia which facilitates CO2 refixation following decarboxylation by mitochondrial NAD-ME. The close relationship between Allionia and Boerhavia could provide insights into why NADP-ME versus NAD-ME subtypes evolve, particularly when coupled to analysis of their respective genomes. As such, the group is an excellent system to dissect the organizational hierarchy of convergent versus divergent traits produced by C4 evolution, enabling us to understand when convergence is favored versus when divergent modifications can result in a common phenotype.

The Development of Crassulacean Acid Metabolism (CAM) Photosynthesis in Cotyledons of the C4 Species, Portulaca grandiflora (Portulacaceae).

Portulaca grandiflora simultaneously utilizes both the C4 and Crassulacean acid metabolism (CAM) photosynthetic pathways. Our goal was to determine whether CAM developed and was functional simultaneously with the C4 pathway in cotyledons of P. grandiflora. We studied during development whether CAM would be induced with water stress by monitoring the enzyme activity, leaf structure, JO2 (rate of O2 evolution calculated by fluorescence analysis), and the changes in titratable acidity of 10 and 25 days old cotyledons. In the 10 days old cotyledons, C4 and CAM anatomy were evident within the leaf tissue. The cotyledons showed high titratable acid levels but a small CAM induction. In the 25 days old cotyledons, there was a significant acid fluctuation under 7 days of water stress. The overall enzyme activity was reduced in the 10 days old plants, while in the 25 days old plants CAM activity increased under water-stressed conditions. In addition to CAM, the research showed the presence of glycine decarboxylase in the CAM tissue. Thus, it appears both pathways develop simultaneously in the cotyledons but the CAM pathway, due to anatomical constraints, may be slower to develop than the C4 pathway. Cotyledons showed the ancestral Atriplicoid leaf anatomy, which leads to the question: Could a CAM cell be the precursor to the C4 pathway? Further study of this may lead to understanding into the evolution of C4 photosynthesis in the Portulaca.

Trichoderma-Azotobacter biofilm inoculation improves soil nutrient availability and plant growth in wheat and cotton.

Microbial biofilms are gaining importance in agriculture, due to their multifaceted agronomic benefits and resilience to environmental fluctuations. This study focuses on comparing the influence of single inoculation-Azotobacter chroococcum (Az) or Trichoderma viride (Tv) and their biofilm (Tv-Az), on soil and plant metabolic activities in wheat and cotton grown under Phytotron conditions. Tv-Az proved superior to all the other treatments in terms of better colonisation, plant growth attributes and 10-40% enhanced availability of macronutrients and micronutrients in the soil, over control. Confocal and scanning electron microscopy showed that the cells attached to the root tips initially, followed by their proliferation along the surface of the roots. Soil polysaccharides, proteins and dehydrogenase activity showed several fold enhancement in Tv-Az biofilm inoculated samples. Time course studies revealed that the population of Az and Tv in the rhizoplane and rhizosphere was significantly higher with a 0.14-0.31 log colony-forming unit (CFU) increase in the biofilm-inoculated treatment in both crops. Enhancement in soil biological activities was facilitated by the improved colonisation of the biofilm, due to the synergistic association between Tv and Az. This demonstrates the utility of Tv-Az biofilm as a multifunctional plant growth promoting and soil fertility enhancing option in agriculture.

Overexpression of the Wheat (Triticum aestivum L.) TaPEPKR2 Gene Enhances Heat and Dehydration Tolerance in Both Wheat and Arabidopsis.

