Complete chloroplast genome of the medicinal plant Cleome paradoxa R.Br. Ex DC: Comparative analysis, and phylogenetic relationships among the members of Cleomaceae.

The cleome species of the Cleomaceae family have several medical uses, including applications such as antioxidants and insecticides. In the present study, we sequenced the complete chloroplast genome (cp genome) of Cleome paradoxa. The chloroplast genome is 159,393 bp long, with a typical four-region structure: a large single copy (LSC) region of 88,191 bp, a small single copy (SSC) region of 18,620 bp, and inverted repeat regions (IRa and IRb) of 26,291 bp each. The proportion of GC content was 35.79 %. The chloroplast genome of C. paradoxa contains 133 genes, 81 of which encode proteins, 29 encode tRNA, and 4 encode rRNA. We noticed a divergence in the location and number of certain genes at the IR-LSC and IR-SSC boundaries. The phylogenetic tree constructed from the complete chloroplast genome data broadly supported the taxonomic situation of Cleome paradoxa as belonging to the Cleomaceae family and Cleome species. The cp genome of C. paradoxa was rich in single sequence repeats (SSRs), with a total of 314 SSRs. Additionally, several genes were under positive selection. These results could be useful for determining the genetic variations and resolving conflicting relationships among Cleomaceae species.

Substrate Specificity of LACCASE8 Facilitates Polymerization of Caffeyl Alcohol for C-Lignin Biosynthesis in the Seed Coat of Cleome hassleriana.

Catechyl lignin (C-lignin) is a linear homopolymer of caffeyl alcohol found in the seed coats of diverse plant species. Its properties make it a natural source of carbon fibers and high-value chemicals, but the mechanism of in planta polymerization of caffeyl alcohol remains unclear. In the ornamental plant Cleome hassleriana, lignin biosynthesis in the seed coat switches from guaiacyl lignin to C-lignin at ∼12 d after pollination. Here we found that the transcript profile of the laccase gene ChLAC8 parallels the accumulation of C-lignin during seed coat development. Recombinant ChLAC8 oxidizes caffeyl and sinapyl alcohols, generating their corresponding dimers or trimers in vitro, but cannot oxidize coniferyl alcohol. We propose a basis for this substrate preference based on molecular modeling/docking experiments. Suppression of ChLAC8 expression led to significantly reduced C-lignin content in the seed coats of transgenic Cleome plants. Feeding of 13C-caffeyl alcohol to the Arabidopsis (Arabidopsis thaliana) caffeic acid o-methyltransferase mutant resulted in no incorporation of 13C into C-lignin, but expressing ChLAC8 in this genetic background led to appearance of C-lignin with >40% label incorporation. These results indicate that ChLAC8 is required for C-lignin polymerization and determines lignin composition when caffeyl alcohol is available.

PISTILLATA paralogs in Tarenaya hassleriana have diverged in interaction specificity.

BACKGROUND: Floral organs are specified by MADS-domain transcription factors that act in a combinatorial manner, as summarized in the (A)BCE model. However, this evolutionarily conserved model is in contrast to a remarkable amount of morphological diversity in flowers. One of the mechanisms suggested to contribute to this diversity is duplication of floral MADS-domain transcription factors. Although gene duplication is often followed by loss of one of the copies, sometimes both copies are retained. If both copies are retained they will initially be redundant, providing freedom for one of the paralogs to change function. Here, we examine the evolutionary fate and functional consequences of a transposition event at the base of the Brassicales that resulted in the duplication of the floral regulator PISTILLATA (PI), using Tarenaya hassleriana (Cleomaceae) as a model system. RESULTS: The transposition of a genomic region containing a PI gene led to two paralogs which are located at different positions in the genome. The original PI copy is syntenic in position with most angiosperms, whereas the transposed copy is syntenic with the PI genes in Brassicaceae. The two PI paralogs of T. hassleriana have very similar expression patterns. However, they may have diverged in function, as only one of these PI proteins was able to act heterologously in the first whorl of A. thaliana flowers. We also observed differences in protein complex formation between the two paralogs, and the two paralogs exhibit subtle differences in DNA-binding specificity. Sequence analysis indicates that most of the protein sequence divergence between the two T. hassleriana paralogs emerged in a common ancestor of the Cleomaceae and the Brassicaceae. CONCLUSIONS: We found that the PI paralogs in T. hassleriana have similar expression patterns, but may have diverged at the level of protein function. Data suggest that most protein sequence divergence occurred rapidly, prior to the origin of the Brassicaceae and Cleomaceae. It is tempting to speculate that the interaction specificities of the Brassicaceae-specific PI proteins are different compared to the PI found in other angiosperms. This could lead to PI regulating partly different genes in the Brassicaceae, and ultimately might result in change floral in morphology.

