The Calvin-Benson-Bassham cycle in C4 and Crassulacean acid metabolism species.

The Calvin-Benson-Bassham (CBB) cycle is the ancestral CO2 assimilation pathway and is found in all photosynthetic organisms. Biochemical extensions to the CBB cycle have evolved that allow the resulting pathways to act as CO2 concentrating mechanisms, either spatially in the case of C4 photosynthesis or temporally in the case of Crassulacean acid metabolism (CAM). While the biochemical steps in the C4 and CAM pathways are known, questions remain on their integration and regulation with CBB cycle activity. The application of omic and transgenic technologies is providing a more complete understanding of the biochemistry of C4 and CAM species and will also provide insight into the CBB cycle in these plants. As the global population increases, new solutions are required to increase crop yields and meet demands for food and other bioproducts. Previous work in C3 species has shown that increasing carbon assimilation through genetic manipulation of the CBB cycle can increase biomass and yield. There may also be options to improve photosynthesis in species using C4 photosynthesis and CAM through manipulation of the CBB cycle in these plants. This is an underexplored strategy and requires more basic knowledge of CBB cycle operation in these species to enable approaches for increased productivity.

Glyceraldehyde-3-phosphate dehydrogenase subunits A and B are essential to maintain photosynthetic efficiency.

In plants, glyceraldehyde-3-phosphate dehydrogenase (GAPDH; EC 1.2.1.12) reversibly converts 1,3-bisphosphoglycerate to glyceraldehyde-3-phosphate coupled with the reduction of NADPH to NADP+. The GAPDH enzyme that functions in the Calvin-Benson cycle is assembled either from 4 glyceraldehyde-3-phosphate dehydrogenase A (GAPA) subunit proteins forming a homotetramer (A4) or from 2 GAPA and 2 glyceraldehyde-3-phosphate dehydrogenase B (GAPB) subunit proteins forming a heterotetramer (A2B2). The relative importance of these 2 forms of GAPDH in determining the rate of photosynthesis is unknown. To address this question, we measured the photosynthetic rates of Arabidopsis (Arabidopsis thaliana) plants containing reduced amounts of the GAPDH A and B subunits individually and jointly, using T-DNA insertion lines of GAPA and GAPB and transgenic GAPA and GAPB plants with reduced levels of these proteins. Here, we show that decreasing the levels of either the A or B subunits decreased the maximum efficiency of CO2 fixation, plant growth, and final biomass. Finally, these data showed that the reduction in GAPA protein to 9% wild-type levels resulted in a 73% decrease in carbon assimilation rates. In contrast, eliminating GAPB protein resulted in a 40% reduction in assimilation rates. This work demonstrates that the GAPA homotetramer can compensate for the loss of GAPB, whereas GAPB alone cannot compensate fully for the loss of the GAPA subunit.

Fruit ripening: dynamics and integrated analysis of carotenoids and anthocyanins.

BACKGROUND: Fruits are vital food resources as they are loaded with bioactive compounds varying with different stages of ripening. As the fruit ripens, a dynamic color change is observed from green to yellow to red due to the biosynthesis of pigments like chlorophyll, carotenoids, and anthocyanins. Apart from making the fruit attractive and being a visual indicator of the ripening status, pigments add value to a ripened fruit by making them a source of nutraceuticals and industrial products. As the fruit matures, it undergoes biochemical changes which alter the pigment composition of fruits. RESULTS: The synthesis, degradation and retention pathways of fruit pigments are mediated by hormonal, genetic, and environmental factors. Manipulation of the underlying regulatory mechanisms during fruit ripening suggests ways to enhance the desired pigments in fruits by biotechnological interventions. Here we report, in-depth insight into the dynamics of a pigment change in ripening and the regulatory mechanisms in action. CONCLUSIONS: This review emphasizes the role of pigments as an asset to a ripened fruit as they augment the nutritive value, antioxidant levels and the net carbon gain of fruits; pigments are a source for fruit biofortification have tremendous industrial value along with being a tool to predict the harvest. This report will be of great utility to the harvesters, traders, consumers, and natural product divisions to extract the leading nutraceutical and industrial potential of preferred pigments biosynthesized at different fruit ripening stages.

