Single-cell genetic models to evaluate orphan gene function: The case of QQS regulating carbon and nitrogen allocation.

We demonstrate two synthetic single-cell systems that can be used to better understand how the acquisition of an orphan gene can affect complex phenotypes. The Arabidopsis orphan gene, Qua-Quine Starch (QQS) has been identified as a regulator of carbon (C) and nitrogen (N) partitioning across multiple plant species. QQS modulates this important biotechnological trait by replacing NF-YB (Nuclear Factor Y, subunit B) in its interaction with NF-YC. In this study, we expand on these prior findings by developing Chlamydomonas reinhardtii and Saccharomyces cerevisiae strains, to refactor the functional interactions between QQS and NF-Y subunits to affect modulations in C and N allocation. Expression of QQS in C. reinhardtii modulates C (i.e., starch) and N (i.e., protein) allocation by affecting interactions between NF-YC and NF-YB subunits. Studies in S. cerevisiae revealed similar functional interactions between QQS and the NF-YC homolog (HAP5), modulating C (i.e., glycogen) and N (i.e., protein) allocation. However, in S. cerevisiae both the NF-YA (HAP2) and NF-YB (HAP3) homologs appear to have redundant functions to enable QQS and HAP5 to affect C and N allocation. The genetically tractable systems that developed herein exhibit the plasticity to modulate highly complex phenotypes.

Removal of redox-sensitive Rubisco Activase does not alter Rubisco regulation in soybean.

Rubisco activase (Rca) facilitates the catalytic repair of Rubisco, the CO2-fixing enzyme of photosynthesis, following periods of darkness, low to high light transitions or stress. Removal of the redox-regulated isoform of Rubisco activase, Rca-α, enhances photosynthetic induction in Arabidopsis and has been suggested as a strategy for the improvement of crops, which may experience frequent light transitions in the field; however, this has never been tested in a crop species. Therefore, we used RNAi to reduce the Rca-α content of soybean (Glycine max cv. Williams 82) below detectable levels and then characterized the growth, photosynthesis, and Rubisco activity of the resulting transgenics, in both growth chamber and field conditions. Under a 16 h sine wave photoperiod, the reduction of Rca-α contents had no impact on morphological characteristics, leaf expansion rate, or total biomass. Photosynthetic induction rates were unaltered in both chamber-grown and field-grown plants. Plants with reduced Rca-α content maintained the ability to regulate Rubisco activity in low light just as in control plants. This result suggests that in soybean, Rca-α is not as centrally involved in the regulation of Rca oligomer activity as it is in Arabidopsis. The isoform stoichiometry supports this conclusion, as Rca-α comprises only ~ 10% of the Rubisco activase content of soybean, compared to ~ 50% in Arabidopsis. This is likely to hold true in other species that contain a low ratio of Rca-α to Rca-ß isoforms.

CRISPR/Cas9 Based Site-Specific Modification of FAD2 cis-Regulatory Motifs in Peanut (Arachis hypogaea L).

Peanut (Arachis hypogaea L.) seed is a rich source of edible oil, comprised primarily of monounsaturated oleic acid and polyunsaturated linoleic acid, accounting for 80% of its fatty acid repertoire. The conversion of oleic acid to linoleic acid, catalyzed by Fatty Acid Desaturase 2 (FAD2) enzymes, is an important regulatory point linked to improved abiotic stress responses while the ratio of these components is a significant determinant of commercial oil quality. Specifically, oleic acid has better oxidative stability leading to longer shelf life and better taste qualities while also providing nutritional based health benefits. Naturally occurring FAD2 gene knockouts that lead to high oleic acid levels improve oil quality at the potential expense of plant health though. We undertook a CRISPR/Cas9 based site-specific genome modification approach designed to downregulate the expression of two homeologous FAD2 genes in seed while maintaining regulation in other plant tissues. Two cis-regulatory elements the RY repeat motif and 2S seed protein motif in the 5'UTR and associated intron of FAD2 genes are potentially important for regulating seed-specific gene expression. Using hairy root and stable germ line transformation, differential editing efficiencies were observed at both CREs when targeted by single gRNAs using two different gRNA scaffolds. The editing efficiencies also differed when two gRNAs were expressed simultaneously. Additionally, stably transformed seed exhibited an increase in oleic acid levels relative to wild type. Taken together, the results demonstrate the immense potential of CRISPR/Cas9 based approaches to achieve high frequency targeted edits in regulatory sequences for the generation of novel transcriptional alleles, which may lead to fine tuning of gene expression and functional genomic studies in peanut.