Identification and characterization of miRNAs in two closely related C4 and C3 species of Cleome by high-throughput sequencing.

Cleome gynandra and Cleome hassleriana, which are C4 and C3 plants, respectively, are two species of Cleome. The close genetic relationship between C. gynandra and C. hassleriana provides advantages for discovering the differences in leaf development and physiological processes between C3 and C4 plants. MicroRNAs (miRNAs) are a class of important regulators of various biological processes. In this study, we investigate the differences in the characteristics of miRNAs between C. gynandra and C. hassleriana using high-throughput sequencing technology. In total, 94 and 102 known miRNAs were identified in C. gynandra and C. hassleriana, respectively, of which 3 were specific for C. gynandra and 10 were specific for C. hassleriana. Ninety-one common miRNAs were identified in both species. In addition, 4 novel miRNAs were detected, including three in C. gynandra and three in C. hassleriana. Of these miRNAs, 67 were significantly differentially expressed between these two species and were involved in extensive biological processes, such as glycol-metabolism and photosynthesis. Our study not only provided resources for C. gynandra and C. hassleriana research but also provided useful clues for the understanding of the roles of miRNAs in the alterations of biological processes in leaf tissues during the evolution of the C4 pathway.

CRYOPRESERVATION OF ADVENTITIOUS ROOTS OF Cleome rosea Vahl (CLEOMACEAE) USING A VITRIFICATION TECHNIQUE AND ASSESSMENT OF GENETIC STABILITY.

BACKGROUND: Cleome rosea, a Brazilian native species, has medicinal potential. Previously a cryopreservation protocol for in vitro roots using the vitrification solution PVS2 has been developed. However, the genetic stability of the cryopreserved material is yet to be assessed. OBJECTIVES: To evaluate the effects of loading and vitrification solutions (PVS2 and PVS3) on post-cryopreservation recovery of C. rosea roots, and to assess their genetic stability using Random Amplified Polymorphic DNA (RAPD) markers. MATERIALS AND METHODS: Root segments were pretreated with increasing concentrations of sucrose (0.2 to 0.4 M), followed by osmoprotection with loading solution and treatment with one of the vitrification solutions tested. RESULTS: The highest recoveries using PVS2 and PVS3 were obtained when root segments were exposed to these solutions for 15 min, reaching 77% and 100% respectively. The RAPD band profiles were monomorphic with most of the primers used. This molecular analysis revealed high genetic similarity (similarity coefficients among 0.98 and 1.00) between the cryopreserved roots and their mother plants. CONCLUSION: Roots from in vitro-propagated plants of C. rosea, were successfully cryopreserved using the vitrification technique. No major variations were observed on the genetic stability of cryopreserved roots, validating the use of this protocol as an efficient long-term conservation option for this species.

Ancestral light and chloroplast regulation form the foundations for C4 gene expression.

C4 photosynthesis acts as a carbon concentrating mechanism that leads to large increases in photosynthetic efficiency. The C4 pathway is found in more than 60 plant lineages1 but the molecular enablers of this evolution are poorly understood. In particular, it is unclear how non-photosynthetic proteins in the ancestral C3 system have repeatedly become strongly expressed and integrated into photosynthesis gene regulatory networks in C4 leaves. Here, we provide clear evidence that in C3 leaves, genes encoding key enzymes of the C4 pathway are already co-regulated with photosynthesis genes and are controlled by both light and chloroplast-to-nucleus signalling. In C4 leaves this regulation becomes increasingly dependent on the chloroplast. We propose that regulation of C4 cycle genes by light and the chloroplast in the ancestral C3 state has facilitated the repeated evolution of the complex and convergent C4 trait.