Options for the generation of seedless cherry, the ultimate snacking product.

MAIN CONCLUSION: This manuscript identifies cherry orthologues of genes implicated in the development of pericarpic fruit and pinpoints potential options and restrictions in the use of these targets for commercial exploitation of parthenocarpic cherry fruit. Cherry fruit contain a large stone and seed, making processing of the fruit laborious and consumption by the consumer challenging, inconvenient to eat 'on the move' and potentially dangerous for children. Availability of fruit lacking the stone and seed would be potentially transformative for the cherry industry, since such fruit would be easier to process and would increase consumer demand because of the potential reduction in costs. This review will explore the background of seedless fruit, in the context of the ambition to produce the first seedless cherry, carry out an in-depth analysis of the current literature around parthenocarpy in fruit, and discuss the available technology and potential for producing seedless cherry fruit as an 'ultimate snacking product' for the twenty-first century.

The role of photosynthesis related pigments in light harvesting, photoprotection and enhancement of photosynthetic yield in planta.

Photosynthetic pigments are an integral and vital part of all photosynthetic machinery and are present in different types and abundances throughout the photosynthetic apparatus. Chlorophyll, carotenoids and phycobilins are the prime photosynthetic pigments which facilitate efficient light absorption in plants, algae, and cyanobacteria. The chlorophyll family plays a vital role in light harvesting by absorbing light at different wavelengths and allowing photosynthetic organisms to adapt to different environments, either in the long-term or during transient changes in light. Carotenoids play diverse roles in photosynthesis, including light capture and as crucial antioxidants to reduce photodamage and photoinhibition. In the marine habitat, phycobilins capture a wide spectrum of light and have allowed cyanobacteria and red algae to colonise deep waters where other frequencies of light are attenuated by the water column. In this review, we discuss the potential strategies that photosynthetic pigments provide, coupled with development of molecular biological techniques, to improve crop yields through enhanced light harvesting, increased photoprotection and improved photosynthetic efficiency.

Field-grown ictB tobacco transformants show no difference in photosynthetic efficiency for biomass relative to the wild type.

In this study, four tobacco transformants overexpressing the inorganic carbon transporter B gene (ictB) were screened for photosynthetic performance relative to the wild type (WT) in field-based conditions. The WT and transgenic tobacco plants were evaluated for photosynthetic performance to determine the maximum rate of carboxylation (Vc, max), maximum rate of electron transport (Jmax), the photosynthetic compensation point (Γ*), quantum yield of PSII (ΦPSII), and mesophyll conductance (gm). Additionally, all plants were harvested to compare differences in above-ground biomass. Overall, transformants did not perform better than the WT on photosynthesis-, biomass-, and leaf composition-related traits. This is in contrast to previous studies that have suggested significant increases in photosynthesis and yield with the overexpression of ictB, although not widely evaluated under field conditions. These findings suggest that the benefit of ictB is not universal and may only be seen under certain growth conditions. While there is certainly still potential benefit to utilizing ictB in the future, further effort must be concentrated on understanding the underlying function of the gene and in which environmental conditions it offers the greatest benefit to crop performance. As it stands at present, it is possible that ictB overexpression may be largely favorable in controlled environments, such as greenhouses.

Photosynthesis in non-foliar tissues: implications for yield.