Application of CRISPR/Cas9 System for Efficient Gene Editing in Peanut.

Peanuts are an economically important crop cultivated worldwide. However, several limitations restrained its productivity, including biotic/abiotic stresses. CRISPR/Cas9-based gene-editing technology holds a promising approach to developing new crops with improved agronomic and nutritional traits. Its application has been successful in many important crops. However, the application of this technology in peanut research is limited, probably due to the lack of suitable constructs and protocols. In this study, two different constructs were generated to induce insertion/deletion mutations in the targeted gene for a loss of function study. The first construct harbors the regular gRNA scaffold, while the second construct has the extended scaffold plus terminator. The designed gRNA targeting the coding sequence of the FAD2 genes was cloned into both constructs, and their functionality and efficiency were validated using the hairy root transformation system. Both constructs displayed insertions and deletions as the types of edits. The construct harboring the extended plus gRNA terminator showed a higher editing efficiency than the regular scaffold for monoallelic and biallelic mutations. These two constructs can be used for gene editing in peanuts and could provide tools for improving peanut lines for the benefit of peanut breeders, farmers, and industry.

Co-targeting strategy for precise, scarless gene editing with CRISPR/Cas9 and donor ssODNs in Chlamydomonas.

Programmable site-specific nucleases, such as the clustered regularly interspaced short palindromic repeat (CRISPR)/ CRISPR-associated protein 9 (Cas9) ribonucleoproteins (RNPs), have allowed creation of valuable knockout mutations and targeted gene modifications in Chlamydomonas (Chlamydomonas reinhardtii). However, in walled strains, present methods for editing genes lacking a selectable phenotype involve co-transfection of RNPs and exogenous double-stranded DNA (dsDNA) encoding a selectable marker gene. Repair of the dsDNA breaks induced by the RNPs is usually accompanied by genomic insertion of exogenous dsDNA fragments, hindering the recovery of precise, scarless mutations in target genes of interest. Here, we tested whether co-targeting two genes by electroporation of pairs of CRISPR/Cas9 RNPs and single-stranded oligodeoxynucleotides (ssODNs) would facilitate the recovery of precise edits in a gene of interest (lacking a selectable phenotype) by selection for precise editing of another gene (creating a selectable marker)-in a process completely lacking exogenous dsDNA. We used PPX1 (encoding protoporphyrinogen IX oxidase) as the generated selectable marker, conferring resistance to oxyfluorfen, and identified precise edits in the homolog of bacterial ftsY or the WD and TetratriCopeptide repeats protein 1 genes in ∼1% of the oxyfluorfen resistant colonies. Analysis of the target site sequences in edited mutants suggested that ssODNs were used as templates for DNA synthesis during homology directed repair, a process prone to replicative errors. The Chlamydomonas acetolactate synthase gene could also be efficiently edited to serve as an alternative selectable marker. This transgene-free strategy may allow creation of individual strains containing precise mutations in multiple target genes, to study complex cellular processes, pathways, or structures.

Arabidopsis plants expressing only the redox-regulated Rca-α isoform have constrained photosynthesis and plant growth.

Rubisco activase (Rca) facilitates the release of sugar-phosphate inhibitors from the active sites of Rubisco and thereby plays a central role in initiating and sustaining Rubisco activation. In Arabidopsis, alternative splicing of a single Rca gene results in two Rca isoforms, Rca-α and Rca-ß. Redox modulation of Rca-α regulates the function of Rca-α and Rca-ß acting together to control Rubisco activation. Although Arabidopsis Rca-α alone less effectively activates Rubisco in vitro, it is not known how CO2 assimilation and plant growth are impacted. Here, we show that two independent transgenic Arabidopsis lines expressing Rca-α in the absence of Rca-ß ('Rca-α only' lines) grew more slowly in various light conditions, especially under low light or fluctuating light intensity, and in a short day photoperiod compared to wildtype. Photosynthetic induction was slower in the Rca-α only lines, and they maintained a lower rate of CO2 assimilation during both photoperiod types. Our findings suggest Rca oligomers composed of Rca-α only are less effective in initiating and sustaining the activation of Rubisco than when Rca-ß is also present. Currently there are no examples of any plant species that naturally express Rca-α only but numerous examples of species expressing Rca-ß only. That Rca-α exists in most plant species, including many C3 and C4 food and bioenergy crops, implies its presence is adaptive under some circumstances.