Wheat (Triticum aestivum L.) yield and quality are adversely affected by heat, drought, or the combination of these two stresses in many regions of the world. A phosphoenolpyruvate carboxylase kinase-related kinase gene, TaPEPKR2, was identified from our previous heat stress-responsive transcriptome analysis of heat susceptible and tolerant wheat cultivars. Based on the wheat cultivar Chinese Spring genome sequence, TaPEPKR2 was mapped to chromosome 5B. Expression analysis revealed that TaPEPKR2 was induced by heat and polyethylene glycol treatment. To analyze the function of TaPEPKR2 in wheat, we transformed it into the wheat cultivar Liaochun10, and observed that the transgenic lines exhibited enhanced heat and dehydration stress tolerance. To examine whether TaPEPKR2 exhibits the same function in dicotyledonous plants, we transformed it into Arabidopsis, and found that its overexpression functionally enhanced tolerance to heat and dehydration stresses. Our results imply that TaPEPKR2 plays an important role in both heat and dehydration stress tolerance, and could be utilized as a candidate gene in transgenic breeding.

Diversity of CO2-concentrating mechanisms and responses to CO2 concentration in marine and freshwater diatoms.

The presence of CO2-concentrating mechanisms (CCMs) is believed to be one of the characteristics that allows diatoms to thrive in many environments and to be major contributors to global productivity. Here, the type of CCM and the responses to variable CO2 concentration were studied in marine and freshwater diatoms. At 400 ppm, there was a large diversity in physiological and biochemical mechanisms among the species. While Phaeodactylum tricornutum mainly used HCO3-, Thalassiosira pseudonana mainly used CO2. Carbonic anhydrase was an important component of the CCM in all species and C4 metabolism was absent, even with T. weissflogii. For all species, at 20 000 ppm, the affinity for dissolved inorganic carbon was lower than at 400 ppm CO2 and the reliance on CO2 was higher. Despite the difference in availability of inorganic carbon in marine and fresh waters, there were only small differences in CCMs between species from the two environments, and Navicula pelliculosa behaved similarly when grown in the two environments. The results suggest that species-specific differences are great, and more important than environmental differences in determining the nature and effectiveness of the CCM in diatoms.

Walking the C4 pathway: past, present, and future.

The year 2016 marks 50 years since the publication of the seminal paper by Hatch and Slack describing the biochemical pathway we now know as C4 photosynthesis. This review provides insight into the initial discovery of this pathway, the clues which led Hatch and Slack and others to these definitive experiments, some of the intrigue which surrounds the international activities which led up to the discovery, and personal insights into the future of this research field. While the biochemical understanding of the basic pathways came quickly, the role of the bundle sheath intermediate CO2 pool was not understood for a number of years, and the nature of C4 as a biochemical CO2 pump then linked the unique Kranz anatomy of C4 plants to their biochemical specialization. Decades of "grind and find biochemistry" and leaf physiology fleshed out the regulation of the pathway and the differences in physiological response to the environment between C3 and C4 plants. The more recent advent of plant transformation then high-throughput RNA and DNA sequencing and synthetic biology has allowed us both to carry out biochemical experiments and test hypotheses in planta and to better understand the evolution-driven molecular and genetic changes which occurred in the genomes of plants in the transition from C3 to C4 Now we are using this knowledge in attempts to engineer C4 rice and improve the C4 engine itself for enhanced food security and to provide novel biofuel feedstocks. The next 50 years of photosynthesis will no doubt be challenging, stimulating, and a drawcard for the best young minds in plant biology.

Walking the C4 pathway: past, present, and future.

The year 2016 marks 50 years since the publication of the seminal paper by Hatch and Slack describing the biochemical pathway we now know as C4 photosynthesis. This review provides insight into the initial discovery of this pathway, the clues which led Hatch and Slack and others to these definitive experiments, some of the intrigue which surrounds the international activities which led up to the discovery, and personal insights into the future of this research field. While the biochemical understanding of the basic pathways came quickly, the role of the bundle sheath intermediate CO2 pool was not understood for a number of years, and the nature of C4 as a biochemical CO2 pump then linked the unique Kranz anatomy of C4 plants to their biochemical specialization. Decades of "grind and find biochemistry" and leaf physiology fleshed out the regulation of the pathway and the differences in physiological response to the environment between C3 and C4 plants. The more recent advent of plant transformation then high-throughput RNA and DNA sequencing and synthetic biology has allowed us both to carry out biochemical experiments and test hypotheses in planta and to better understand the evolution-driven molecular and genetic changes which occurred in the genomes of plants in the transition from C3 to C4 Now we are using this knowledge in attempts to engineer C4 rice and improve the C4 engine itself for enhanced food security and to provide novel biofuel feedstocks. The next 50 years of photosynthesis will no doubt be challenging, stimulating, and a drawcard for the best young minds in plant biology.