Photosynthesis is currently a focus for crop improvement. The majority of this work has taken place and been assessed in leaves, and limited consideration has been given to the contribution that other green tissues make to whole-plant carbon assimilation. The major focus of this review is to evaluate the impact of non-foliar photosynthesis on carbon-use efficiency and total assimilation. Here we appraise and summarize past and current literature on the substantial contribution of different photosynthetically active organs and tissues to productivity in a variety of different plant types, with an emphasis on fruit and cereal crops. Previous studies provide evidence that non-leaf photosynthesis could be an unexploited potential target for crop improvement. We also briefly examine the role of stomata in non-foliar tissues, gas exchange, maintenance of optimal temperatures and thus photosynthesis. In the final section, we discuss possible opportunities to manipulate these processes and provide evidence that Triticum aestivum (wheat) plants genetically manipulated to increase leaf photosynthesis also displayed higher rates of ear assimilation, which translated to increased grain yield. By understanding these processes, we can start to provide insights into manipulating non-foliar photosynthesis and stomatal behaviour to identify novel targets for exploitation in continuing breeding programmes.

Feeding the world: improving photosynthetic efficiency for sustainable crop production.

A number of recent studies have provided strong support demonstrating that improving the photosynthetic processes through genetic engineering can provide an avenue to improve yield potential. The major focus of this review is on improvement of the Calvin-Benson cycle and electron transport. Consideration is also given to how altering regulatory process may provide an additional route to increase photosynthetic efficiency. Here we summarize some of the recent successes that have been observed through genetic manipulation of photosynthesis, showing that, in both the glasshouse and the field, yield can be increased by >40%. These results provide a clear demonstration of the potential for increasing yield through improvements in photosynthesis. In the final section, we consider the need to stack improvement in photosynthetic traits with traits that target the yield gap in order to provide robust germplasm for different crops across the globe.

Overexpression of the RieskeFeS Protein Increases Electron Transport Rates and Biomass Yield.

In this study, we generated transgenic Arabidopsis (Arabidopsis thaliana) plants overexpressing the Rieske FeS protein (PetC), a component of the cytochrome b6f (cyt b6f) complex. Increasing the levels of this protein resulted in concomitant increases in the levels of cyt f (PetA) and cyt b6 (PetB), core proteins of the cyt b6f complex. Interestingly, an increase in the levels of proteins in both the photosystem I (PSI) and PSII complexes also was seen in the Rieske FeS overexpression plants. Although the mechanisms leading to these changes remain to be identified, the transgenic plants presented here provide novel tools to explore this. Importantly, overexpression of the Rieske FeS protein resulted in substantial and significant impacts on the quantum efficiency of PSI and PSII, electron transport, biomass, and seed yield in Arabidopsis plants. These results demonstrate the potential for manipulating electron transport processes to increase crop productivity.

Importance of Fluctuations in Light on Plant Photosynthetic Acclimation.

The acclimation of plants to light has been studied extensively, yet little is known about the effect of dynamic fluctuations in light on plant phenotype and acclimatory responses. We mimicked natural fluctuations in light over a diurnal period to examine the effect on the photosynthetic processes and growth of Arabidopsis (Arabidopsis thaliana). High and low light intensities, delivered via a realistic dynamic fluctuating or square wave pattern, were used to grow and assess plants. Plants subjected to square wave light had thicker leaves and greater photosynthetic capacity compared with fluctuating light-grown plants. This, together with elevated levels of proteins associated with electron transport, indicates greater investment in leaf structural components and photosynthetic processes. In contrast, plants grown under fluctuating light had thinner leaves, lower leaf light absorption, but maintained similar photosynthetic rates per unit leaf area to square wave-grown plants. Despite high light use efficiency, plants grown under fluctuating light had a slow growth rate early in development, likely due to the fact that plants grown under fluctuating conditions were not able to fully utilize the light energy absorbed for carbon fixation. Diurnal leaf-level measurements revealed a negative feedback control of photosynthesis, resulting in a decrease in total diurnal carbon assimilated of at least 20%. These findings highlight that growing plants under square wave growth conditions ultimately fails to predict plant performance under realistic light regimes and stress the importance of considering fluctuations in incident light in future experiments that aim to infer plant productivity under natural conditions in the field.