LCI1, a Chlamydomonas reinhardtii plasma membrane protein, functions in active CO2 uptake under low CO2.

In response to high CO2 environmental variability, green algae, such as Chlamydomonas reinhardtii, have evolved multiple physiological states dictated by external CO2 concentration. Genetic and physiological studies demonstrated that at least three CO2 physiological states, a high CO2 (0.5-5% CO2 ), a low CO2 (0.03-0.4% CO2 ) and a very low CO2 (< 0.02% CO2 ) state, exist in Chlamydomonas. To acclimate in the low and very low CO2 states, Chlamydomonas induces a sophisticated strategy known as a CO2 -concentrating mechanism (CCM) that enables proliferation and survival in these unfavorable CO2 environments. Active uptake of Ci from the environment is a fundamental aspect in the Chlamydomonas CCM, and consists of CO2 and HCO3- uptake systems that play distinct roles in low and very low CO2 acclimation states. LCI1, a putative plasma membrane Ci transporter, has been linked through conditional overexpression to active Ci uptake. However, both the role of LCI1 in various CO2 acclimation states and the species of Ci , HCO3- or CO2 , that LCI1 transports remain obscure. Here we report the impact of an LCI1 loss-of-function mutant on growth and photosynthesis in different genetic backgrounds at multiple pH values. These studies show that LCI1 appears to be associated with active CO2 uptake in low CO2 , especially above air-level CO2 , and that any LCI1 role in very low CO2 is minimal.

Structure and function of LCI1: a plasma membrane CO2 channel in the Chlamydomonas CO2 concentrating mechanism.

Microalgae and cyanobacteria contribute roughly half of the global photosynthetic carbon assimilation. Faced with limited access to CO2 in aquatic environments, which can vary daily or hourly, these microorganisms have evolved use of an efficient CO2 concentrating mechanism (CCM) to accumulate high internal concentrations of inorganic carbon (Ci ) to maintain photosynthetic performance. For eukaryotic algae, a combination of molecular, genetic and physiological studies using the model organism Chlamydomonas reinhardtii, have revealed the function and molecular characteristics of many CCM components, including active Ci uptake systems. Fundamental to eukaryotic Ci uptake systems are Ci transporters/channels located in membranes of various cell compartments, which together facilitate the movement of Ci from the environment into the chloroplast, where primary CO2 assimilation occurs. Two putative plasma membrane Ci transporters, HLA3 and LCI1, are reportedly involved in active Ci uptake. Based on previous studies, HLA3 clearly plays a meaningful role in HCO3- transport, but the function of LCI1 has not yet been thoroughly investigated so remains somewhat obscure. Here we report a crystal structure of the full-length LCI1 membrane protein to reveal LCI1 structural characteristics, as well as in vivo physiological studies in an LCI1 loss-of-function mutant to reveal the Ci species preference for LCI1. Together, these new studies demonstrate LCI1 plays an important role in active CO2 uptake and that LCI1 likely functions as a plasma membrane CO2 channel, possibly a gated channel.

In vivo evidence for a regulatory role of phosphorylation of Arabidopsis Rubisco activase at the Thr78 site.

Arabidopsis Rubisco activase (Rca) is phosphorylated at threonine-78 (Thr78) in low light and in the dark, suggesting a potential regulatory role in photosynthesis, but this has not been directly tested. To do so, we transformed an rca-knockdown mutant largely lacking redox regulation with wild-type Rca-ß or Rca-ß with Thr78-to-Ala (T78A) or Thr78-to-Ser (T78S) site-directed mutations. Interestingly, the T78S mutant was hyperphosphorylated at the Ser78 site relative to Thr78 of the Rca-ß wild-type control, as evidenced by immunoblotting with custom antibodies and quantitative mass spectrometry. Moreover, plants expressing the T78S mutation had reduced photosynthesis and quantum efficiency of photosystem II (ϕPSII) and reduced growth relative to control plants expressing wild-type Rca-ß under all conditions tested. Gene expression was also altered in a manner consistent with reduced growth. In contrast, plants expressing Rca-ß with the phospho-null T78A mutation had faster photosynthetic induction kinetics and increased ϕPSII relative to Rca-ß controls. While expression of the wild-type Rca-ß or the T78A mutant fully rescued the slow-growth phenotype of the rca-knockdown mutant grown in a square-wave light regime, the T78A mutants grew faster than the Rca-ß control plants at low light (30 µmol photons m-2 s-1) and in a fluctuating low-light/high-light environment. Collectively, these results suggest that phosphorylation of Thr78 (or Ser78 in the T78S mutant) plays a negative regulatory role in vivo and provides an explanation for the absence of Ser at position 78 in terrestrial plant species.

