Emerging microalgal feed additives for ruminant production and sustainability.

The global demand for animal-derived foods has led to a substantial expansion in ruminant production, which has raised concerns regarding methane emissions. To address these challenges, microalgal species that are nutritionally-rich and contain bioactive compounds in their biomass have been explored as attractive feed additives for ruminant livestock production. In this review, we discuss the different microalgal species used for this purpose in recent studies, and review the effects of microalgal feed supplements on ruminant growth, performance, health, and product quality, as well as their potential contributions in reducing methane emissions. We also examine the potential complexities of adopting microalgae as feed additives in the ruminant industry.

Elucidation of Physiological, Transcriptomic and Metabolomic Salinity Response Mechanisms in Medicago sativa.

Alfalfa (Medicago sativa L.) is a widely grown perennial leguminous forage crop with a number of positive attributes. However, despite its moderate ability to tolerate saline soils, which are increasing in prevalence worldwide, it suffers considerable yield declines under these growth conditions. While a general framework of the cascade of events involved in plant salinity response has been unraveled in recent years, many gaps remain in our understanding of the precise molecular mechanisms involved in this process, particularly in non-model yet economically important species such as alfalfa. Therefore, as a means of further elucidating salinity response mechanisms in this species, we carried out in-depth physiological assessments of M. sativa cv. Beaver, as well as transcriptomic and untargeted metabolomic evaluations of leaf tissues, following extended exposure to salinity (grown for 3-4 weeks under saline treatment) and control conditions. In addition to the substantial growth and photosynthetic reductions observed under salinity treatment, we identified 1233 significant differentially expressed genes between growth conditions, as well as 60 annotated differentially accumulated metabolites. Taken together, our results suggest that changes to cell membranes and walls, cuticular and/or epicuticular waxes, osmoprotectant levels, antioxidant-related metabolic pathways, and the expression of genes encoding ion transporters, protective proteins, and transcription factors are likely involved in alfalfa's salinity response process. Although some of these alterations may contribute to alfalfa's modest salinity resilience, it is feasible that several may be disadvantageous in this context and could therefore provide valuable targets for the further improvement of tolerance to this stress in the future.

Eliciting Targeted Mutations in Medicago sativa Using CRISPR/Cas9-Mediated Genome Editing: A Potential Tool for the Improvement of Disease Resistance.

CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9) has become a breeding tool of choice for eliciting targeted genetic alterations in crop species as a means of improving a wide range of agronomic traits, including disease resistance, in recent years. With the recent development of CRISPR/Cas9 technology in Medicago sativa (alfalfa), which is an important perennial forage legume grown worldwide, its use for the enhancement of pathogen resistance is almost certainly on the horizon. In this chapter, we present detailed procedures for the generation of a single nonhomologous end-joining-derived indel at a precise genomic locus of alfalfa via CRISPR/Cas9. This method encompasses crucial steps in this process, including guide RNA design, binary CRISPR vector construction, Agrobacterium-mediated transformation of alfalfa explants, and molecular assessments of transformed genotypes for transgene and edit identification.

Genome-wide characterization of plant CTP:phosphocholine cytidylyltransferases through evolutionary, biochemical, and structural analyses.

Phosphatidylcholine has essential functions in many eukaryotic cells, and its de novo biosynthesis is rate-limited by cytidine triphosphate:phosphocholine cytidylyltransferase (CCT). Although the biological and biochemical functions of CCT have been reported in mammals and several plants, this key enzyme has yet to be examined at a genome-wide level. As such, certain fundamental questions remain unanswered, including the evolutionary history, genetic and functional relationships, and structural variations among CCTs in the green lineage. In the current study, in-depth phylogenetic analysis, as well as the conservation and diversification in CCT gene structure and motif patterns, indicated that CCTs exist broadly in chlorophytes, bryophytes, lycophytes, monilophytes, gymnosperms, early-diverging angiosperms, monocots, and eudicots, and form eight relatively conserved clades. To further explore the potential function of selection pressure, we conducted extensive selection pressure analysis with a representative CCT gene, CCT1 from the model plant Arabidopsis thaliana (AthCCT1), and identified two positive selection sites, L59 and Q156. Site-directed mutagenesis and in vitro enzyme assays demonstrated that these positively selected sites were indeed important for the activity and substrate affinity of AthCCT1, and subsequent 3D structure analyses explained the potential biochemical mechanism. Taken together, our results unraveled the evolution and diversity of CCTs in the green lineage, as well as their association with the enzyme's biochemical and structural properties, and expanded our understanding of this important enzyme at the genome-wide level.