Simultaneous stimulation of sedoheptulose 1,7-bisphosphatase, fructose 1,6-bisphophate aldolase and the photorespiratory glycine decarboxylase-H protein increases CO2 assimilation, vegetative biomass and seed yield in Arabidopsis.

In this article, we have altered the levels of three different enzymes involved in the Calvin-Benson cycle and photorespiratory pathway. We have generated transgenic Arabidopsis plants with altered combinations of sedoheptulose 1,7-bisphosphatase (SBPase), fructose 1,6-bisphophate aldolase (FBPA) and the glycine decarboxylase-H protein (GDC-H) gene identified as targets to improve photosynthesis based on previous studies. Here, we show that increasing the levels of the three corresponding proteins, either independently or in combination, significantly increases the quantum efficiency of PSII. Furthermore, photosynthetic measurements demonstrated an increase in the maximum efficiency of CO2 fixation in lines over-expressing SBPase and FBPA. Moreover, the co-expression of GDC-H with SBPase and FBPA resulted in a cumulative positive impact on leaf area and biomass. Finally, further analysis of transgenic lines revealed a cumulative increase of seed yield in SFH lines grown in high light. These results demonstrate the potential of multigene stacking for improving the productivity of food and energy crops.

Multigene manipulation of photosynthetic carbon assimilation increases CO2 fixation and biomass yield in tobacco.

Over the next 40 years it has been estimated that a 50% increase in the yield of grain crops such as wheat and rice will be required to meet the food and fuel demands of the increasing world population. Transgenic tobacco plants have been generated with altered combinations of sedoheptulose-1,7-bisphosphatase, fructose-1,6-bisphosphate aldolase, and the cyanobacterial putative-inorganic carbon transporter B, ictB, of which have all been identified as targets to improve photosynthesis based on empirical studies. It is shown here that increasing the levels of the three proteins individually significantly increases the rate of photosynthetic carbon assimilation, leaf area, and biomass yield. Furthermore, the daily integrated measurements of photosynthesis showed that mature plants fixed between 12-19% more CO2 than the equivalent wild-type plants. Further enhancement of photosynthesis and yield was observed when sedoheptulose-1,7-bisphosphatase, fructose-1,6-bisphosphate aldolase, and ictB were over-expressed together in the same plant. These results demonstrate the potential for the manipulation of photosynthesis, using multigene-stacking approaches, to increase crop yields.

Subcellular localization of the histidine kinase receptors Sln1p, Nik1p and Chk1p in the yeast CTG clade species Candida guilliermondii.

Fungal histidine kinase receptors (HKR) sense and transduce many intra- and extracellular signals that regulate a wide range of physiological processes. Candida CTG clade species commonly possess three types of HKR namely Sln1p (type VI), Nik1p (type III) and Chk1p (type X). Although some recent work has demonstrated the potential involvement of HKR in osmoregulation, morphogenesis, sexual development, adaptation to osmotic stresses and drug resistance in distinct Candida species, little data is available in relation to their subcellular distribution within yeast cells. We describe in this work the comparative subcellular localization of class III, VI, and X HKRs in Candida guilliermondii, a yeast CTG clade species of clinical and biotechnological interest. Using a fluorescent protein fusion approach, we showed that C. guilliermondii Sln1p fused to the yellow fluorescent protein (Sln1p-YFP) appeared to be anchored in the plasma membrane. By contrast, both Chk1p-YFP and YFP-Chk1p were localized in the nucleocytosol of C. guilliermondii transformed cells. Furthermore, while Nik1p-YFP fusion protein always displayed a nucleocytosolic localization, we noted that most of the cells expressing YFP-Nik1p fusion protein displayed an aggregated pattern of fluorescence in the cytosol but not in the nucleus. Interestingly, Sln1p-YFP and Nik1p-YFP fusion protein localization changed in response to hyperosmotic stress by rapidly clustering into punctuated structures that could be associated to osmotic stress signaling. To date, this work provides the first insight into the subcellular localization of the three classes of HKR encoded by CTG clade yeast genomes and constitutes original new data concerning this family of receptors. This represents also an essential prerequisite to open a window into the understanding of the global architecture of HKR-mediated signaling pathways in CTG clade species.