Microfluidic chip for automated screening of carbon dioxide conditions for microalgal cell growth.

This paper reports on a microfluidic device capable of screening carbon dioxide (CO2) conditions for microalgal cell growth. The device mainly consists of a microfluidic cell culture (MCC) unit, a gas concentration gradient generator (CGG), and an in-line cell growth optical measurement unit. The MCC unit is structured with multiple aqueous-filled cell culture channels at the top layer, multiple CO2 flow channels at the bottom layer, and a commercial hydrophobic gas semipermeable membrane sandwiched between the two channel layers. The CGG unit provides different CO2 concentrations to support photosynthesis of microalgae in the culture channels. The integration of the commercial gas semipermeable membrane into the cell culture device allows rapid mass transport and uniform distribution of CO2 inside the culture medium without using conventional agitation-assisted convection methods, because the diffusion of CO2 from the gas flow channels to the culture channels is fast over a small length scale. In addition, automated in-line monitoring of microalgal cell growth is realized via the optical measurement unit that is able to detect changes in the light intensity transmitted through the cell culture in the culture channels. The microfluidic device also allows a simple grayscale analysis method to quantify the cell growth. The utility of the system is validated by growing Chlamydomonas reinhardtii cells under different low or very-low CO2 levels below the nominal ambient CO2 concentration.

An Agrobacterium-delivered CRISPR/Cas9 system for high-frequency targeted mutagenesis in maize.

CRISPR/Cas9 is a powerful genome editing tool in many organisms, including a number of monocots and dicots. Although the design and application of CRISPR/Cas9 is simpler compared to other nuclease-based genome editing tools, optimization requires the consideration of the DNA delivery and tissue regeneration methods for a particular species to achieve accuracy and efficiency. Here, we describe a public sector system, ISU Maize CRISPR, utilizing Agrobacterium-delivered CRISPR/Cas9 for high-frequency targeted mutagenesis in maize. This system consists of an Escherichia coli cloning vector and an Agrobacterium binary vector. It can be used to clone up to four guide RNAs for single or multiplex gene targeting. We evaluated this system for its mutagenesis frequency and heritability using four maize genes in two duplicated pairs: Argonaute 18 (ZmAgo18a and ZmAgo18b) and dihydroflavonol 4-reductase or anthocyaninless genes (a1 and a4). T0 transgenic events carrying mono- or diallelic mutations of one locus and various combinations of allelic mutations of two loci occurred at rates over 70% mutants per transgenic events in both Hi-II and B104 genotypes. Through genetic segregation, null segregants carrying only the desired mutant alleles without the CRISPR transgene could be generated in T1 progeny. Inheritance of an active CRISPR/Cas9 transgene leads to additional target-specific mutations in subsequent generations. Duplex infection of immature embryos by mixing two individual Agrobacterium strains harbouring different Cas9/gRNA modules can be performed for improved cost efficiency. Together, the findings demonstrate that the ISU Maize CRISPR platform is an effective and robust tool to targeted mutagenesis in maize.

The Plastid Casein Kinase 2 Phosphorylates Rubisco Activase at the Thr-78 Site but Is Not Essential for Regulation of Rubisco Activation State.

Rubisco activase (RCA) is essential for the activation of Rubisco, the carboxylating enzyme of photosynthesis. In Arabidopsis, RCA is composed of a large RCAα and small RCAß isoform that are formed by alternative splicing of a single gene (At2g39730). The activity of Rubisco is controlled in response to changes in irradiance by regulation of RCA activity, which is known to involve a redox-sensitive disulfide bond located in the carboxy-terminal extension of the RCAα subunit. Additionally, phosphorylation of RCA threonine-78 (Thr-78) has been reported to occur in the dark suggesting that phosphorylation may also be associated with dark-inactivation of RCA and deactivation of Rubisco. In the present study, we developed site-specific antibodies to monitor phosphorylation of RCA at the Thr-78 site and used non-reducing SDS-PAGE to monitor the redox status of the RCAα subunit. By immunoblotting, phosphorylation of both RCA isoforms occurred at low light and in the dark and feeding peroxide or DTT to leaf segments indicated that redox status of the chloroplast stroma was a critical factor controlling RCA phosphorylation. Use of a knockout mutant identified the plastid-targeted casein kinase 2 (cpCK2α) as the major protein kinase involved in RCA phosphorylation. Studies with recombinant cpCK2α and synthetic peptide substrates identified acidic residues at the -1, +2, and +3 positions surrounding Thr-78 as strong positive recognition elements. The cpck2 knockout mutant had strongly reduced phosphorylation at the Thr-78 site but was similar to wild type plants in terms of induction kinetics of photosynthesis following transfer from darkness or low light to high light, suggesting that if phosphorylation of RCA Thr-78 plays a direct role it would be redundant to redox regulation for control of Rubisco activation state under normal conditions.

