A K homology (KH) domain protein identified by a forward genetic screen affects bundle sheath anatomy in Arabidopsis thaliana.

Because of their photosynthetic capacity, leaves function as solar panels providing the basis for the growth of the entire plant. Although the molecular mechanisms of leaf development have been well studied in model dicot and monocot species, a lot of information is still needed about the interplay of the genes that regulate cell division and differentiation and thereby affect the photosynthetic performance of the leaf. We were specifically interested in understanding the differentiation of mesophyll and bundle sheath cells in Arabidopsis thaliana and aimed to identify genes that are involved in determining bundle sheath anatomy. To this end, we established a forward genetic screen by using ethyl methanesulfonate (EMS) for mutagenizing a reporter line expressing a chloroplast-targeted green fluorescent protein (sGFP) under the control of a bundle sheath-specific promoter. Based on the GFP fluorescence phenotype, numerous mutants were produced, and by pursuing a mapping-by-sequencing approach, the genomic segments containing mutated candidate genes were identified. One of the lines with an enhanced GFP fluorescence phenotype (named ELEVATED BUNDLE SHEATH CELLS SIGNAL 1 [ebss1]) was selected for further study, and the responsible gene was verified by CRISPR/Cas9-based mutagenesis of candidate genes located in the mapped genomic segment. The verified gene, At2g25970, encodes a K homology (KH) domain-containing protein.

Improving crop yield potential: Underlying biological processes and future prospects.

The growing world population and global increases in the standard of living both result in an increasing demand for food, feed and other plant-derived products. In the coming years, plant-based research will be among the major drivers ensuring food security and the expansion of the bio-based economy. Crop productivity is determined by several factors, including the available physical and agricultural resources, crop management, and the resource use efficiency, quality and intrinsic yield potential of the chosen crop. This review focuses on intrinsic yield potential, since understanding its determinants and their biological basis will allow to maximize the plant's potential in food and energy production. Yield potential is determined by a variety of complex traits that integrate strictly regulated processes and their underlying gene regulatory networks. Due to this inherent complexity, numerous potential targets have been identified that could be exploited to increase crop yield. These encompass diverse metabolic and physical processes at the cellular, organ and canopy level. We present an overview of some of the distinct biological processes considered to be crucial for yield determination that could further be exploited to improve future crop productivity.

Shedding light on AT1G29480 of Arabidopsis thaliana-An enigmatic locus restricted to Brassicacean genomes.

A key feature of C4 Kranz anatomy is the presence of an enlarged, photosynthetically highly active bundle sheath whose cells contain large numbers of chloroplasts. With the aim to identify novel candidate regulators of C4 bundle sheath development, we performed an activation tagging screen with Arabidopsis thaliana. The reporter gene used encoded a chloroplast-targeted GFP protein preferentially expressed in the bundle sheath, and the promoter of the C4 phosphoenolpyruvate carboxylase gene from Flaveria trinervia served as activation tag because of its activity in all chlorenchymatous tissues of A. thaliana. Primary mutants were selected based on their GFP signal intensity, and one stable mutant named kb-1 with a significant increase in GFP fluorescence intensity was obtained. Despite the increased GFP signal, kb-1 showed no alterations to bundle sheath anatomy. The causal locus, AT1G29480, is specific to the Brassicaceae with its second exon being conserved. Overexpression and reconstitution studies confirmed that AT1G29480, and specifically its second exon, were sufficient for the enhanced GFP phenotype, which was not dependent on translation of the locus or its parts into protein. We conclude, therefore, that the AT1G29480 locus enhances the GFP reporter gene activity via an RNA-based mechanism.

The coordination of major events in C4 photosynthesis evolution in the genus Flaveria.

C4 photosynthesis is a remarkable complex trait, elucidations of the evolutionary trajectory of C4 photosynthesis from its ancestral C3 pathway can help us better understand the generic principles of the evolution of complex traits and guide the engineering of C3 crops for higher yields. Here, we used the genus Flaveria that contains C3, C3-C4, C4-like and C4 species as a system to study the evolution of C4 photosynthesis. We first mapped transcript abundance, protein sequence and morphological features onto the phylogenetic tree of the genus Flaveria, and calculated the evolutionary correlation of different features; we then predicted the relative changes of ancestral nodes of those features to illustrate the major events during the evolution of C4 photosynthesis. We found that gene expression and protein sequence showed consistent modification patterns in the phylogenetic tree. High correlation coefficients ranging from 0.46 to 0.9 among gene expression, protein sequence and morphology were observed. The greatest modification of those different features consistently occurred at the transition between C3-C4 species and C4-like species. Our results show highly coordinated changes in gene expression, protein sequence and morphological features, which support evolutionary major events during the evolution of C4 metabolism.