Disrupting the methionine biosynthetic pathway in Candida guilliermondii: characterization of the MET2 gene as counter-selectable marker.

Candida guilliermondii (teleomorph Meyerozyma guilliermondii) is an ascomycetous species belonging to the fungal CTG clade. This yeast remains actively studied as a result of its moderate clinical importance and most of all for its potential uses in biotechnology. The aim of the present study was to establish a convenient transformation system for C. guilliermondii by developing both a methionine auxotroph recipient strain and a functional MET gene as selection marker. We first disrupted the MET2 and MET15 genes encoding homoserine-O-acetyltransferase and O-acetylserine O-acetylhomoserine sulphydrylase, respectively. The met2 mutant was shown to be a methionine auxotroph in contrast to met15 which was not. Interestingly, met2 and met15 mutants formed brown colonies when cultured on lead-containing medium, contrary to the wild-type strain, which develop as white colonies on this medium. The MET2 wild-type allele was successfully used to transfer a yellow fluorescent protein (YFP) gene-expressing vector into the met2 recipient strain. In addition, we showed that the loss of the MET2-containing YFP-expressing plasmid can be easily observed on lead-containing medium. The MET2 wild-type allele, flanked by two short repeated sequences, was then used to disrupt the LYS2 gene (encoding the α-aminoadipate reductase) in the C. guilliermondii met2 recipient strain. The resulting lys2 mutants displayed, as expected, auxotrophy for lysine. Unfortunately, all our attempts to pop-out the MET2 marker (following the recombination of the bordering repeat sequences) from a target lys2 locus were unsuccessful using white/brown colony colour screening. Nevertheless, this MET2 transformation/disruption system represents a new versatile genetic tool for C. guilliermondii.

Mesophyll photosynthesis and guard cell metabolism impacts on stomatal behaviour.

Stomata control gaseous fluxes between the internal leaf air spaces and the external atmosphere. Guard cells determine stomatal aperture and must operate to ensure an appropriate balance between CO2 uptake for photosynthesis (A) and water loss, and ultimately plant water use efficiency (WUE). A strong correlation between A and stomatal conductance (gs ) is well documented and often observed, but the underlying mechanisms, possible signals and metabolites that promote this relationship are currently unknown. In this review we evaluate the current literature on mesophyll-driven signals that may coordinate stomatal behaviour with mesophyll carbon assimilation. We explore a possible role of various metabolites including sucrose and malate (from several potential sources; including guard cell photosynthesis) and new evidence that improvements in WUE have been made by manipulating sucrose metabolism within the guard cells. Finally we discuss the new tools and techniques available for potentially manipulating cell-specific metabolism, including guard and mesophyll cells, in order to elucidate mesophyll-derived signals that coordinate mesophyll CO2 demands with stomatal behaviour, in order to provide a mechanistic understanding of these processes as this may identify potential targets for manipulations in order to improve plant WUE and crop yield.

Candida guilliermondii: biotechnological applications, perspectives for biological control, emerging clinical importance and recent advances in genetics.

