Evaluation of Photosynthetic Efficiency in Photorespiratory Mutants by Chlorophyll Fluorescence Analysis.

Photosynthesis and photorespiration represent the largest carbon fluxes in plant primary metabolism and are necessary for plant survival. Many of the enzymes and genes important for photosynthesis and photorespiration have been well studied for decades, but some aspects of these biochemical pathways and their crosstalk with several subcellular processes are not yet fully understood. Much of the work that has identified the genes and proteins important in plant metabolism has been conducted under highly controlled environments that may not best represent how photosynthesis and photorespiration function under natural and farming environments. Considering that abiotic stress results in impaired photosynthetic efficiency, the development of a high-throughput screen that can monitor both abiotic stress and its impact on photosynthesis is necessary. Therefore, we have developed a relatively fast method to screen for abiotic stress-induced changes to photosynthetic efficiency that can identify uncharacterized genes with roles in photorespiration using chlorophyll fluorescence analysis and low CO2 screening. This paper describes a method to study changes in photosynthetic efficiency in transferred DNA (T-DNA) knockout mutants in Arabidopsis thaliana. The same method can be used for screening ethyl methanesulfonate (EMS)-induced mutants or suppressor screening. Utilizing this method can identify gene candidates for further study in plant primary metabolism and abiotic stress responses. Data from this method can provide insight into gene function that may not be recognized until exposure to increased stress environments.

Design and Analysis of Native Photorespiration Gene Motifs of Promoter Untranslated Region Combinations Under Short Term Abiotic Stress Conditions.

Quantitative traits are rarely controlled by a single gene, thereby making multi-gene transformation an indispensable component of modern synthetic biology approaches. However, the shortage of unique gene regulatory elements (GREs) for the robust simultaneous expression of multiple nuclear transgenes is a major bottleneck that impedes the engineering of complex pathways in plants. In this study, we compared the transcriptional efficacies of a comprehensive list of well-documented promoter and untranslated region (UTR) sequences side by side. The strength of GREs was examined by a dual-luciferase assay in conjunction with transient expression in tobacco. In addition, we created suites of new GREs with higher transcriptional efficacies by combining the best performing promoter-UTR sequences. We also tested the impact of elevated temperature and high irradiance on the effectiveness of these GREs. While constitutive promoters ensure robust expression of transgenes, they lack spatiotemporal regulations exhibited by native promoters. Here, we present a proof-of-principle study on the characterization of synthetic promoters based on cis-regulatory elements of three key photorespiratory genes. This conserved biochemical process normally increases under elevated temperature, low CO2, and high irradiance stress conditions and results in ∼25% loss in fixed CO2. To select stress-responsive cis-regulatory elements involved in photorespiration, we analyzed promoters of two chloroplast transporters (AtPLGG1 and AtBASS6) and a key plastidial enzyme, AtPGLP using PlantPAN3.0 and AthaMap. Our results suggest that these motifs play a critical role for PLGG1, BASS6, and PGLP in mediating response to elevated temperature and high-intensity light stress. These findings will not only enable the advancement of metabolic and genetic engineering of photorespiration but will also be instrumental in related synthetic biology approaches.

Alternative pathway to photorespiration protects growth and productivity at elevated temperatures in a model crop.

Adapting crops to warmer growing season temperatures is a major challenge in mitigating the impacts of climate change on crop production. Warming temperatures drive greater evaporative demand and can directly interfere with both reproductive and vegetative physiological processes. Most of the world's crop species have C3 photosynthetic metabolism for which increasing temperature means higher rates of photorespiration, wherein the enzyme responsible for fixing CO2 fixes O2 instead followed by an energetically costly recycling pathway that spans several cell compartments. In C3 crops like wheat, rice and soybean, photorespiration translates into large yield losses that are predicted to increase as global temperature warms. Engineering less energy-intensive alternative photorespiratory pathways into crop chloroplasts drives increases in C3 biomass production under agricultural field conditions, but the efficacy of these pathways in mitigating the impact of warmer growing temperatures has not been tested. We grew tobacco plants expressing an alternative photorespiratory pathway under current and elevated temperatures (+5 °C) in agricultural field conditions. Engineered plants exhibited higher photosynthetic quantum efficiency under heated conditions than the control plants, and produced 26% (between 16% and 37%) more total biomass than WT plants under heated conditions, compared to 11% (between 5% and 17%) under ambient conditions. That is, engineered plants sustained 19% (between 11% and 21%) less yield loss under heated conditions compared to non-engineered plants. These results support the theoretical predictions of temperature impacts on photorespiratory losses and provide insight toward the optimisation strategies required to help sustain or improve C3 crop yields in a warming climate.

Synthetic glycolate metabolism pathways stimulate crop growth and productivity in the field.