Dynamic changes of genome sizes and gradual gain of cell-specific distribution of C4 enzymes during C4 evolution in genus Flaveria.

C4 plants are believed to have evolved from C3 plants through various C3 -C4 intermediate stages in which a photorespiration-dependent CO2 concentration system known as C2 photosynthesis operates. Genes involved in the C4 cycle were thought to be recruited from orthologs present in C3 species and developed cell-specific expression during C4 evolution. To understand the process of establishing C4 photosynthesis, we performed whole-genome sequencing and investigated expression and mesophyll- or bundle-sheath-cell-specific localization of phosphoenolpyruvate carboxylase (PEPC), NADP-malic enzyme (NADP-ME), pyruvate, orthophosphate dikinase (PPDK) in C3 , C3 -C4 intermediate, C4 -like, and C4 Flaveria species. While genome sizes vary greatly, the number of predicted protein-coding genes was similar among C3 , C3 -C4 intermediate, C4 -like, and C4 Flaveria species. Cell-specific localization of the PEPC, NADP-ME, and PPDK transcripts was insignificant or weak in C3 -C4 intermediate species, whereas these transcripts were expressed cell-type specific in C4 -like species. These results showed that elevation of gene expression and cell-specific control of pre-existing C4 cycle genes in C3 species was involved in C4 evolution. Gene expression was gradually enhanced during C4 evolution, whereas cell-specific control was gained independently of quantitative transcriptional activation during evolution from C3 -C4 intermediate to C4 photosynthesis in genus Flaveria.

The C4 Ppc promoters of many C4 grass species share a common regulatory mechanism for gene expression in the mesophyll cell.

C4 photosynthetic plants have evolved from C3 ancestors and are characterized by differential expression of several hundred genes. Strict compartmentalization of key C4 enzymes either to mesophyll (M) or bundle sheath cells is considered a crucial step towards the evolution of C4 photosynthesis. In this study, we demonstrate that the 5'-flanking sequences of the C4 type phosphoenolpyruvate carboxylase (Ppc) gene from three C4 grass species could drive M-cell-specific expression of a reporter gene in rice. In addition to that, we identified about 450 bp (upstream of their transcription start site) of the analyzed C4 Ppc promoters contain all the essential regulatory elements for driving M-cell-specific expression in rice leaves. Importantly, four motifs of conserved nucleotide sequences (CNSs) were also determined, which are essential for the activity of the promoter. A putative interaction between the CNSs and an unknown upstream element(s) is required for driving M-cell-specific expression. This work identifies the evolutionary conservation of C4 Ppc regulatory mechanisms of multiple closely related C4 grass species.

Targeted misexpression of NAC052, acting in H3K4 demethylation, alters leaf morphological and anatomical traits in Arabidopsis thaliana.

In an effort to identify genetic regulators for the cell ontogeny around the veins in Arabidopsis thaliana leaves, an activation-tagged mutant line with altered leaf morphology and altered bundle sheath anatomy was characterized. This mutant had a small rosette area with wrinkled leaves and chlorotic leaf edges, as well as enhanced chloroplast numbers in the (pre-)bundle sheath tissue. It had a bundle-specific promoter from the gene GLYCINE DECARBOXYLASE SUBUNIT-T from the C4 species Flaveria trinervia (GLDTFt promoter) inserted in the coding region of the transcriptional repressor NAC052, functioning in H3K4 demethylation, in front of an alternative start codon in-frame with the natural start codon. Reconstruction of the mutation event of our activation-tagged line by creating a line expressing an N-terminally truncated sequence of NAC052 under control of the GLDTFt promoter confirmed the involvement of NAC052 in leaf development. Our study not only reveals leaf anatomic and transcriptomic effects of an N-terminally truncated NAC052 under control of the GLDTFt promoter, but also identifies NAC052 as a novel genetic regulator of leaf development.

