The genome of Salsola soda allows a transition from C3 to C4 photosynthesis. A developmental transcriptome series revealed novel genes showing expression patterns similar to those encoding C4 proteins.

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.

SummaryThe 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.

C4 species have evolved more than 60 times independently from C3 ancestors. This multiple and parallel evolution of the complex C4 trait indicates common underlying evolutionary mechanisms that might be identified by comparative analysis of closely related C3 and C4 species. Efficient C4 function depends on a distinctive leaf anatomy that is characterized by enlarged, chloroplast rich bundle sheath cells and a narrow vein spacing. To elucidate molecular mechanisms generating this so called Kranz anatomy, we analyzed a developmental series of leaves from the C4 plant Flaveria bidentis and the closely related C3 species Flaveria robusta using leaf clearing and whole transcriptome sequencing. Applying non-negative matrix factorization on the data identified four different zones with distinct transcriptome patterns in growing leaves of both species. Comparing these transcriptome patterns revealed an important role of auxin metabolism and especially auxin homeostasis for establishing the high vein density typical for C4 leaves.2012). Under current ambient CO2 concentrations (405 ppm) at 25°C, photorespiration is estimated to decrease the yield of soybean or wheat in the US by 36% and 20%, respectively (Walker et al., 2016). Environmental constrains such as high temperatures and drought further increases Rubisco oxygenase activity (Laing et al., 1974; Jordan and Ogren, 1984; Brooks and Farquhar, 1985; Parry et al., 2007).In most C4 plants CO2 fixation is compartmentalized between two cell types, the bundle sheath (BS) and the mesophyll (M) cells. In the mesophyll phosphoenolpyruvate (PEP) is carboxylated by phosphoenolpyruvate carboxylase (PEPC), resulting in the 4-carbon compound oxaloacetate (OAA). OAA is converted to malate and/or aspartate, which is then transferred to the bundle sheath. Here the 4-carbon compounds are decarboxylated and the released CO2 is assimilated in the Calvin-Benson-Bassham cycle (CBB). The resulting pyruvate is transferred back to the mesophyll where the primary CO2 acceptor PEP is regenerated by pyruvate orthophosphate dikinase (PPDK) (Hatch, 1987).C4 photosynthesis requires a particular leaf anatomy. As BS and M cells operate as a photosynthetic unit, direct contact of both cell types is necessary to ensure efficient photosynthesis.The BS is composed of the cells directly adjacent to the vasculature. Therefore, leaves of most C4 species exhibit high vein densities with a characteristic pattern in which two veins, each surrounded by BS cells, are separated by only two layers of M cells in a vein-bundle sheathmesophyll-mesophyll-bundle sheath-vein layout. Bundle sheath cells of C4 plants often appear larger in cross-section compared to C3 species and contain more chloroplasts. This character-

This article comments on: Chen J, Zhu M, Liu R, Zhang M, Lv Y, Liu Y, Xiao X, Yuan J, Cai H. 2020. BIOMASS YIELD 1 regulates Sorghum biomass and grain yield via the shikimate pathway. Journal of Experimental Botany 71, 5506–5520.

Background It has been proposed that engineering the C4 photosynthetic pathway into C3 crops could significantly increase yield. This goal requires an increase in the chloroplast compartment of bundle sheath cells in C3 species. To facilitate large-scale testing of candidate regulators of chloroplast development in the rice bundle sheath, a simple and robust method to phenotype this tissue in C3 species is required. Results We established a leaf ablation method to accelerate phenotyping of rice bundle sheath cells. The bundle sheath cells and chloroplasts were visualized using light and confocal laser microscopy. Bundle sheath cell dimensions, chloroplast area and chloroplast number per cell were measured from the images obtained by confocal laser microscopy. Bundle sheath cell dimensions of maize were also measured and compared with rice. Our data show that bundle sheath width but not length significantly differed between C3 rice and C4 maize. Comparison of paradermal versus transverse bundle sheath cell width indicated that bundle sheath cells were intact after leaf ablation. Moreover, comparisons of planar chloroplast areas and chloroplast numbers per bundle sheath cell between wild-type and transgenic rice lines expressing the maize GOLDEN-2 (ZmG2) showed that the leaf ablation method allowed differences in chloroplast parameters to be detected. Conclusions Leaf ablation is a simple approach to accessing bundle sheath cell files in C3 species. We show that this method is suitable for obtaining parameters associated with bundle sheath cell size, chloroplast area and chloroplast number per cell.

A key feature of C 4 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 C 4 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 C 4 phospho enol pyruvate 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.

Background: It has been proposed that engineering the C4 photosynthetic pathway into C3 crops could significantly increase yield. This goal requires an increase in the chloroplast compartment of bundle sheath cells in C3 species. To facilitate large-scale testing of candidate regulators of chloroplast development in the rice bundle sheath, a simple and robust method to phenotype this tissue in C3 species is required. Results: We established a leaf ablation method to accelerate phenotyping of rice bundle sheath cells. The approach allowed bundle sheath cell dimensions, chloroplast area and chloroplast number per cell to be measured. Using this method, bundle sheath cell dimensions of maize were also measured and compared with rice. Our data show that bundle sheath width but not length significantly differed between C3 rice and C4 maize. Comparison of paradermal versus transverse bundle sheath cell width indicated that bundle sheath cells were intact after leaf ablation. Moreover, comparisons of planar chloroplast areas and chloroplast numbers per bundle sheath cell between wild-type and transgenic rice lines expressing the maize GOLDEN-2 (ZmG2) showed that the leaf ablation method allowed differences in chloroplast parameters to be detected. Conclusions: Leaf ablation is a simple approach to accessing bundle sheath cell files in C3 species. We show that this method is suitable for obtaining parameters associated with bundle sheath cell size, chloroplast area and chloroplast number per cell.