Protein interactomes for plant lipid biosynthesis and their biotechnological applications.

Plant lipids have essential biological roles in plant development and stress responses through their functions in cell membrane formation, energy storage and signalling. Vegetable oil, which is composed mainly of the storage lipid triacylglycerol, also has important applications in food, biofuel and oleochemical industries. Lipid biosynthesis occurs in multiple subcellular compartments and involves the coordinated action of various pathways. Although biochemical and molecular biology research over the last few decades has identified many proteins associated with lipid metabolism, our current understanding of the dynamic protein interactomes involved in lipid biosynthesis, modification and channelling is limited. This review examines advances in the identification and characterization of protein interactomes involved in plant lipid biosynthesis, with a focus on protein complexes consisting of different subunits for sequential reactions such as those in fatty acid biosynthesis and modification, as well as transient or dynamic interactomes formed from enzymes in cooperative pathways such as assemblies of membrane-bound enzymes for triacylglycerol biosynthesis. We also showcase a selection of representative protein interactome structures predicted using AlphaFold2, and discuss current and prospective strategies involving the use of interactome knowledge in plant lipid biotechnology. Finally, unresolved questions in this research area and possible approaches to address them are also discussed.

CW198 acts as a genetic insulator to block enhancer-promoter interaction in plants.

Insulators in vertebrates play a role in genome architecture and orchestrate temporo-spatial enhancer-promoter interactions. In plants, insulators and their associated binding factors have not been documented as of yet, largely as a result of a lack of characterized insulators. In this study, we took a comprehensive strategy to identify and validate the enhancer-blocking insulator CW198. We show that a 1.08-kb CW198 fragment from Arabidopsis can, when interposed between an enhancer and a promoter, efficiently abrogate the activation function of both constitutive and floral organ-specific enhancers in transgenic Arabidopsis and tobacco plants. In plants, both transcriptional crosstalk and spreading of histone modifications were rarely detectable across CW198, which resembles the insulation property observed across the CTCF insulator in the mammalian genome. Taken together, our findings support that CW198 acts as an enhancer-blocking insulator in both Arabidopsis and tobacco. The significance of the present findings and their relevance to the mitigation of mutual interference between enhancers and promoters, as well as multiple promoters in transgenes, is discussed.

Current progress in lipid-based biofuels: Feedstocks and production technologies.

The expanding use of fossil fuels has caused concern in terms of both energy security and environmental issues. Therefore, attempts have been made worldwide to promote the development of renewable energy sources, among which biofuel is especially attractive. Compared to other biofuels, lipid-derived biofuels have a higher energy density and better compatibility with existing infrastructure, and their performance can be readily improved by adjusting the chemical composition of lipid feedstocks. This review thus addresses the intrinsic interactions between lipid feedstocks and lipid-based biofuels, including biodiesel, and renewable equivalents to conventional gasoline, diesel, and jet fuel. Advancements in lipid-associated biofuel technology, as well as the properties and applicability of various lipid sources in terms of biofuel production, are also discussed. Furthermore, current progress in lipid production and profile optimization in the context of plant lipids, microbial lipids, and animal fats are presented to provide a wider context of lipid-based biofuel technology.

Rice RS2-9, which is bound by transcription factor OSH1, blocks enhancer-promoter interactions in plants.

Insulators characterized in Drosophila and mammals have been shown to play a key role in the restriction of promiscuous enhancer-promoter interactions, as well as reshaping the topological landscape of chromosomes. Yet the role of insulators in plants remains poorly understood, in large part because of a lack of well-characterized insulators and binding factor(s). In this study, we isolated a 1.2-kb RS2-9 insulator from the Oryza sativa (rice) genome that can, when interposed between an enhancer and promoter, efficiently block the activation function of both constitutive and floral organ-specific enhancers in transgenic Arabidopsis and Nicotiana tabacum (tobacco). In the rice genome, the genes flanking RS2-9 exhibit an absence of mutual transcriptional interactions, as well as a lack of histone modification spread. We further determined that O. sativa Homeobox 1 (OSH1) bound two regions of RS2-9, as well as over 50 000 additional sites in the rice genome, the majority of which resided in intergenic regions. Mutation of one of the two OSH1-binding sites in RS2-9 impaired insulation activity by up to 60%, whereas the mutation of both binding sites virtually abolished insulator function. We also demonstrated that OSH1 binding sites were associated with 72% of the boundaries of topologically associated domains (TADs) identified in the rice genome, which is comparable to the 77% of TAD boundaries bound by the insulator CCCTC-binding factor (CTCF) in mammals. Taken together, our findings indicate that OSH1-RS2-9 acts as a true insulator in plants, and highlight a potential role for OSH1 in gene insulation and topological organization in plant genomes.