Reporter-based forward genetic screen to identify bundle sheath anatomy mutants in A. thaliana.

The evolution of C4 photosynthesis proceeded stepwise with each small step increasing the fitness of the plant. An important pre-condition for the introduction of a functional C4 cycle is the photosynthetic activation of the C3 bundle sheath by increasing its volume and organelle number. Therefore, to engineer C4 photosynthesis into existing C3 crops, information about genes that control the bundle sheath cell size and organelle content is needed. However, very little information is known about the genes that could be manipulated to create a more C4 -like bundle sheath. To this end, an ethylmethanesulfonate (EMS)-based forward genetic screen was established in the Brassicaceae C3  species Arabidopsis thaliana. To ensure a high-throughput primary screen, the bundle sheath cells of A. thaliana were labeled using a luciferase (LUC68) or by a chloroplast-targeted green fluorescent protein (sGFP) reporter using a bundle sheath specific promoter. The signal strengths of the reporter genes were used as a proxy to search for mutants with altered bundle sheath anatomy. Here, we show that our genetic screen predominantly identified mutants that were primarily affected in the architecture of the vascular bundle, and led to an increase in bundle sheath volume. By using a mapping-by-sequencing approach the genomic segments that contained mutated candidate genes were identified.

Efficient 2-phosphoglycolate degradation is required to maintain carbon assimilation and allocation in the C4 plant Flaveria bidentis.

Photorespiration is indispensable for oxygenic photosynthesis since it detoxifies and recycles 2-phosphoglycolate (2PG), which is the primary oxygenation product of Rubisco. However, C4 plant species typically display very low rates of photorespiration due to their efficient biochemical carbon-concentrating mechanism. Thus, the broader relevance of photorespiration in these organisms remains unclear. In this study, we assessed the importance of a functional photorespiratory pathway in the C4 plant Flaveria bidentis using knockdown of the first enzymatic step, namely 2PG phosphatase (PGLP). The isolated RNAi lines showed strongly reduced amounts of PGLP protein, but distinct signs of the photorespiratory phenotype only emerged below 5% residual PGLP protein. Lines with this characteristic were stunted in growth, had strongly increased 2PG content, exhibited accelerated leaf senescence, and accumulated high amounts of branched-chain and aromatic amino acids, which are both characteristics of incipient carbon starvation. Oxygen-dependent gas-exchange measurements consistently suggested the cumulative impairment of ribulose-1,5-bisphosphate regeneration with increased photorespiratory pressure. Our results indicate that photorespiration is essential for maintaining high rates of C4 photosynthesis by preventing the 2PG-mediated inhibition of carbon utilization efficiency. However, considerably higher 2PG accumulation can be tolerated compared to equivalent lines of C3 plants due to the differential distribution of specific enzymatic steps between the mesophyll and bundle sheath cells.

Expression of SULTR2;2, encoding a low-affinity sulphur transporter, in the Arabidopsis bundle sheath and vein cells is mediated by a positive regulator.

The bundle sheath provides a conduit linking veins and mesophyll cells. In the C3 plant Arabidopsis thaliana, it also plays important roles in oxidative stress and sulphur metabolism. However, the mechanisms responsible for the patterns of gene expression that underpin these metabolic specializations are poorly understood. Here, we used the Arabidopsis SULTR2;2 gene as a model to better understand mechanisms that restrict expression to the bundle sheath. Deletion analysis indicated that the SULTR2;2 promoter contains a short region necessary for expression in the bundle sheath and veins. This sequence acts as a positive regulator and is tolerant to multiple consecutive deletions indicating considerable redundancy in the cis-elements involved. It is highly conserved in SULTR2;2 genes of the Brassicaceae and is functional in the distantly related C4 species Flaveria bidentis that belongs to the Asteraceae. We conclude that expression of SULTR2;2 in the bundle sheath and veins is underpinned by a highly redundant sequence that likely represents an ancient and conserved mechanism found in families as diverse as the Asteraceae and Brassicaceae.

SHORTROOT-Mediated Increase in Stomatal Density Has No Impact on Photosynthetic Efficiency.