Photorespiration is required in C3 plants to metabolize toxic glycolate formed when ribulose-1,5-bisphosphate carboxylase-oxygenase oxygenates rather than carboxylates ribulose-1,5-bisphosphate. Depending on growing temperatures, photorespiration can reduce yields by 20 to 50% in C3 crops. Inspired by earlier work, we installed into tobacco chloroplasts synthetic glycolate metabolic pathways that are thought to be more efficient than the native pathway. Flux through the synthetic pathways was maximized by inhibiting glycolate export from the chloroplast. The synthetic pathways tested improved photosynthetic quantum yield by 20%. Numerous homozygous transgenic lines increased biomass productivity by >40% in replicated field trials. These results show that engineering alternative glycolate metabolic pathways into crop chloroplasts while inhibiting glycolate export into the native pathway can drive increases in C3 crop yield under agricultural field conditions.

Overexpressing the H-protein of the glycine cleavage system increases biomass yield in glasshouse and field-grown transgenic tobacco plants.

Photorespiration is essential for C3 plants, enabling oxygenic photosynthesis through the scavenging of 2-phosphoglycolate. Previous studies have demonstrated that overexpression of the L- and H-proteins of the photorespiratory glycine cleavage system results in an increase in photosynthesis and growth in Arabidopsis thaliana. Here, we present evidence that under controlled environment conditions an increase in biomass is evident in tobacco plants overexpressing the H-protein. Importantly, the work in this paper provides a clear demonstration of the potential of this manipulation in tobacco grown in field conditions, in two separate seasons. We also demonstrate the importance of targeted overexpression of the H-protein using the leaf-specific promoter ST-LS1. Although increases in the H-protein driven by this promoter have a positive impact on biomass, higher levels of overexpression of this protein driven by the constitutive CaMV 35S promoter result in a reduction in the growth of the plants. Furthermore in these constitutive overexpressor plants, carbon allocation between soluble carbohydrates and starch is altered, as is the protein lipoylation of the enzymes pyruvate dehydrogenase and alpha-ketoglutarate complexes. Our data provide a clear demonstration of the positive effects of overexpression of the H-protein to improve yield under field conditions.

Optimizing photorespiration for improved crop productivity.

In C3 plants, photorespiration is an energy-expensive process, including the oxygenation of ribulose-1,5-bisphosphate (RuBP) by ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) and the ensuing multi-organellar photorespiratory pathway required to recycle the toxic byproducts and recapture a portion of the fixed carbon. Photorespiration significantly impacts crop productivity through reducing yields in C3 crops by as much as 50% under severe conditions. Thus, reducing the flux through, or improving the efficiency of photorespiration has the potential of large improvements in C3 crop productivity. Here, we review an array of approaches intended to engineer photorespiration in a range of plant systems with the goal of increasing crop productivity. Approaches include optimizing flux through the native photorespiratory pathway, installing non-native alternative photorespiratory pathways, and lowering or even eliminating Rubisco-catalyzed oxygenation of RuBP to reduce substrate entrance into the photorespiratory cycle. Some proposed designs have been successful at the proof of concept level. A plant systems-engineering approach, based on new opportunities available from synthetic biology to implement in silico designs, holds promise for further progress toward delivering more productive crops to farmer's fields.

Bile Acid Sodium Symporter BASS6 Can Transport Glycolate and Is Involved in Photorespiratory Metabolism in Arabidopsis thaliana.

Photorespiration is an energy-intensive process that recycles 2-phosphoglycolate, a toxic product of the Rubisco oxygenation reaction. The photorespiratory pathway is highly compartmentalized, involving the chloroplast, peroxisome, cytosol, and mitochondria. Though the soluble enzymes involved in photorespiration are well characterized, very few membrane transporters involved in photorespiration have been identified to date. In this work, Arabidopsis thaliana plants containing a T-DNA disruption of the bile acid sodium symporter BASS6 show decreased photosynthesis and slower growth under ambient, but not elevated CO2 Exogenous expression of BASS6 complemented this photorespiration mutant phenotype. In addition, metabolite analysis and genetic complementation of glycolate transport in yeast showed that BASS6 was capable of glycolate transport. This is consistent with its involvement in the photorespiratory export of glycolate from Arabidopsis chloroplasts. An Arabidopsis double knockout line of both BASS6 and the glycolate/glycerate transporter PLGG1 (bass6, plgg1) showed an additive growth defect, an increase in glycolate accumulation, and reductions in photosynthetic rates compared with either single mutant. Our data indicate that BASS6 and PLGG1 partner in glycolate export from the chloroplast, whereas PLGG1 alone accounts for the import of glycerate. BASS6 and PLGG1 therefore balance the export of two glycolate molecules with the import of one glycerate molecule during photorespiration.

