Deep evolutionary comparison of gene expression identifies parallel recruitment of trans-factors in two independent origins of C4 photosynthesis

With at least 60 independent origins spanning monocotyledons and dicotyledons, the C(4) photosynthetic pathway represents one of the most remarkable examples of convergent evolution. The recurrent evolution of this highly complex trait involving alterations to leaf anatomy, cell biology and biochemistry allows an increase in productivity by ∼50% in tropical and subtropical areas. The extent to which separate lineages of C(4) plants use the same genetic networks to maintain C(4) photosynthesis is unknown. We developed a new informatics framework to enable deep evolutionary comparison of gene expression in species lacking reference genomes. We exploited this to compare gene expression in species representing two independent C(4) lineages (Cleome gynandra and Zea mays) whose last common ancestor diverged ∼140 million years ago. We define a cohort of 3,335 genes that represent conserved components of leaf and photosynthetic development in these species. Furthermore, we show that genes encoding proteins of the C(4) cycle are recruited into networks defined by photosynthesis-related genes. Despite the wide evolutionary separation and independent origins of the C(4) phenotype, we report that these species use homologous transcription factors to both induce C(4) photosynthesis and to maintain the cell specific gene expression required for the pathway to operate. We define a core molecular signature associated with leaf and photosynthetic maturation that is likely shared by angiosperm species derived from the last common ancestor of the monocotyledons and dicotyledons. We show that deep evolutionary comparisons of gene expression can reveal novel insight into the molecular convergence of highly complex phenotypes and that parallel evolution of trans-factors underpins the repeated appearance of C(4) photosynthesis. Thus, exploitation of extant natural variation associated with complex traits can be used to identify regulators. Moreover, the transcription factors that are shared by independent C(4) lineages are key targets for engineering the C(4) pathway into C(3) crops such as rice

Dual expression and anatomy lines allow simultaneous visualization of gene expression and anatomy

Studying the developmental genetics of plant organs, requires following gene expression in specific tissues. To facilitate this, we have developed the Dual Expression Anatomy Lines (DEAL), which incorporate a red plasma membrane marker alongside a fluorescent reporter for a gene of interest in the same vector. Here, we adapted the GreenGate cloning vectors to create two destination vectors showing strong marking of cell membranes in either the whole root or specifically in the lateral roots. This system can also be used in both embryos and whole seedlings. As proof of concept, we follow both gene expression and anatomy in Arabidopsis (Arabidopsis thaliana) during lateral root organogenesis for a period of over 24h,. and cCoupled with the development of a flow cell and perfusion system, we follow changes in activity of the DII auxin sensor following application of auxin

Correction: Deep Evolutionary Comparison of Gene Expression Identifies Parallel Recruitment of Trans-Factors in Two Independent Origins of C4 Photosynthesis.

[This corrects the article DOI: 10.1371/journal.pgen.1004365.]

The significance of meristic changes in the flowers of Sapotaceae

Sapotaceae belongs to the heterogeneous order Ericales and exhibits extensive diversity in floral morphology. Although pentamery is widespread and probably the ancestral condition, some clades are extremely variable in merism, with fluctuations between tetramery to hexamery and octomery, affecting different floral organs to different degrees. We assessed the different states of merism in Sapotaceae to determine the evolution of this character among different clades. The floral morphology and development of nine species from eight genera were investigated using scanning electron microscopy (SEM). Furthermore, floral characters related to merism were mapped onto a phylogenetic tree to analyse the distribution and evolutionary significance of merism in the family. Developmental evidence shows that changes in merism are linked to a concerted multiplication of organs among whorls and an increase in whorls through the displacement of organs. Although pentamery is reconstructed as the ancestral condition, a reduction to tetramery or an increase to a higher merism (mainly hexamery or octomery) has evolved at least five times in the family. Fluctuations in merism between different whorls are not random but occur in a coordinated pattern, presenting strong synapomorphies for selected clades. Octomery has evolved at least twice, in Isonandreae from tetramery and in Sapoteae-Mimusopinae from pentamery. Hexamery has evolved at least three times, independently in Northia, the Palaquium clade of Isonandreae and derived from octomery in Sapoteae-Mimusopinae. Three possibilities of merism increase have been identified in Sapotaceae: (1) a concerted increase affecting all organs more or less equally (Palaquium clade of Isonandreae, Sapoteae); (2) a coordinated increase in petals, stamens and mostly carpels without effect on sepals (Labourdonnaisia, Payena-Madhuca clade of Isonandreae); (3) an increase in carpels independently of other organs (Burckella, Letestua, Labramia, etc.). A major shift affecting all Sapotaceae, except Isonandreae, is the sterilization or loss of the antesepalous stamen whorl. The presence of two fertile stamen whorls in Isonandreae indicates a possible reversal or a retained plesiomorphy. In a number of genera, stamens are secondarily increased independently of changes in merism. Descriptions of flowers listing only organ numbers are thus misleading in the inference of evolutionary relationships, as they do not differentiate between changes in merism affecting the number of perianth whorls and other changes affecting the androecium, such as sterilization, loss or occasional doubling of antepetalous stamens. © 2016 The Linnean Society of London