Identification of Differential Drought Response Mechanisms in Medicago sativa subsp. sativa and falcata through Comparative Assessments at the Physiological, Biochemical, and Transcriptional Levels.

Alfalfa (Medicago sativa L.) is an extensively grown perennial forage legume, and although it is relatively drought tolerant, it consumes high amounts of water and depends upon irrigation in many regions. Given the progressive decline in water available for irrigation, as well as an escalation in climate change-related droughts, there is a critical need to develop alfalfa cultivars with improved drought resilience. M. sativa subsp. falcata is a close relative of the predominantly cultivated M. sativa subsp. sativa, and certain accessions have been demonstrated to exhibit superior performance under drought. As such, we endeavoured to carry out comparative physiological, biochemical, and transcriptomic evaluations of an as of yet unstudied drought-tolerant M. sativa subsp. falcata accession (PI 641381) and a relatively drought-susceptible M. sativa subsp. sativa cultivar (Beaver) to increase our understanding of the molecular mechanisms behind the enhanced ability of falcata to withstand water deficiency. Our findings indicate that unlike the small number of falcata genotypes assessed previously, falcata PI 641381 may exploit smaller, thicker leaves, as well as an increase in the baseline transcriptional levels of genes encoding particular transcription factors, protective proteins, and enzymes involved in the biosynthesis of stress-related compounds. These findings imply that different falcata accessions/genotypes may employ distinct drought response mechanisms, and the study provides a suite of candidate genes to facilitate the breeding of alfalfa with enhanced drought resilience in the future.

Improving the Production of Punicic Acid in Baker's Yeast by Engineering Genes in Acyl Channeling Processes and Adjusting Precursor Supply.

Punicic acid (PuA) is a high-value edible conjugated fatty acid with strong bioactivities and has important potential applications in nutraceutical, pharmaceutical, feeding, and oleochemical industries. Since the production of PuA is severely limited by the fact that its natural source (pomegranate seed oil) is not readily available on a large scale, there is considerable interest in understanding the biosynthesis and accumulation of this plant-based unusual fatty acid in transgenic microorganisms to support the rational design of biotechnological approaches for PuA production via fermentation. Here, we tested the effectiveness of genetic engineering and precursor supply in PuA production in the model yeast strain Saccharomyces cerevisiae. The results revealed that the combination of precursor feeding and co-expression of selected genes in acyl channeling processes created an effective "push-pull" approach to increase PuA content, which could prove valuable in future efforts to produce PuA in industrial yeast and other microorganisms via fermentation.

The effect of AINTEGUMENTA-LIKE 7 over-expression on seed fatty acid biosynthesis, storage oil accumulation and the transcriptome in Arabidopsis thaliana.

KEY MESSAGE: AIL7 over-expression modulates fatty acid biosynthesis and triacylglycerol accumulation in Arabidopsis developing seeds through the transcriptional regulation of associated genes. Seed fatty acids (FAs) and triacylglycerol (TAG) contribute to many functions in plants, and seed lipids have broad food, feed and industrial applications. As a result, an enormous amount of attention has been dedicated towards uncovering the regulatory cascade responsible for the fine-tuning of the lipid biosynthetic pathway in seeds, which is regulated in part through the action of LEAFY COTYLEDON1, ABSCISSIC ACID INSENSITIVE 3, FUSCA3 and LEC2 (LAFL) transcription factors. Although AINTEGUMENTA-LIKE 7 (AIL7) is involved in meristematic function and shoot phyllotaxy, its effect in the context of lipid biosynthesis has yet to be assessed. Here, we generated AIL7 seed-specific over-expression lines and found that they exhibited significant alterations in FA composition and decreased total lipid accumulation in seeds. Seeds and seedlings from transgenic lines also exhibited morphological deviations compared to wild type. Correspondingly, RNA-Seq analysis demonstrated that the expression of many genes related to FA biosynthesis and TAG breakdown were significantly altered in developing siliques from transgenic lines compared to wild-type plants. The seed-specific over-expression of AIL7 also altered the expression profiles of many genes related to starch metabolism, photosynthesis and stress response, suggesting further roles for AIL7 in plants. These findings not only advance our understanding of the lipid biosynthetic pathway in seeds, but also provide evidence for additional functions of AIL7, which could prove valuable in downstream breeding and/or metabolic engineering endeavors.