Physiological evidence for plasticity in glycolate/glycerate transport during photorespiration.

Photorespiration recycles fixed carbon following the oxygenation reaction of Ribulose, 1-5, carboxylase oxygenase (Rubisco). The recycling of photorespiratory C2 to C3 intermediates is not perfectly efficient and reduces photosynthesis in C3 plants. Recently, a plastidic glycolate/glycerate transporter (PLGG1) in photorespiration was identified in Arabidopsis thaliana, but it is not known how critical this transporter is for maintaining photorespiratory efficiency. We examined a mutant deficient in PLGG1 (plgg1-1) using modeling, gas exchange, and Rubisco biochemistry. Under low light (under 65 µmol m(-2) s(-1) PAR), there was no difference in the quantum efficiency of CO2 assimilation or in the photorespiratory CO2 compensation point of plgg1-1, indicating that photorespiration proceeded with wild-type efficiency under sub-saturating light irradiances. Under saturating light irradiance (1200 µmol m(-2) s(-1) PAR), plgg1-1 showed decreased CO2 assimilation that was explained by decreases in the maximum rate of Rubisco carboxylation and photosynthetic linear electron transport. Decreased rates of Rubisco carboxylation resulted from probable decreases in the Rubisco activation state. These results suggest that glycolate/glycerate transport during photorespiration can proceed in moderate rates through an alternative transport process with wild-type efficiencies. These findings also suggest that decreases in net CO2 assimilation that occur due to disruption to photorespiration can occur by decreases in Rubisco activity and not necessarily decreases in the recycling efficiency of photorespiration.

Standards for plant synthetic biology: a common syntax for exchange of DNA parts.

Inventors in the field of mechanical and electronic engineering can access multitudes of components and, thanks to standardization, parts from different manufacturers can be used in combination with each other. The introduction of BioBrick standards for the assembly of characterized DNA sequences was a landmark in microbial engineering, shaping the field of synthetic biology. Here, we describe a standard for Type IIS restriction endonuclease-mediated assembly, defining a common syntax of 12 fusion sites to enable the facile assembly of eukaryotic transcriptional units. This standard has been developed and agreed by representatives and leaders of the international plant science and synthetic biology communities, including inventors, developers and adopters of Type IIS cloning methods. Our vision is of an extensive catalogue of standardized, characterized DNA parts that will accelerate plant bioengineering.

Immediate chromatin immunoprecipitation and on-bead quantitative PCR analysis: a versatile and rapid ChIP procedure.

Genome-wide chromatin immunoprecipitation (ChIP) studies have brought significant insight into the genomic localization of chromatin-associated proteins and histone modifications. The large amount of data generated by these analyses, however, require approaches that enable rapid validation and analysis of biological relevance. Furthermore, there are still protein and modification targets that are difficult to detect using standard ChIP methods. To address these issues, we developed an immediate chromatin immunoprecipitation procedure which we call ZipChip. ZipChip significantly reduces the time and increases sensitivity allowing for rapid screening of multiple loci. Here we describe how ZipChIP enables detection of histone modifications (H3K4 mono- and trimethylation) and two yeast histone demethylases, Jhd2 and Rph1, which were previously difficult to detect using standard methods. Furthermore, we demonstrate the versatility of ZipChIP by analyzing the enrichment of the histone deacetylase Sir2 at heterochromatin in yeast and enrichment of the chromatin remodeler, PICKLE, at euchromatin in Arabidopsis thaliana.

H3K4 methyltransferase Set1 is involved in maintenance of ergosterol homeostasis and resistance to Brefeldin A.

Set1 is a conserved histone H3 lysine 4 (H3K4) methyltransferase that exists as a multisubunit complex. Although H3K4 methylation is located on many actively transcribed genes, few studies have established a direct connection showing that loss of Set1 and H3K4 methylation results in a phenotype caused by disruption of gene expression. In this study, we determined that cells lacking Set1 or Set1 complex members that disrupt H3K4 methylation have a growth defect when grown in the presence of the antifungal drug Brefeldin A (BFA), indicating that H3K4 methylation is needed for BFA resistance. To determine the role of Set1 in BFA resistance, we discovered that Set1 is important for the expression of genes in the ergosterol biosynthetic pathway, including the rate-limiting enzyme HMG-CoA reductase. Consequently, deletion of SET1 leads to a reduction in HMG-CoA reductase protein and total cellular ergosterol. In addition, the lack of Set1 results in an increase in the expression of DAN1 and PDR11, two genes involved in ergosterol uptake. The increase in expression of uptake genes in set1Δ cells allows sterols such as cholesterol and ergosterol to be actively taken up under aerobic conditions. Interestingly, when grown in the presence of ergosterol set1Δ cells become resistant to BFA, indicating that proper ergosterol levels are needed for antifungal drug resistance. These data show that H3K4 methylation impacts gene expression and output of a biologically and medically relevant pathway and determines why cells lacking H3K4 methylation have antifungal drug sensitivity.