Opportunistic proteolytic processing of carbonic anhydrase 1 from Chlamydomonas in Arabidopsis reveals a novel route for protein maturation.

Proteolytic processing of secretory proteins to yield an active form generally involves specific proteolytic cleavage of a pre-protein. Multiple specific proteases have been identified that target specific pre-protein processing sites in animals. However, characterization of site-specific proteolysis of plant pre-proteins is still evolving. In this study, we characterized proteolytic processing of Chlamydomonas periplasmic carbonic anhydrase 1 (CAH1) in Arabidopsis. CAH1 pre-protein undergoes extensive post-translational modification in the endomembrane system, including glycosylation, disulfide bond formation and proteolytic removal of a peptide 'spacer' region, resulting in a mature, heterotetrameric enzyme with two large and two small subunits. We generated a series of small-scale and large-scale modifications to the spacer and flanking regions to identify potential protease target motifs. Surprisingly, we found that the endoproteolytic removal of the spacer from the CAH1 pre-protein proceeded via an opportunistic process apparently followed by further maturation via amino and carboxy peptidases. We also discovered that the spacer itself is not required for processing, which appears to be dependent only on the number of amino acids separating two key disulfide-bond-forming cysteines. Our data suggest a novel, opportunistic route for pre-protein processing of CAH1.

Use of designer nucleases for targeted gene and genome editing in plants.

The ability to efficiently inactivate or replace genes in model organisms allowed a rapid expansion of our understanding of many of the genetic, biochemical, molecular and cellular mechanisms that support life. With the advent of new techniques for manipulating genes and genomes that are applicable not only to single-celled organisms, but also to more complex organisms such as animals and plants, the speed with which scientists and biotechnologists can expand fundamental knowledge and apply that knowledge to improvements in medicine, industry and agriculture is set to expand in an exponential fashion. At the heart of these advancements will be the use of gene editing tools such as zinc finger nucleases, modified meganucleases, hybrid DNA/RNA oligonucleotides, TAL effector nucleases and modified CRISPR/Cas9. Each of these tools has the ability to precisely target one specific DNA sequence within a genome and (except for DNA/RNA oligonucleotides) to create a double-stranded DNA break. DNA repair to such breaks sometimes leads to gene knockouts or gene replacement by homologous recombination if exogenously supplied homologous DNA fragments are made available. Genome rearrangements are also possible to engineer. Creation and use of such genome rearrangements, gene knockouts and gene replacements by the plant science community is gaining significant momentum. To document some of this progress and to explore the technology's longer term potential, this review highlights present and future uses of designer nucleases to greatly expedite research with model plant systems and to engineer genes and genomes in major and minor crop species for enhanced food production.

Biocatalytic role of potato starch synthase III for α-glucan biosynthesis in Synechocystis sp. PCC6803 mutants.

A potato starch synthase III (PSSIII) was expressed in the Synechocystis mutants deficient in either glycogen synthase I (M1) or II (M2) to replenish α-(1,4) linkage synthesizing activity, resulting in new mutants, PM1 and PM2, respectively. These mutants were applied to study the role of exogenous plant starch synthase for starch/glycogen biosynthesis mechanism established in the cyanobacteria. The remaining glycogen synthase genes in PM1 and PM2 were further disrupted to make the mutants PM12 and PM21 which contained PSSIII as the sole glycogen/starch synthase. Among wild type and mutants, there were no significant differences in the amount of α-glucan produced. All the mutants harboring active PSSIII produced α-glucans with relatively much shorter and less longer α-1,4 chains than wild-type glycogen, which was exactly in accordance with the increase in glycogen branching enzyme activity. In fact, α-glucan structure of PM1 was very similar to those of PM12 and PM21, and PM2 had more intermediate chains than M2. This result suggests PSSIII may have distributive elongation property during α-glucan synthesis. In conclusion, the Synechocystis as an expression model system of plant enzymes can be applied to determine the role of starch synthesizing enzymes and their association during α-glucan synthesis.

