The C4 photosynthetic pathway accounts for ∼25% of primary productivity on the planet despite bei... more The C4 photosynthetic pathway accounts for ∼25% of primary productivity on the planet despite being used by only 3% of species. Because C4 plants are higher yielding than C3 plants, efforts are underway to introduce the C4 pathway into the C3 crop rice. This is an ambitious endeavor; however, the C4 pathway evolved from C3 on multiple independent occasions over the last 30 million years, and steps along the trajectory are evident in extant species. One approach toward engineering C4 rice is to recapitulate this trajectory, one of the first steps of which was a change in leaf anatomy. The transition from C3 to so-called "proto-Kranz" anatomy requires an increase in organelle volume in sheath cells surrounding leaf veins. Here we induced chloroplast and mitochondrial development in rice vascular sheath cells through constitutive expression of maize GOLDEN2-LIKE genes. Increased organelle volume was accompanied by the accumulation of photosynthetic enzymes and by increased in...
Agriculture is a panacea during all emergencies except the emergencies in agriculture itself. The... more Agriculture is a panacea during all emergencies except the emergencies in agriculture itself. The recent novel coronavirus that caused COVID-19 pandemic has once again rejuvenated this realization. In Nepal, where the agrarian economy is still predominant, the effect of the pandemic differs from that of industrialized and developed countries where commercial agriculture is in practice. The fear of the outbreak of COVID-19, lockdown for its prevention, and diversion of state resources to health and other welfare sector have both direct, and indirect as well as short-term, mid-term, and long-term effects on the agriculture sector. Until now there is no complete assessment of the loss caused by the lockdown in the agriculture sector. Therefore, this study was conducted to collect representative data and information regarding the loss caused at different stages of various agricultural commodities. The data were collected using a semi-structured questionnaire survey from 60 respondents a...
Every day almost one billion people suffer from chronic hunger, and the situation is expected to ... more Every day almost one billion people suffer from chronic hunger, and the situation is expected to deteriorate with a projected population growth to 9 billion worldwide by 2050. In order to provide adequate nutrition into the future, rice yields in Asia need to increase by 60%, a change that may be achieved by introduction of the C4 photosynthetic cycle into rice. The international C4 Rice Consortium was founded in order to test the feasibility of installing the C4 engine into rice. This review provides an update on two of the many approaches employed by the C4 Rice Consortium: namely, metabolic C4 engineering and identification of determinants of leaf anatomy by mutant screens. The aim of the metabolic C4 engineering approach is to generate a two-celled C4 shuttle in rice by expressing the classical enzymes of the NADP-ME C4 cycle in a cell-appropriate manner. The aim is also to restrict RuBisCO and glycine decarboxylase expression to the bundle sheath (BS) cells of rice in a C4-like...
Introduction of a C4photosynthetic pathway into C3rice (Oryza sativa) requires installation of a ... more Introduction of a C4photosynthetic pathway into C3rice (Oryza sativa) requires installation of a biochemical pump that concentrates CO2at the site of carboxylation in modified bundle sheath cells. To investigate the feasibility of this, we generated a quadruple line that simultaneously expresses four of the core C4photosynthetic enzymes from the NADP-malic enzyme subtype, phosphoenolpyruvate carboxylase (ZmPEPC), NADP-malate dehydrogenase (ZmNADP-MDH), NADP-malic enzyme (ZmNADP-ME) and pyruvate phosphate dikinase (ZmPPDK), in a cell-specific manner. This led to enhanced enzyme activity but was largely neutral in its effects on photosynthetic rate and growth. Measurements of the flux of13CO2through photosynthetic metabolism revealed a significant increase in the incorporation of13C into malate, consistent with increased fixation of13CO2via PEP carboxylase in lines expressing the maize PEPC enzyme. We also showed13C labelling of aspartate indicating additional13CO2fixation into oxaloa...
The chloroplastic 2-oxaloacetate (OAA)/malate transporter (OMT1 or DiT1) takes part in the malate... more The chloroplastic 2-oxaloacetate (OAA)/malate transporter (OMT1 or DiT1) takes part in the malate valve that protects chloroplasts from excessive redox poise through export of malate and import of OAA. Together with the glutamate/malate transporter (DCT1 or DiT2), it connects carbon with nitrogen assimilation, by providing 2-oxoglutarate for the GS/GOGAT (glutamine synthetase/glutamate synthase) reaction and exporting glutamate to the cytoplasm. OMT1 further plays a prominent role in C4 photosynthesis: OAA resulting from phosphoenolpyruvate carboxylation is imported into the chloroplast, reduced to malate by plastidic NADP-malate dehydrogenase, and then exported for transport to bundle sheath cells. Both transport steps are catalyzed by OMT1, at the rate of net carbon assimilation. To engineer C4 photosynthesis into C3 crops, OMT1 must be expressed in high amounts on top of core C4 metabolic enzymes. We report here high-level expression of ZmOMT1 from maize in rice (Oryza sativa ssp...