The coordinated positioning of veins, mesophyll cells, and stomata across a leaf is crucial for efficient gas exchange and transpiration and, therefore, for overall function. In monocot leaves, stomatal cell files are positioned at the flanks of underlying longitudinal leaf veins, rather than directly above or below. This pattern suggests either that stomatal formation is inhibited in epidermal cells directly in contact with the vein or that specification is induced in cell files beyond the vein. The SHORTROOT pathway specifies distinct cell types around the vasculature in subepidermal layers of both root and shoots, with cell type identity determined by distance from the vein. To test whether the pathway has the potential to similarly pattern epidermal cell types, we expanded the expression domain of the rice (Oryza sativa ssp japonica) OsSHR2 gene, which we show is restricted to developing leaf veins, to include bundle sheath cells encircling the vein. In transgenic lines, which were generated using the orthologous ZmSHR1 gene to avoid potential silencing of OsSHR2, stomatal cell files were observed both in the normal position and in more distant positions from the vein. Contrary to theoretical predictions, and to phenotypes observed in eudicot leaves, the increase in stomatal density did not enhance photosynthetic capacity or increase mesophyll cell density. Collectively, these results suggest that the SHORTROOT pathway may coordinate the positioning of veins and stomata in monocot leaves and that distinct mechanisms may operate in monocot and eudicot leaves to coordinate stomatal patterning with the development of underlying mesophyll cells.

De novo Transcriptome Assembly and Comparison of C3, C3-C4, and C4 Species of Tribe Salsoleae (Chenopodiaceae).

C4 photosynthesis is a carbon-concentrating mechanism that evolved independently more than 60 times in a wide range of angiosperm lineages. Among other alterations, the evolution of C4 from ancestral C3 photosynthesis requires changes in the expression of a vast number of genes. Differential gene expression analyses between closely related C3 and C4 species have significantly increased our understanding of C4 functioning and evolution. In Chenopodiaceae, a family that is rich in C4 origins and photosynthetic types, the anatomy, physiology and phylogeny of C4, C2, and C3 species of Salsoleae has been studied in great detail, which facilitated the choice of six samples of five representative species with different photosynthetic types for transcriptome comparisons. mRNA from assimilating organs of each species was sequenced in triplicates, and sequence reads were de novo assembled. These novel genetic resources were then analyzed to provide a better understanding of differential gene expression between C3, C2 and C4 species. All three analyzed C4 species belong to the NADP-ME type as most genes encoding core enzymes of this C4 cycle are highly expressed. The abundance of photorespiratory transcripts is decreased compared to the C3 and C2 species. Like in other C4 lineages of Caryophyllales, our results suggest that PEPC1 is the C4-specific isoform in Salsoleae. Two recently identified transporters from the PHT4 protein family may not only be related to the C4 syndrome, but also active in C2 photosynthesis in Salsoleae. In the two populations of the C2 species S. divaricata transcript abundance of several C4 genes are slightly increased, however, a C4 cycle is not detectable in the carbon isotope values. Most of the core enzymes of photorespiration are highly increased in the C2 species compared to both C3 and C4 species, confirming a successful establishment of the C2 photosynthetic pathway. Furthermore, a function of PEP-CK in C2 photosynthesis appears likely, since PEP-CK gene expression is not only increased in S. divaricata but also in C2 species of other groups.

The DnaJ-Like Zinc-Finger Protein HCF222 Is Required for Thylakoid Membrane Biogenesis in Plants.

To understand the biogenesis of the thylakoid membrane in higher plants and to identify auxiliary proteins required to build up this highly complex membrane system, we have characterized the allelic nuclear mutants high chlorophyll fluorescence222-1 (hcf222-1) and hcf222-2 and isolated the causal gene by map-based cloning. In the ethyl methanesulfonate-induced mutant hcf222-1, the accumulation of the cytochrome b6f (Cytb6f) complex was reduced to 30% compared with the wild type. Other thylakoid membrane complexes accumulated to normal levels. The T-DNA knockout mutant hcf222-2 showed a more severe defect with respect to thylakoid membrane proteins and accumulated only 10% of the Cytb6f complex, accompanied by a reduction in photosystem II, the photosystem II light-harvesting complex, and photosystem I. HCF222 encodes a protein of 99 amino acids in Arabidopsis (Arabidopsis thaliana) that has similarities to the cysteine-rich zinc-binding domain of DnaJ chaperones. The insulin precipitation assay demonstrated that HCF222 has disulfide reductase activity in vitro. The protein is conserved in higher plants and bryophytes but absent in algae and cyanobacteria. Confocal fluorescence microscopy showed that a fraction of HCF222-green fluorescent protein was detectable in the endoplasmic reticulum but that it also could be recognized in chloroplasts. A fusion construct of HCF222 containing a plastid transit peptide targets the protein into chloroplasts and was able to complement the mutational defect. These findings indicate that the chloroplast-targeted HCF222 is indispensable for the maturation and/or assembly of the Cytb6f complex and is very likely involved in thiol-disulfide biochemistry at the thylakoid membrane.