Redesigning photosynthesis to sustainably meet global food and bioenergy demand.

The world's crop productivity is stagnating whereas population growth, rising affluence, and mandates for biofuels put increasing demands on agriculture. Meanwhile, demand for increasing cropland competes with equally crucial global sustainability and environmental protection needs. Addressing this looming agricultural crisis will be one of our greatest scientific challenges in the coming decades, and success will require substantial improvements at many levels. We assert that increasing the efficiency and productivity of photosynthesis in crop plants will be essential if this grand challenge is to be met. Here, we explore an array of prospective redesigns of plant systems at various scales, all aimed at increasing crop yields through improved photosynthetic efficiency and performance. Prospects range from straightforward alterations, already supported by preliminary evidence of feasibility, to substantial redesigns that are currently only conceptual, but that may be enabled by new developments in synthetic biology. Although some proposed redesigns are certain to face obstacles that will require alternate routes, the efforts should lead to new discoveries and technical advances with important impacts on the global problem of crop productivity and bioenergy production.

The CO2 concentrating mechanism and photosynthetic carbon assimilation in limiting CO2 : how Chlamydomonas works against the gradient.

The CO2 concentrating mechanism (CCM) represents an effective strategy for carbon acquisition that enables microalgae to survive and proliferate when the CO2 concentration limits photosynthesis. The CCM improves photosynthetic performance by raising the CO2 concentration at the site of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), simultaneously enhancing carbon fixation and suppressing photorespiration. Active inorganic carbon (Ci) uptake, Rubisco sequestration and interconversion between different Ci species catalyzed by carbonic anhydrases (CAs) are key components in the CCM, and an array of molecular regulatory elements is present to facilitate the sensing of CO2 availability, to regulate the expression of the CCM and to coordinate interplay between photosynthetic carbon metabolism and other metabolic processes in response to limiting CO2 conditions. This review intends to integrate our current understanding of the eukaryotic algal CCM and its interaction with carbon assimilation, based largely on Chlamydomonas as a model, and to illustrate how Chlamydomonas acclimates to limiting CO2 conditions and how its CCM is regulated.

Heritable site-specific mutagenesis using TALENs in maize.

Transcription activator-like effector nuclease (TALEN) technology has been utilized widely for targeted gene mutagenesis, especially for gene inactivation, in many organisms, including agriculturally important plants such as rice, wheat, tomato and barley. This report describes application of this technology to generate heritable genome modifications in maize. TALENs were employed to generate stable, heritable mutations at the maize glossy2 (gl2) locus. Transgenic lines containing mono- or di-allelic mutations were obtained from the maize genotype Hi-II at a frequency of about 10% (nine mutated events in 91 transgenic events). In addition, three of the novel alleles were tested for function in progeny seedlings, where they were able to confer the glossy phenotype. In a majority of the events, the integrated TALEN T-DNA segregated independently from the new loss of function alleles, producing mutated null-segregant progeny in T1 generation. Our results demonstrate that TALENs are an effective tool for genome mutagenesis in maize, empowering the discovery of gene function and the development of trait improvement.

Expression activation and functional analysis of HLA3, a putative inorganic carbon transporter in Chlamydomonas reinhardtii.

The CO2 concentrating mechanism (CCM) is a key component of the carbon assimilation strategy of aquatic microalgae. Induced by limiting CO2 and tightly regulated, the CCM enables these microalgae to respond rapidly to varying environmental CO2 supplies and to perform photosynthetic CO2 assimilation in a cost-effective way. A functional CCM in eukaryotic algae requires Rubisco sequestration, rapid interconversion between CO2 and HCO3(-) catalyzed by carbonic anhydrases (CAs), and active inorganic carbon (Ci) uptake. In the model microalga Chlamydomonas reinhardtii, a membrane protein HLA3 is proposed to be involved in active Ci uptake across the plasma membrane. In this study, we use an artificially designed transcription activator-like effector (dTALE) to activate the expression of HLA3. The successful activation of HLA3 expression demonstrates dTALE as a promising tool for gene-specific activation and investigation of gene function in Chlamydomonas. Activation of HLA3 expression in high CO2 acclimated cells, where HLA3 is not expressed, resulted in increased Ci accumulation and Ci-dependent photosynthetic O2 evolution specifically in very low CO2 concentrations, which confirms that HLA3 is indeed involved in Ci uptake, and suggests it is mainly associated with HCO3(-) transport in very low CO2 concentrations, conditions in which active CO2 uptake is highly limited.