The chloroplastic oxaloacetate/malate transporter (OMT1 or DiT1) takes part in the malate valve t... more The chloroplastic oxaloacetate/malate transporter (OMT1 or DiT1) takes part in the malate valve that protects chloroplasts from excessive redox poise through export of malate and import of oxaloacetate (OAA). Together with the glutamate/malate transporter (DCT1 or DiT2), it connects carbon with nitrogen assimilation, by providing α-ketoglutarate for the GS/GOGAT reaction and exporting glutamate to the cytoplasm. OMT1 further plays a prominent role in C4 photosynthesis. OAA resulting from PEP-carboxylation is imported into the chloroplast, reduced to malate by plastidic NADP-MDH, and then exported for transport to bundle sheath cells. Both transport steps are catalyzed by OMT1, at the rate of net carbon assimilation. Therefore, to engineer C4 photosynthesis into C3 crops, OMT1 must be expressed in high amounts on top of core C4 metabolic enzymes. We report here high-level expression of ZmOMT1 from maize in rice (Oryza sativa ssp. indica IR64). Increased activity of the transporter in...
All grass leaves are strap-shaped with a series of parallel veins running from base to tip, but t... more All grass leaves are strap-shaped with a series of parallel veins running from base to tip, but the distance between each pair of veins, and the cell-types that develop between them, differs depending on whether the plant performs C3 or C4 photosynthesis. As part of a multinational effort to introduce C4 traits into rice to boost crop yield, candidate regulators of C4 leaf anatomy were previously identified through an analysis of maize leaf transcriptomes. Here we tested the potential of 60 of those candidate genes to alter leaf anatomy in rice. In each case, transgenic rice lines were generated in which the maize gene was constitutively expressed. Lines grouped into three phenotypic classes: (1) indistinguishable from wild-type; (2) aberrant shoot and/or root growth indicating possible perturbations to hormone homeostasis; and (3) altered secondary cell wall formation. One of the genes in class 3 defines a novel monocot-specific family. None of the genes were individually sufficien...
An efficient method for crossing green foxtail (Setaria viridis) is currently lacking. S. viridis... more An efficient method for crossing green foxtail (Setaria viridis) is currently lacking. S. viridis is considered to be the new model plant for the study of C4 system in monocots and so an effective crossing protocol is urgently needed. S. viridis is a small grass with C4-NADP (ME) type of photosynthesis and has the advantage of having small genome of about 515 Mb, small plant stature, short life cycle, multiple tillers, and profuse seed set, and hence is an ideal model species for research. The objectives of this project were to develop efficient methods of emasculation and pollination, and to speed up generation advancement. We assessed the response of S. viridis flowers to hot water treatment (48°C) and to different concentrations of gibberellic acid, abscisic acid, maleic hydrazide (MH), and kinetin. We found that 500 μM of MH was effective in the emasculation of S. viridis, whilst still retaining the receptivity of the stigma to pollination. We also report effective ways to accel...
Convergent trait evolution is a recurrent phenomenon in all domains of the tree of life. While so... more Convergent trait evolution is a recurrent phenomenon in all domains of the tree of life. While some convergent traits are caused by simple sequence changes, many are associated with extensive changes to the sequence and regulation of large cohorts of genes. It is unknown how organisms traverse this expansive genotype space to assemble such complex convergent phenotypes. C4photosynthesis is a paradigm of large-scale phenotypic convergence. Conceptual and mathematical models propose that C4photosynthesis evolved from ancestral C3photosynthesis through sequential adaptive changes. These adaptive changes could have been rapidly assembled if modifications to the activity and abundance of enzymes of the C4cycle was neutral in C3plants. This neutrality would enable populations of C3plants to maintain genotypes with expression levels of C4enzymes analogous to those in C4species and thus enable rapid assembly of a functional C4cycle from naturally occurring genotypes given shared environment...