Direct and selective small-molecule inhibition of photosynthetic PEP carboxylase: New approach to combat C4 weeds in arable crops.

Phosphoenolpyruvate carboxylase (PEPC) is a key enzyme of C4 photosynthesis. Besides, non-photosynthetic isoforms of PEPC are found in bacteria and all types of plants, although not in animals or fungi. A single residue in the allosteric feedback inhibitor site of PEPC was shown to adjust the affinity of the photosynthetic and non-photosynthetic isoforms for feedback inhibition by metabolites of the C4 pathway. Here, we applied computational screening and biochemical analyses to identify molecules that selectively inhibit C4 PEPC, but have no effect on the activity of non-photosynthetic PEPCs. We found two types of selective inhibitors, catechins and quinoxalines. Binding constants in the lower µM range and a strong preference for C4 PEPC qualify the quinoxaline compounds as potential selective herbicides to combat C4 weeds.

Cell maturity gradient determines light regulated accumulation of proteins in pearl millet leaves.

Developing pearl millet leaves possess cells of increasing maturity from the leaf base to the tip with oldest cells at the leaf tip. This natural developmental gradient was used to analyze the photoregulation of enzymes located in cytosolic, peroxisomal, and plastidic compartments of the leaf in relation to the cell age. In dark-grown leaves, the level of plastidic protochlorophyllide oxidoreductase A (PORA) (EC 1.3.1.33) protein increased from the leaf base to tip. Exposure to light reduced the level of PORA protein and stiumulated accmulation of PORB protein with increasing level from the base to the leaf tip. Light induced formation of cytosolic PEP carboxylase (EC 4.1.1.31) in the leaf with induction being maximal in the leaf tip. The levels of peroxisomal protein catalase (EC 1.11.1.6) showed gradual increase from the base to leaf tip in dark and light-grown leaves. By contrast, the distribution profile of cytosolic enzyme peroxidase (EC 1.11.1.7) followed reverse pattern in the dark-and light-grown leaves. In dark-grown leaves, peroxidase level showed increase towards the leaf tip, whereas light exposure lowered peroxidase level near the leaf tip and stimulated near the leaf base. These results indicate that the cell maturity gradient in cooperation with plastids modulates in a dual fashion the magnitude and pattern of photoregulation of protein levels in pearl millet leaves. It promotes the level of proteins functionally related to plastids towards the leaf tip and at the same time suppresses the level of cytosolic proteins, restricting them to the leaf base.

PEP carboxylase kinase is a novel protein kinase controlled at the level of expression.

Phosphoenolpyruvate (PEP) carboxylase plays a number of key roles in the central metabolism of higher plants. The enzyme is regulated by reversible phosphorylation in response to a range of signals in many different plant tissues. The data discussed here illustrate several novel features of this system. The phosphorylation state of PEP carboxylase is controlled largely by the activity of PEP carboxylase kinase. This enzyme comprises a protein kinase catalytic domain with no regulatory regions. In many systems it is controlled at the level of expression. In C4 plants, expression of PEP carboxylase kinase is light-regulated and involves changes in cytosolic pH, InsP3 and Ca2+ levels. Expression of PEP carboxylase kinase in CAM plants is regulated by a circadian oscillator, perhaps via metabolite control. Some plants contain multiple PEP carboxylase kinase genes, probably with different expression patterns and roles. A newly discovered PEP carboxylase kinase inhibitor protein might facilitate the net dephosphorylation of PEP carboxylase under conditions in which flux through this enzyme is not required.