Phylogenetic analysis, genetic diversity and relationships between the recently segregated species of Corynandra and Cleoserrata from the genus Cleome using DNA barcoding and molecular markers.

Cleome is the largest genus in the family Cleomaceae and it is known for its various medicinal properties. Recently, some species from the Cleome genus (Cleome viscosa, Cleome chelidonii, Cleome felina and Cleome speciosa) are split into genera Corynandra (Corynandra viscosa, Corynandra chelidonii, Corynandra felina), and Cleoserrata (Cleoserrata speciosa). The objective of this study was to obtain DNA barcodes for these species for their accurate identification and determining phylogenetic relationships. Out of 10 screened barcoding regions, rbcL, matK and ITS1 regions showed higher PCR efficiency and sequencing success. This study added matK, rbcL and ITS1 barcodes for the identification of Corynandra chelidonii, Corynandra felina, Cleome simplicifolia and Cleome aspera species in existing barcode data. Corynandra chelidonii and Corynandra felina species belong to the Corynandra genus, but they are not grouped with the Corynandra viscosa species, however clustered with the Cleome species. Molecular marker analysis showed 100% polymorphism among the studied plant samples. Diversity indices for molecular markers were ranged from He=0.1115-0.1714 and I=0.2268-0.2700, which indicates a significant amount of genetic diversity among studied species. Discrimination of the Cleome and Corynandra species from Cleoserrata speciosa was obtained by two RAPD primers (OPA-4 and RAPD-17) and two ISSR primers (ISSR-1 and ISSR-2). RAPD and ISSR markers are useful for the genetic characterization of these studied species. The present investigation will be helpful to understand the relationships of Cleome lineages with Corynandra and Cleoserrata species.

A Specific Transcriptome Signature for Guard Cells from the C4 Plant Gynandropsis gynandra.

C4 photosynthesis represents an excellent example of convergent evolution that results in the optimization of both carbon and water usage by plants. In C4 plants, a carbon-concentrating mechanism divided between bundle sheath and mesophyll cells increases photosynthetic efficiency. Compared with C3 leaves, the carbon-concentrating mechanism of C4 plants allows photosynthetic operation at lower stomatal conductance, and as a consequence, transpiration is reduced. Here, we characterize transcriptomes from guard cells in C3 Tareneya hassleriana and C4 Gynandropsis gynandra belonging to the Cleomaceae. While approximately 60% of Gene Ontology terms previously associated with guard cells from the C3 model Arabidopsis (Arabidopsis thaliana) are conserved, there is much less overlap between patterns of individual gene expression. Most ion and CO2 signaling modules appear unchanged at the transcript level in guard cells from C3 and C4 species, but major variations in transcripts associated with carbon-related pathways known to influence stomatal behavior were detected. Genes associated with C4 photosynthesis were more highly expressed in guard cells of C4 compared with C3 leaves. Furthermore, we detected two major patterns of cell-specific C4 gene expression within the C4 leaf. In the first, genes previously associated with preferential expression in the bundle sheath showed continually decreasing expression from bundle sheath to mesophyll to guard cells. In the second, expression was maximal in the mesophyll compared with both guard cells and bundle sheath. These data imply that at least two gene regulatory networks act to coordinate gene expression across the bundle sheath, mesophyll, and guard cells in the C4 leaf.

An Untranslated cis-Element Regulates the Accumulation of Multiple C4 Enzymes in Gynandropsis gynandra Mesophyll Cells.