A MEM1-like motif directs mesophyll cell-specific expression of the gene encoding the C4 carbonic anhydrase in Flaveria.

The first two reactions of C4 photosynthesis are catalysed by carbonic anhydrase (CA) and phosphoenolpyruvate carboxylase (PEPC) in the leaf mesophyll (M) cell cytosol. Translatome experiments using a tagged ribosomal protein expressed under the control of M and bundle-sheath (BS) cell-specific promoters showed transcripts encoding CA3 from the C4 species Flaveria bidentis were highly enriched in polysomes from M cells relative to those of the BS. Localisation experiments employing a CA3-green fluorescent protein fusion protein showed F. bidentis CA3 is a cytosolic enzyme. A motif showing high sequence homology to that of the Flaveria M expression module 1 (MEM1) element was identified approximately 2 kb upstream of the F. bidentis and F. trinervia ca3 translation start sites. MEM1 is located in the promoter of C4 Flaveria ppcA genes, which encode the C4-associated PEPC, and is necessary for M-specific expression. No MEM1-like sequence was found in the 4 kb upstream of the C3 species F. pringlei ca3 translation start site. Promoter-reporter fusion experiments demonstrated the region containing the ca3 MEM1-like element also directs M-specific expression. These results support the idea that a common regulatory switch drives the expression of the C4 Flaveria ca3 and ppcA1 genes specifically in M cells.

C3 cotyledons are followed by C4 leaves: intra-individual transcriptome analysis of Salsola soda (Chenopodiaceae).

Some species of Salsoleae (Chenopodiaceae) convert from C3 photosynthesis during the seedling stage to the C4 pathway in adult leaves. This unique developmental transition of photosynthetic pathways offers the exceptional opportunity to follow the development of the derived C4 syndrome from the C3 condition within individual plants, avoiding phylogenetic noise. Here we investigate Salsola soda, a little-studied species from tribe Salsoleae, using an ontogenetic approach. Anatomical sections, carbon isotope (δ13C) values, transcriptome analysis by means of mRNA sequencing, and protein levels of the key C4 enzyme phosphoenolpyruvate carboxylase (PEPC) were examined from seed to adult plant stages. Despite a previous report, our results based on δ13C values, anatomy and transcriptomics clearly indicate a C3 phase during the cotyledon stage. During this stage, the entire transcriptional repertoire of the C4 NADP-malic enzyme type is detected at low levels compared to a significant increase in true leaves. In contrast, abundance of transcripts encoding most of the major photorespiratory enzymes is not significantly decreased in leaves compared to cotyledons. PEPC polypeptide was detected only in leaves, correlating with increased PEPC transcript abundance from the cotyledon to leaf stage.

Glycine decarboxylase in C3, C4 and C3-C4 intermediate species.

The glycine decarboxylase complex (GDC) plays a central role in photorespiration. GDC is localized in the mitochondria and together with serine hydroxymethyltransferase it converts two molecules of glycine to one molecule of serine, CO2 and NH3. Overexpression of GDC subunits in the C3 species Arabidopsis thaliana can increase the metabolic flux through the photorespiratory pathway leading to enhanced photosynthetic efficiency and consequently to an enhanced biomass production of the transgenic plants. Changing the spatial expression patterns of GDC subunits was an important step during the evolution of C3-C4 intermediate and likely also C4 plants. Restriction of the GDC activity to the bundle sheath cells led to the establishment of a photorespiratory CO2 pump.

Recent advances in understanding photosynthesis.