The CRISPR/Cas9-Mediated Modulation of SQUAMOSA PROMOTER-BINDING PROTEIN-LIKE 8 in Alfalfa Leads to Distinct Phenotypic Outcomes.

Alfalfa (Medicago sativa L.) is the most widely grown perennial leguminous forage and is an essential component of the livestock industry. Previously, the RNAi-mediated down-regulation of alfalfa SQUAMOSA PROMOTER-BINDING PROTEIN-LIKE 8 (MsSPL8) was found to lead to increased branching, regrowth and biomass, as well as enhanced drought tolerance. In this study, we aimed to further characterize the function of MsSPL8 in alfalfa using CRISPR/Cas9-induced mutations in this gene. We successfully generated alfalfa genotypes with small insertions/deletions (indels) at the target site in up to three of four MsSPL8 alleles in the first generation. The efficiency of editing appeared to be tightly linked to the particular gRNA used. The resulting genotypes displayed consistent morphological alterations, even with the presence of up to two wild-type MsSPL8 alleles, including reduced leaf size and early flowering. Other phenotypic effects appeared to be dependent upon mutational dosage, with those plants with the highest number of mutated MsSPL8 alleles also exhibiting significant decreases in internode length, plant height, shoot and root biomass, and root length. Furthermore, MsSPL8 mutants displayed improvements in their ability to withstand water-deficit compared to empty vector control genotypes. Taken together, our findings suggest that allelic mutational dosage can elicit phenotypic gradients in alfalfa, and discrepancies may exist in terms of MsSPL8 function between alfalfa genotypes, growth conditions, or specific alleles. In addition, our results provide the foundation for further research exploring drought tolerance mechanisms in a forage crop.

Physaria fendleri and Ricinus communis lecithin:cholesterol acyltransferase-like phospholipases selectively cleave hydroxy acyl chains from phosphatidylcholine.

Production of hydroxy fatty acids (HFAs) in transgenic crops represents a promising strategy to meet our demands for specialized plant oils with industrial applications. The expression of Ricinus communis (castor) OLEATE 12-HYDROXYLASE (RcFAH12) in Arabidopsis has resulted in only limited accumulation of HFAs in seeds, which probably results from inefficient transfer of HFAs from their site of synthesis (phosphatidylcholine; PC) to triacylglycerol (TAG), especially at the sn-1/3 positions of TAG. Phospholipase As (PLAs) may be directly involved in the liberation of HFAs from PC, but the functions of their over-expression in HFA accumulation and distribution at TAG in transgenic plants have not been well studied. In this work, the functions of lecithin:cholesterol acyltransferase-like PLAs (LCAT-PLAs) in HFA biosynthesis were characterized. The LCAT-PLAs were shown to exhibit homology to LCAT and mammalian lysosomal PLA2 , and to contain a conserved and functional Ser/His/Asp catalytic triad. In vitro assays revealed that LCAT-PLAs from the HFA-accumulating plant species Physaria fendleri (PfLCAT-PLA) and castor (RcLCAT-PLA) could cleave acyl chains at both the sn-1 and sn-2 positions of PC, and displayed substrate selectivity towards sn-2-ricinoleoyl-PC over sn-2-oleoyl-PC. Furthermore, co-expression of RcFAH12 with PfLCAT-PLA or RcLCAT-PLA, but not Arabidopsis AtLCAT-PLA, resulted in increased occupation of HFA at the sn-1/3 positions of TAG as well as small but insignificant increases in HFA levels in Arabidopsis seeds compared with RcFAH12 expression alone. Therefore, PfLCAT-PLA and RcLCAT-PLA may contribute to HFA turnover on PC, and represent potential candidates for engineering the production of unusual fatty acids in crops.

The Potential of Genome Editing for Improving Seed Oil Content and Fatty Acid Composition in Oilseed Crops.