The C4 photosynthetic pathway accounts for ∼25% of primary productivity on the planet despite bei... more The C4 photosynthetic pathway accounts for ∼25% of primary productivity on the planet despite being used by only 3% of species. Because C4 plants are higher yielding than C3 plants, efforts are underway to introduce the C4 pathway into the C3 crop rice. This is an ambitious endeavor; however, the C4 pathway evolved from C3 on multiple independent occasions over the last 30 million years, and steps along the trajectory are evident in extant species. One approach toward engineering C4 rice is to recapitulate this trajectory, one of the first steps of which was a change in leaf anatomy. The transition from C3 to so-called "proto-Kranz" anatomy requires an increase in organelle volume in sheath cells surrounding leaf veins. Here we induced chloroplast and mitochondrial development in rice vascular sheath cells through constitutive expression of maize GOLDEN2-LIKE genes. Increased organelle volume was accompanied by the accumulation of photosynthetic enzymes and by increased in...
Agriculture is a panacea during all emergencies except the emergencies in agriculture itself. The... more Agriculture is a panacea during all emergencies except the emergencies in agriculture itself. The recent novel coronavirus that caused COVID-19 pandemic has once again rejuvenated this realization. In Nepal, where the agrarian economy is still predominant, the effect of the pandemic differs from that of industrialized and developed countries where commercial agriculture is in practice. The fear of the outbreak of COVID-19, lockdown for its prevention, and diversion of state resources to health and other welfare sector have both direct, and indirect as well as short-term, mid-term, and long-term effects on the agriculture sector. Until now there is no complete assessment of the loss caused by the lockdown in the agriculture sector. Therefore, this study was conducted to collect representative data and information regarding the loss caused at different stages of various agricultural commodities. The data were collected using a semi-structured questionnaire survey from 60 respondents a...
Every day almost one billion people suffer from chronic hunger, and the situation is expected to ... more Every day almost one billion people suffer from chronic hunger, and the situation is expected to deteriorate with a projected population growth to 9 billion worldwide by 2050. In order to provide adequate nutrition into the future, rice yields in Asia need to increase by 60%, a change that may be achieved by introduction of the C4 photosynthetic cycle into rice. The international C4 Rice Consortium was founded in order to test the feasibility of installing the C4 engine into rice. This review provides an update on two of the many approaches employed by the C4 Rice Consortium: namely, metabolic C4 engineering and identification of determinants of leaf anatomy by mutant screens. The aim of the metabolic C4 engineering approach is to generate a two-celled C4 shuttle in rice by expressing the classical enzymes of the NADP-ME C4 cycle in a cell-appropriate manner. The aim is also to restrict RuBisCO and glycine decarboxylase expression to the bundle sheath (BS) cells of rice in a C4-like...
Introduction of a C4photosynthetic pathway into C3rice (Oryza sativa) requires installation of a ... more Introduction of a C4photosynthetic pathway into C3rice (Oryza sativa) requires installation of a biochemical pump that concentrates CO2at the site of carboxylation in modified bundle sheath cells. To investigate the feasibility of this, we generated a quadruple line that simultaneously expresses four of the core C4photosynthetic enzymes from the NADP-malic enzyme subtype, phosphoenolpyruvate carboxylase (ZmPEPC), NADP-malate dehydrogenase (ZmNADP-MDH), NADP-malic enzyme (ZmNADP-ME) and pyruvate phosphate dikinase (ZmPPDK), in a cell-specific manner. This led to enhanced enzyme activity but was largely neutral in its effects on photosynthetic rate and growth. Measurements of the flux of13CO2through photosynthetic metabolism revealed a significant increase in the incorporation of13C into malate, consistent with increased fixation of13CO2via PEP carboxylase in lines expressing the maize PEPC enzyme. We also showed13C labelling of aspartate indicating additional13CO2fixation into oxaloa...
The chloroplastic 2-oxaloacetate (OAA)/malate transporter (OMT1 or DiT1) takes part in the malate... more The chloroplastic 2-oxaloacetate (OAA)/malate transporter (OMT1 or DiT1) takes part in the malate valve that protects chloroplasts from excessive redox poise through export of malate and import of OAA. Together with the glutamate/malate transporter (DCT1 or DiT2), it connects carbon with nitrogen assimilation, by providing 2-oxoglutarate for the GS/GOGAT (glutamine synthetase/glutamate synthase) reaction and exporting glutamate to the cytoplasm. OMT1 further plays a prominent role in C4 photosynthesis: OAA resulting from phosphoenolpyruvate carboxylation is imported into the chloroplast, reduced to malate by plastidic NADP-malate dehydrogenase, and then exported for transport to bundle sheath cells. Both transport steps are catalyzed by OMT1, at the rate of net carbon assimilation. To engineer C4 photosynthesis into C3 crops, OMT1 must be expressed in high amounts on top of core C4 metabolic enzymes. We report here high-level expression of ZmOMT1 from maize in rice (Oryza sativa ssp...