Candida guilliermondii (teleomorph Meyerozyma guilliermondii) is an ascomycetous species belonging to the Saccharomycotina CTG clade which has been studied over the last 40 years due to its biotechnological interest, biological control potential and clinical importance. Such a wide range of applications in various areas of fundamental and applied scientific research has progressively made C. guilliermondii an attractive model for exploring the potential of yeast metabolic engineering as well as for elucidating new molecular events supporting pathogenicity and antifungal resistance. All these research fields now take advantage of the establishment of a useful molecular toolbox specifically dedicated to C. guilliermondii genetics including the construction of recipient strains, the development of selectable markers and reporter genes and optimization of transformation protocols. This area of study is further supported by the availability of the complete genome sequence of the reference strain ATCC 6260 and the creation of numerous databases dedicated to gene ontology annotation (metabolic pathways, virulence, and morphogenesis). These genetic tools and genomic resources represent essential prerequisites for further successful development of C. guilliermondii research in medical mycology and in biological control by facilitating the identification of the multiple factors that contribute to its pathogenic potential. These genetic and genomic advances should also expedite future practical uses of C. guilliermondii strains of biotechnological interest by opening a window into a better understanding of the biosynthetic pathways of valuable metabolites.

Transformation of Candida guilliermondii wild-type strains using the Staphylococcus aureus MRSA 252 ble gene as a phleomycin-resistant marker.

We designed an efficient transformation system for Candida guilliermondii wild-type strains. We demonstrated that the Staphylococcus aureus MRSA 252 ble coding sequence placed under the control of the yeast phosphoglycerate kinase gene transcription-regulating regions confers phleomycin resistance to transformed C. guilliermondii cells. To illustrate the potential of this drug-resistant cassette, we carried out the disruption of the C. guilliermondii ADE2 gene. This new dominant selectable marker represents a powerful tool to study the function of various genes in this yeast of clinical and biotechnological interest.

Efficient gene targeting in a Candida guilliermondii non-homologous end-joining pathway-deficient strain.

The yeast, Candida guilliermondii, has been widely studied due to its biotechnological interest as well as its biological control potential. It integrates foreign DNA predominantly via ectopic events, likely through the well-known non-homologous end-joining (NHEJ) pathway involving the Ku70p/Ku80p heterodimer, Lig4p, Nej1p and Lif1p. This phenomenon remains highly deleterious for targeted gene knock-out strategies that require the homologous recombination process. Here, we have constructed a ku70 mutant strain derived from the ATCC 6260 reference strain of C. guilliermondii. Following a series of disruption attempts of various genes (FCY1, ADE2 and TRP5), using several previously described dominant selectable markers (URA5, SAT-1 and HPH#), we demonstrated that the efficiencies of homologous gene targeting in such a NHEJ-deficient strain was very high compared to the wild type strain. The C. guilliermondii ku70 deficient mutant thus represents a powerful recipient strain to knock-out genes efficiently in this yeast.

Feeding the world: impacts of elevated [CO2] on nutrient content of greenhouse grown fruit crops and options for future yield gains.

Several long-term studies have provided strong support demonstrating that growing crops under elevated [CO2] can increase photosynthesis and result in an increase in yield, flavour and nutritional content (including but not limited to Vitamins C, E and pro-vitamin A). In the case of tomato, increases in yield by as much as 80% are observed when plants are cultivated at 1000 ppm [CO2], which is consistent with current commercial greenhouse production methods in the tomato fruit industry. These results provide a clear demonstration of the potential for elevating [CO2] for improving yield and quality in greenhouse crops. The major focus of this review is to bring together 50 years of observations evaluating the impact of elevated [CO2] on fruit yield and fruit nutritional quality. In the final section, we consider the need to engineer improvements to photosynthesis and nitrogen assimilation to allow plants to take greater advantage of elevated CO2 growth conditions.

Machine learning in photosynthesis: Prospects on sustainable crop development.

Improving photosynthesis is a promising avenue to increase food security. Studying photosynthetic traits with the aim to improve efficiency has been one of many strategies to increase crop yield but analyzing large data sets presents an ongoing challenge. Machine learning (ML) represents a ubiquitous tool that can provide a more elaborate data analysis. Here we review the application of ML in various domains of photosynthetic research, as well as in photosynthetic pigment studies. We highlight how correlating hyperspectral data with photosynthetic parameters to improve crop yield could be achieved through various ML algorithms. We also propose strategies to employ ML in promoting photosynthetic pigment research for furthering crop yield.