A continuous rise in demand for vegetable oils, which comprise mainly the storage lipid triacylglycerol, is fueling a surge in research efforts to increase seed oil content and improve fatty acid composition in oilseed crops. Progress in this area has been achieved using both conventional breeding and transgenic approaches to date. However, further advancements using traditional breeding methods will be complicated by the polyploid nature of many oilseed crops and associated time constraints, while public perception and the prohibitive cost of regulatory processes hinders the commercialization of transgenic oilseed crops. As such, genome editing using CRISPR/Cas is emerging as a breakthrough breeding tool that could provide a platform to keep pace with escalating demand while potentially minimizing regulatory burden. In this review, we discuss the technology itself and progress that has been made thus far with respect to its use in oilseed crops to improve seed oil content and quality. Furthermore, we examine a number of genes that may provide ideal targets for genome editing in this context, as well as new CRISPR-related tools that have the potential to be applied to oilseed plants and may allow additional gains to be made in the future.

Characterization of the diversification of phospholipid:diacylglycerol acyltransferases in the green lineage.

Triacylglycerols have important physiological roles in photosynthetic organisms, and are widely used as food, feed and industrial materials in our daily life. Phospholipid:diacylglycerol acyltransferase (PDAT) is the pivotal enzyme catalyzing the acyl-CoA-independent biosynthesis of triacylglycerols, which is unique in plants, algae and fungi, but not in animals, and has essential functions in plant and algal growth, development and stress responses. Currently, this enzyme has yet to be examined in an evolutionary context at the level of the green lineage. Some fundamental questions remain unanswered, such as how PDATs evolved in photosynthetic organisms and whether the evolution of terrestrial plant PDATs from a lineage of charophyte green algae diverges in enzyme function. As such, we used molecular evolutionary analysis and biochemical assays to address these questions. Our results indicated that PDAT underwent divergent evolution in the green lineage: PDATs exist in a wide range of plants and algae, but not in cyanobacteria. Although PDATs exhibit the conservation of several features, phylogenetic and selection-pressure analyses revealed that overall they evolved to be highly divergent, driven by different selection constraints. Positive selection, as one major driving force, may have resulted in enzymes with a higher functional importance in land plants than green algae. Further structural and mutagenesis analyses demonstrated that some amino acid sites under positive selection are critically important to PDAT structure and function, and may be central in lecithin:cholesterol acyltransferase family enzymes in general.

Seed-specific down-regulation of Arabidopsis CELLULOSE SYNTHASE 1 or 9 reduces seed cellulose content and differentially affects carbon partitioning.

KEY MESSAGE: Seed-specific down-regulation of AtCESA1 and AtCESA9, which encode cellulose synthase subunits, differentially affects seed storage compound accumulation in Arabidopsis. High amounts of cellulose can negatively affect crop seed quality, and, therefore, diverting carbon partitioning from cellulose to oil, protein and/or starch via molecular breeding may improve seed quality. To determine the effect of seed cellulose content reduction on levels of storage compounds, Arabidopsis thaliana CELLULOSE SYNTHASE1 (AtCESA1) and AtCESA9 genes, which both encode cellulose synthase subunits, were individually down-regulated using seed-specific intron-spliced hairpin RNA (hpRNAi) constructs. The selected seed-specific AtCESA1 and AtCESA9 Arabidopsis RNAi lines displayed reduced cellulose contents in seeds, and exhibited no obvious visual phenotypic growth defects with the exception of a minor effect on early root development in AtCESA1 RNAi seedlings and early hypocotyl elongation in the dark in both types of RNAi line. The seed-specific down-regulation of AtCESA9 resulted in a reduction in seed weight compared to empty vector controls, which was not observed in AtCESA1 RNAi lines. In terms of effects on carbon partitioning, AtCESA1 and AtCESA9 RNAi lines exhibited distinct effects. The down-regulation of AtCESA1 led to a ~ 3% relative increase in seed protein content (P = 0.04) and a ~ 3% relative decrease in oil content (P = 0.02), but caused no alteration in soluble glucose levels. On the contrary, AtCESA9 RNAi lines did not display a significant reduction in seed oil, protein or soluble glucose content. Taken together, our results indicate that the seed-specific down-regulation of AtCESA1 causes alterations in seed storage compound accumulation, while the effect of AtCESA9 on carbon partitioning is absent or minor in Arabidopsis.

The Role of Triacylglycerol in Plant Stress Response.