Charge-based interaction conserved within histone H3 lysine 4 (H3K4) methyltransferase complexes is needed for protein stability, histone methylation, and gene expression.

Histone H3 lysine 4 (H3K4) methyltransferases are conserved from yeast to humans, assemble in multisubunit complexes, and are needed to regulate gene expression. The yeast H3K4 methyltransferase complex, Set1 complex or complex of proteins associated with Set1 (COMPASS), consists of Set1 and conserved Set1-associated proteins: Swd1, Swd2, Swd3, Spp1, Bre2, Sdc1, and Shg1. The removal of the WD40 domain-containing subunits Swd1 and Swd3 leads to a loss of Set1 protein and consequently a complete loss of H3K4 methylation. However, until now, how these WD40 domain-containing proteins interact with Set1 and contribute to the stability of Set1 and H3K4 methylation has not been determined. In this study, we identified small basic and acidic patches that mediate protein interactions between the C terminus of Swd1 and the nSET domain of Set1. Absence of either the basic or acidic patches of Set1 and Swd1, respectively, disrupts the interaction between Set1 and Swd1, diminishes Set1 protein levels, and abolishes H3K4 methylation. Moreover, these basic and acidic patches are also important for cell growth, telomere silencing, and gene expression. We also show that the basic and acidic patches of Set1 and Swd1 are conserved in their human counterparts SET1A/B and RBBP5, respectively, and are needed for the protein interaction between SET1A and RBBP5. Therefore, this charge-based interaction is likely important for maintaining the protein stability of the human SET1A/B methyltransferase complexes so that proper H3K4 methylation, cell growth, and gene expression can also occur in mammals.

A conserved interaction between the SDI domain of Bre2 and the Dpy-30 domain of Sdc1 is required for histone methylation and gene expression.

In Saccharomyces cerevisiae, lysine 4 on histone H3 (H3K4) is methylated by the Set1 complex (Set1C or COMPASS). Besides the catalytic Set1 subunit, several proteins that form the Set1C (Swd1, Swd2, Swd3, Spp1, Bre2, and Sdc1) are also needed to mediate proper H3K4 methylation. Until this study, it has been unclear how individual Set1C members interact and how this interaction may impact histone methylation and gene expression. In this study, Bre2 and Sdc1 are shown to directly interact, and it is shown that the association of this heteromeric complex is needed for proper H3K4 methylation and gene expression to occur. Interestingly, mutational and biochemical analysis identified the C terminus of Bre2 as a critical protein-protein interaction domain that binds to the Dpy-30 domain of Sdc1. Using the human homologs of Bre2 and Sdc1, ASH2L and DPY-30, respectively, we demonstrate that the C terminus of ASH2L also interacts with the Dpy-30 domain of DPY-30, suggesting that this protein-protein interaction is maintained from yeast to humans. Because of the functionally conserved nature of the C terminus of Bre2 and ASH2L, this region was named the SDI (Sdc1 Dpy-30 interaction) domain. Finally, we show that the SDI-Dpy-30 domain interaction is physiologically important for the function of Set1 in vivo, because specific disruption of this interaction prevents Bre2 and Sdc1 association with Set1, resulting in H3K4 methylation defects and decreases in gene expression. Overall, these and other mechanistic studies on how H3K4 methyltransferase complexes function will likely provide insights into how human MLL and SET1-like complexes or overexpression of ASH2L leads to oncogenesis.

Polyubiquitination of the demethylase Jhd2 controls histone methylation and gene expression.

The identification of histone methyltransferases and demethylases has uncovered a dynamic methylation system needed to modulate appropriate levels of gene expression. Gene expression levels of various histone demethylases, such as the JARID1 family, show distinct patterns of embryonic and adult expression and respond to different environmental cues, suggesting that histone demethylase protein levels must be tightly regulated for proper development. In our study, we show that the protein level of the yeast histone H3 Lys 4 (H3 K4) demethylase Jhd2/Kdm5 is modulated through polyubiquitination by the E3 ubiquitin ligase Not4 and turnover by the proteasome. We determine that polyubiquitin-mediated degradation of Jhd2 controls in vivo H3 K4 trimethylation and gene expression levels. Finally, we show that human NOT4 can polyubiquitinate human JARID1C/SMCX, a homolog of Jhd2, suggesting that this is likely a conserved mechanism. We propose that Not4 is an E3 ubiquitin ligase that monitors and controls a precise amount of Jhd2 protein so that the proper balance between histone demethylase and histone methyltransferase activities occur in the cell, ensuring appropriate levels of H3 K4 trimethylation and gene expression.