The chloroplastic oxaloacetate/malate transporter (OMT1 or DiT1) takes part in the malate valve t... more The chloroplastic oxaloacetate/malate transporter (OMT1 or DiT1) takes part in the malate valve that protects chloroplasts from excessive redox poise through export of malate and import of oxaloacetate (OAA). Together with the glutamate/malate transporter (DCT1 or DiT2), it connects carbon with nitrogen assimilation, by providing α-ketoglutarate for the GS/GOGAT reaction and exporting glutamate to the cytoplasm. OMT1 further plays a prominent role in C4 photosynthesis. OAA resulting from PEP-carboxylation is imported into the chloroplast, reduced to malate by plastidic NADP-MDH, and then exported for transport to bundle sheath cells. Both transport steps are catalyzed by OMT1, at the rate of net carbon assimilation. Therefore, to engineer C4 photosynthesis into C3 crops, OMT1 must be expressed in high amounts on top of core C4 metabolic enzymes. We report here high-level expression of ZmOMT1 from maize in rice (Oryza sativa ssp. indica IR64). Increased activity of the transporter in...
All grass leaves are strap-shaped with a series of parallel veins running from base to tip, but t... more All grass leaves are strap-shaped with a series of parallel veins running from base to tip, but the distance between each pair of veins, and the cell-types that develop between them, differs depending on whether the plant performs C3 or C4 photosynthesis. As part of a multinational effort to introduce C4 traits into rice to boost crop yield, candidate regulators of C4 leaf anatomy were previously identified through an analysis of maize leaf transcriptomes. Here we tested the potential of 60 of those candidate genes to alter leaf anatomy in rice. In each case, transgenic rice lines were generated in which the maize gene was constitutively expressed. Lines grouped into three phenotypic classes: (1) indistinguishable from wild-type; (2) aberrant shoot and/or root growth indicating possible perturbations to hormone homeostasis; and (3) altered secondary cell wall formation. One of the genes in class 3 defines a novel monocot-specific family. None of the genes were individually sufficien...
An efficient method for crossing green foxtail (Setaria viridis) is currently lacking. S. viridis... more An efficient method for crossing green foxtail (Setaria viridis) is currently lacking. S. viridis is considered to be the new model plant for the study of C4 system in monocots and so an effective crossing protocol is urgently needed. S. viridis is a small grass with C4-NADP (ME) type of photosynthesis and has the advantage of having small genome of about 515 Mb, small plant stature, short life cycle, multiple tillers, and profuse seed set, and hence is an ideal model species for research. The objectives of this project were to develop efficient methods of emasculation and pollination, and to speed up generation advancement. We assessed the response of S. viridis flowers to hot water treatment (48°C) and to different concentrations of gibberellic acid, abscisic acid, maleic hydrazide (MH), and kinetin. We found that 500 μM of MH was effective in the emasculation of S. viridis, whilst still retaining the receptivity of the stigma to pollination. We also report effective ways to accel...
Convergent trait evolution is a recurrent phenomenon in all domains of the tree of life. While so... more Convergent trait evolution is a recurrent phenomenon in all domains of the tree of life. While some convergent traits are caused by simple sequence changes, many are associated with extensive changes to the sequence and regulation of large cohorts of genes. It is unknown how organisms traverse this expansive genotype space to assemble such complex convergent phenotypes. C4photosynthesis is a paradigm of large-scale phenotypic convergence. Conceptual and mathematical models propose that C4photosynthesis evolved from ancestral C3photosynthesis through sequential adaptive changes. These adaptive changes could have been rapidly assembled if modifications to the activity and abundance of enzymes of the C4cycle was neutral in C3plants. This neutrality would enable populations of C3plants to maintain genotypes with expression levels of C4enzymes analogous to those in C4species and thus enable rapid assembly of a functional C4cycle from naturally occurring genotypes given shared environment...
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