Vegetable oil is mainly composed of triacylglycerol (TAG), a storage lipid that serves as a major commodity for food and industrial purposes, as well as an alternative biofuel source. While TAG is typically not produced at significant levels in vegetative tissues, emerging evidence suggests that its accumulation in such tissues may provide one mechanism by which plants cope with abiotic stress. Different types of abiotic stress induce lipid remodeling through the action of specific lipases, which results in various alterations in membrane lipid composition. This response induces the formation of toxic lipid intermediates that cause membrane damage or cell death. However, increased levels of TAG under stress conditions are believed to function, at least in part, as a means of sequestering these toxic lipid intermediates. Moreover, the lipid droplets (LDs) in which TAG is enclosed also function as a subcellular factory to provide binding sites and substrates for the biosynthesis of bioactive compounds that protect against insects and fungi. Though our knowledge concerning the role of TAG in stress tolerance is expanding, many gaps in our understanding of the mechanisms driving these processes are still evident. In this review, we highlight progress that has been made to decipher the role of TAG in plant stress response, and we discuss possible ways in which this information could be utilized to improve crops in the future.

Potential effects of a high CO2 future on leguminous species.

Legumes provide an important source of food and feed due to their high protein levels and many health benefits, and also impart environmental and agronomic advantages as a consequence of their ability to fix nitrogen through their symbiotic relationship with rhizobia. As a result of our growing population, the demand for products derived from legumes will likely expand considerably in coming years. Since there is little scope for increasing production area, improving the productivity of such crops in the face of climate change will be essential. While a growing number of studies have assessed the effects of climate change on legume yield, there is a paucity of information regarding the direct impact of elevated CO2 concentration (e[CO2]) itself, which is a main driver of climate change and has a substantial physiological effect on plants. In this review, we discuss current knowledge regarding the influence of e[CO2] on the photosynthetic process, as well as biomass production, seed yield, quality, and stress tolerance in legumes, and examine how these responses differ from those observed in non-nodulating plants. Although these relationships are proving to be extremely complex, mounting evidence suggests that under limiting conditions, overall declines in many of these parameters could ensue. While further research will be required to unravel precise mechanisms underlying e[CO2] responses of legumes, it is clear that integrating such knowledge into legume breeding programs will be indispensable for achieving yield gains by harnessing the potential positive effects, and minimizing the detrimental impacts, of CO2 in the future.

Characterization of a Type-2 Diacylglycerol Acyltransferase from Haematococcus pluvialis Reveals Possible Allostery of the Recombinant Enzyme.

Haematococcus pluvialis is a green microalga used in the algal biotechnology industry that can accumulate considerable amounts of storage triacylglycerol (TAG) and astaxanthin, which is a high-value carotenoid with strong antioxidant activity, under stress conditions. Diacylglycerol acyltransferase (DGAT) catalyzes the last step of the acyl-CoA-dependent TAG biosynthesis and appears to represent a bottleneck in algal TAG formation. In this study, putative H. pluvialis DGAT2 cDNA (HpDGAT2A, B, D and E) were identified from a transcriptome database and were subjected to sequence-based in silico analyses. The coding sequences of HpDGAT2B, D, and E were then isolated and characterized through heterologous expression in a TAG-deficient Saccharomyces cerevisiae strain H1246. The expression of HpDGAT2D allowed the recovery of TAG biosynthesis in this yeast mutant, and further in vitro enzymatic assays confirmed that the recombinant HpDGAT2D possessed strong DGAT activity. Interestingly, the recombinant HpDGAT2D displayed sigmoidal kinetics in response to increasing acyl-CoA concentrations, which has not been reported in plant or algal DGAT2 in previous studies.

Biotechnological strategies for improved photosynthesis in a future of elevated atmospheric CO2.

MAIN CONCLUSION: The improvement of photosynthesis using biotechnological approaches has been the focus of much research. It is now vital that these strategies be assessed under future atmospheric conditions. The demand for crop products is expanding at an alarming rate due to population growth, enhanced affluence, increased per capita calorie consumption, and an escalating need for plant-based bioproducts. While solving this issue will undoubtedly involve a multifaceted approach, improving crop productivity will almost certainly provide one piece of the puzzle. The improvement of photosynthetic efficiency has been a long-standing goal of plant biotechnologists as possibly one of the last remaining means of achieving higher yielding crops. However, the vast majority of these studies have not taken into consideration possible outcomes when these plants are grown long-term under the elevated CO2 concentrations (e[CO2]) that will be evident in the not too distant future. Due to the considerable effect that CO2 levels have on the photosynthetic process, these assessments should become commonplace as a means of ensuring that research in this field focuses on the most effective approaches for our future climate scenarios. In this review, we discuss the main biotechnological research strategies that are currently underway with the aim of improving photosynthetic efficiency and biomass production/yields in the context of a future of e[CO2], as well as alternative approaches that may provide further photosynthetic benefits under these conditions.