Photosynthesis is central to all life on earth, providing not only oxygen but also organic compounds that are synthesized from atmospheric CO 2 and water using light energy as the driving force. The still-increasing world population poses a serious challenge to further enhance biomass production of crop plants. Crop yield is determined by various parameters, inter alia by the light energy conversion efficiency of the photosynthetic machinery. Photosynthesis can be looked at from different perspectives: (i) light reactions and carbon assimilation, (ii) leaves and canopy structure, and (ii) source-sink relationships. In this review, we discuss opportunities and prospects to increase photosynthetic performance at the different layers, taking into account the recent progress made in the respective fields.

RNA-Seq based phylogeny recapitulates previous phylogeny of the genus Flaveria (Asteraceae) with some modifications.

BACKGROUND: The genus Flaveria has been extensively used as a model to study the evolution of C4 photosynthesis as it contains C3 and C4 species as well as a number of species that exhibit intermediate types of photosynthesis. The current phylogenetic tree of the genus Flaveria contains 21 of the 23 known Flaveria species and has been previously constructed using a combination of morphological data and three non-coding DNA sequences (nuclear encoded ETS, ITS and chloroplast encoded trnL-F). RESULTS: Here we developed a new strategy to update the phylogenetic tree of 16 Flaveria species based on RNA-Seq data. The updated phylogeny is largely congruent with the previously published tree but with some modifications. We propose that the data collection method provided in this study can be used as a generic method for phylogenetic tree reconstruction if the target species has no genomic information. We also showed that a "F. pringlei" genotype recently used in a number of labs may be a hybrid between F. pringlei (C3) and F. angustifolia (C3-C4). CONCLUSIONS: We propose that the new strategy of obtaining phylogenetic sequences outlined in this study can be used to construct robust trees in a larger number of taxa. The updated Flaveria phylogenetic tree also supports a hypothesis of stepwise and parallel evolution of C4 photosynthesis in the Flavaria clade.

Expression of a nuclear-encoded psbH gene complements the plastidic RNA processing defect in the PSII mutant hcf107 in Arabidopsis thaliana.

The helical-repeat RNA-binding protein HCF107 is required for processing, stabilization and translation of plastid-encoded psbH mRNA. The psbH gene encodes a small, hydrophilic subunit of the PSII complex and is part of the plastidic psbB-psbT-psbH-petB-petD transcription unit. In Arabidopsis hcf107 mutants, only trace amounts of PSII proteins can be detected. Beside drastically reduced synthesis of PsbH, the synthesis of CP47 was also reduced in these mutants, although the corresponding psbB transcripts accumulate to wild type levels. This situation raises the question, whether the reduction of CP47 is a direct consequence of the mutation, based on targeting of HCF107 to the psbB mRNA, or a secondary affect due to the absent PsbH. To clarify this issue we introduced a chimeric psbH construct comprising a sequence encoding a chloroplast transit peptide into the hcf107-2 background. We found that the nucleus-localized psbH was able to complement the mutant defect resulting in photoautotrophic plants. The PSII proteins CP47 and D1 accumulated to barely half of the wild type level. Further experiments showed that cytosolically synthesized PsbH was imported into chloroplasts and assembled into PSII complexes. Using this approach, we showed that the tetratricopeptide repeat protein HCF107 of Arabidopsis is only responsible for expression of PsbH and not for synthesis of CP47. In addition the data suggest the necessity of the small, one-helix membrane spanning protein PsbH for the accumulation of CP47 in higher plants.

The role of photorespiration during the evolution of C4 photosynthesis in the genus Flaveria.

C4 photosynthesis represents a most remarkable case of convergent evolution of a complex trait, which includes the reprogramming of the expression patterns of thousands of genes. Anatomical, physiological, and phylogenetic and analyses as well as computational modeling indicate that the establishment of a photorespiratory carbon pump (termed C2 photosynthesis) is a prerequisite for the evolution of C4. However, a mechanistic model explaining the tight connection between the evolution of C4 and C2 photosynthesis is currently lacking. Here we address this question through comparative transcriptomic and biochemical analyses of closely related C3, C3-C4, and C4 species, combined with Flux Balance Analysis constrained through a mechanistic model of carbon fixation. We show that C2 photosynthesis creates a misbalance in nitrogen metabolism between bundle sheath and mesophyll cells. Rebalancing nitrogen metabolism requires anaplerotic reactions that resemble at least parts of a basic C4 cycle. Our findings thus show how C2 photosynthesis represents a pre-adaptation for the C4 system, where the evolution of the C2 system establishes important C4 components as a side effect.