Transcriptome dynamics in developing leaves from C₃and C₄Flaveriaspecies reveal determinants of Kranz anatomy

Abstract

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-

The evolution of stomatal traits along the trajectory towards C4photosynthesis

“…Fold change means the ratio between the gene expression in a C 3 Flaveria specie and gene expression in a C 4 Flaveria specie under the same leaf development stage and growth condition as the C 3 Flaveria species. The expression data were from ( 44 ), ( 35 ), ( 34 ) and One Thousand Plants (1KP). d , The volcano plot for the expression of the stomatal development related genes between C 3 and C 4 Flaveria species.…”

“…d , The volcano plot for the expression of the stomatal development related genes between C 3 and C 4 Flaveria species. The expression data were from ( 35 ) and ( 44 ). e , Relative expression levels determined by RT-qPCR and expression level determined by RNA-seq of FSTO (FSTOMAGEN) in rob and bid (n=4, biologically independent replicates).…”

“…The first data source is the One Thousand Plants (1KP) Consortium; we subsequently analyzed the data according to the methods described in previous work ( 39, 40 ). The second and third data sources were Mallman et al (2014) ( 33 ) and Kumari Billakurthi et al (2018) ( 41 ), respectively. For the second data source, the second and fourth visible leaves were sampled; the second leaf was not completely mature, and stomatal developmental related genes were still actively expressed.…”

“…For the Flaveria , the samples data from four source containing the leaves of plants grown under normal condition were analysed. The first data source is the One Thousand Plants (1KP) Consortium, and we subsequently analyzed the data according to the methods described in previous work ( 42, 43 ); The second and third data sources were Mallman et al (2014) ( 35 ) and Udo Gowik et al ( 34 ); The fourth data source was Kumari Billakurthi et al (2018) ( 44 ). The last three data sources are all from Heinrich-Heine-University.…”

“…The second and third data sources were Mallman et al (2014) (41) and Udo Gowik et al (47). The fourth data source was Kumari Billakurthi et al (2018) (66). The last three data sources are all from Heinrich Heine University.…”