The significance of meristic changes in the flowers of Sapotaceae

Sapotaceae belongs to the heterogeneous order Ericales and exhibits extensive diversity in floral morphology. Although pentamery is widespread and probably the ancestral condition, some clades are extremely variable in merism, with fluctuations between tetramery to hexamery and octomery, affecting different floral organs to different degrees. We assessed the different states of merism in Sapotaceae to determine the evolution of this character among different clades. The floral morphology and development of nine species from eight genera were investigated using scanning electron microscopy (SEM). Furthermore, floral characters related to merism were mapped onto a phylogenetic tree to analyse the distribution and evolutionary significance of merism in the family. Developmental evidence shows that changes in merism are linked to a concerted multiplication of organs among whorls and an increase in whorls through the displacement of organs. Although pentamery is reconstructed as the ancestral condition, a reduction to tetramery or an increase to a higher merism (mainly hexamery or octomery) has evolved at least five times in the family. Fluctuations in merism between different whorls are not random but occur in a coordinated pattern, presenting strong synapomorphies for selected clades. Octomery has evolved at least twice, in Isonandreae from tetramery and in Sapoteae-Mimusopinae from pentamery. Hexamery has evolved at least three times, independently in Northia, the Palaquium clade of Isonandreae and derived from octomery in Sapoteae-Mimusopinae. Three possibilities of merism increase have been identified in Sapotaceae: (1) a concerted increase affecting all organs more or less equally (Palaquium clade of Isonandreae, Sapoteae); (2) a coordinated increase in petals, stamens and mostly carpels without effect on sepals (Labourdonnaisia, Payena-Madhuca clade of Isonandreae); (3) an increase in carpels independently of other organs (Burckella, Letestua, Labramia, etc.). A major shift affecting all Sapotaceae, except Isonandreae, is the sterilization or loss of the antesepalous stamen whorl. The presence of two fertile stamen whorls in Isonandreae indicates a possible reversal or a retained plesiomorphy. In a number of genera, stamens are secondarily increased independently of changes in merism. Descriptions of flowers listing only organ numbers are thus misleading in the inference of evolutionary relationships, as they do not differentiate between changes in merism affecting the number of perianth whorls and other changes affecting the androecium, such as sterilization, loss or occasional doubling of antepetalous stamens. © 2016 The Linnean Society of London

Classification of gene expression in the two C₄ species <i>C. gynandra</i> and maize.

<p>As leaves of <i>C. gynandra</i> (A) and maize (B) mature, transcripts were classified into twenty-six behaviours, thirteen ascending (A&B) and thirteen descending. Statistically significant differences between neighbouring tissue types are delineated by red circles in ascending filters. The total number of genes within each behaviour is presented in parentheses and behaviours containing photosynthesis-related genes are annotated by red boxes around each plot (eg A3, A4 and A6). Genes of the core C₄ cycle occupy six and five of the thirteen ascending filters in <i>C. gynandra</i> and maize respectively (transcripts in green). (C) Venn diagram representing transcription factors showing the same behaviours as C₄-related genes in the maize and in <i>C. gynandra</i> leaf gradients. (D) Behaviour of homologous genes in C₃<i>A. thaliana</i>.</p

Convergence in patterns of gene expression in leaf gradients of <i>C. gynandra</i> and maize.

<p>(A) Venn diagram indicating numbers of shared and unique transcripts to each type of <i>C. gynandra</i> leaf tissue. (B) Major bin categories identified using Wilcoxon test implemented in Pageman <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004365#pgen.1004365-Usadel1" target="_blank">[55]</a> tool that alter between the base, middle, tip of 3 mm and mature <i>C. gynandra</i> leaves. (C) Number of genes with ascending (red) and descending (grey) behaviours as leaves of <i>C. gynandra</i> (Cg) and maize (Zm) mature. (D) Venn diagrams depicting the total number of transcript homologues that increase or decrease in abundance as leaves of both <i>C. gynandra</i> and maize mature. The number of genes common to the two gradients is shown in blue, with the number of transcription factors shown in parentheses. Red circles and numbers correspond to genes that increase in abundance, while grey circles represent genes that show reduced abundance.</p

Convergence of mesophyll and bundle sheath transcriptomes in <i>C. gynandra</i> and maize.

<p>(A) Schematic showing M or BS accumulation of transcripts involved in the C₄ cycle. Shared parts of the pathway are annotated in red, while differences between the species are shown in grey. CA, carbonic anhydrase; PPC, phospho<i>enol</i>pyruvate carboxylase; PEPC Kin, phospho<i>enol</i>pyruvate carboxylase kinase, ASPAT, aspartate aminotransferase; ALAAT, alanine aminotransferase; PPDK, pyruvate-orthophosphate dikinase; TPI, triose phosphate isomerase; PGK, phosphoglycerate kinase; FBA, fructose-bisphosphate aldolase; SBP, sedoheptulose-bisphosphatase; TKL, transketolase; PRK, phosphoribulokinase; RbcS, RubisCO small subunit; RCA, RubisCO activase; FBP, fructose 1,6-bisphosphate phosphatase; RPE, D-ribulose-5-phosphate-3-epimerase; NAD-ME, NAD-dependent malic enzyme, MDH malate dehydrogenase. (B) Venn diagrams representing transcripts expressed in M (left panel) and BS (right panel) of <i>C. gynandra</i> and maize. Cell-specific maize data represents the overlap between two independent experiments <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004365#pgen.1004365-Li1" target="_blank">[12]</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004365#pgen.1004365-Chang2" target="_blank">[56]</a>. (C) Venn diagrams of transcription factors expressed in M or BS in maize and <i>C. gynandra</i>. (D–G) Expression in M and BS cells of the 18 homologous transcription factors showing co-ordinated induction with C₄ photosynthesis genes during leaf maturation of both maize and <i>C. gynandra</i>. Abbreviations: Cg data from <i>C. gynandra</i> (this study), while Zm1 data are from Li <i>et al</i> (2010) <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004365#pgen.1004365-Li1" target="_blank">[12]</a> and Chang et al (2012) <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004365#pgen.1004365-Chang1" target="_blank">[35]</a> respectively.</p