C4 photosynthesis is a complex phenotype that allows more efficient carbon capture than the ancestral C3 pathway. In leaves of C4 species, hundreds of transcripts increase in abundance compared with C3 relatives and become restricted to mesophyll (M) or bundle sheath (BS) cells. However, no mechanism has been reported that regulates the compartmentation of multiple enzymes in M or BS cells. We examined mechanisms regulating CARBONIC ANHYDRASE4 (CA4) in C4 Gynandropsis gynandra. Increased abundance is directed by both the promoter region and introns of the G. gynandra gene. A nine-nucleotide motif located in the 5' untranslated region (UTR) is required for preferential accumulation of GUS in M cells. This element is present and functional in three additional 5' UTRs and six 3' UTRs where it determines accumulation of two isoforms of CA and pyruvate,orthophosphate dikinase in M cells. Although the GgCA4 5' UTR is sufficient to direct GUS accumulation in M cells, transcripts encoding GUS are abundant in both M and BS. Mutating the GgCA4 5' UTR abolishes enrichment of protein in M cells without affecting transcript abundance. The work identifies a mechanism that directs cell-preferential accumulation of multiple enzymes required for C4 photosynthesis.

Polyploid evolution of the Brassicaceae during the Cenozoic era.

The Brassicaceae (Cruciferae) family, owing to its remarkable species, genetic, and physiological diversity as well as its significant economic potential, has become a model for polyploidy and evolutionary studies. Utilizing extensive transcriptome pyrosequencing of diverse taxa, we established a resolved phylogeny of a subset of crucifer species. We elucidated the frequency, age, and phylogenetic position of polyploidy and lineage separation events that have marked the evolutionary history of the Brassicaceae. Besides the well-known ancient α (47 million years ago [Mya]) and ß (124 Mya) paleopolyploidy events, several species were shown to have undergone a further more recent (∼7 to 12 Mya) round of genome multiplication. We identified eight whole-genome duplications corresponding to at least five independent neo/mesopolyploidy events. Although the Brassicaceae family evolved from other eudicots at the beginning of the Cenozoic era of the Earth (60 Mya), major diversification occurred only during the Neogene period (0 to 23 Mya). Remarkably, the widespread species divergence, major polyploidy, and lineage separation events during Brassicaceae evolution are clustered in time around epoch transitions characterized by prolonged unstable climatic conditions. The synchronized diversification of Brassicaceae species suggests that polyploid events may have conferred higher adaptability and increased tolerance toward the drastically changing global environment, thus facilitating species radiation.

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 C4 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 C4 plants use the same genetic networks to maintain C4 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 C4 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 C4 cycle are recruited into networks defined by photosynthesis-related genes. Despite the wide evolutionary separation and independent origins of the C4 phenotype, we report that these species use homologous transcription factors to both induce C4 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 C4 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 C4 lineages are key targets for engineering the C4 pathway into C3 crops such as rice.

Endoreduplication is not involved in bundle-sheath formation in the C4 species Cleome gynandra.

There is currently significant interest in engineering the two-celled C4 photosynthesis pathway into crops such as rice in order to increase yield. This will require alterations to the biochemistry of photosynthesis in both mesophyll (M) and bundle-sheath (BS) cells, but also alterations to leaf anatomy. For example, the BS of C4 species is enlarged compared with that in C3 species. Because cell and nucleus size are often correlated, this study investigated whether nuclear endoreduplication is associated with increased differentiation and expansion of BS cells. Nuclei in the BS of C4 Cleome gynandra were tagged with green fluorescent protein. Confocal laser-scanning microscopy and flow cytometry of isolated nuclei were used to quantify size and DNA content in BS cells. The results showed a significant endoreduplication in BS cells of C. gynandra but not in additional C4 lineages from both the monocotyledonous and dicotyledenous plants. Furthermore, in the C3 species Arabidopsis thaliana, BS cells undergo endoreduplication. Due to this significant endoreduplication in the small BS cells of C3 A. thaliana, it was concluded that endoreduplication of BS nuclei in C4 plants is not linked to expansion and differentiation of BS cells, and therefore that alternative strategies to increase this compartment need to be sought in order to engineer C4 traits into C3 crops such as rice.

Getting the most out of natural variation in C4 photosynthesis.

C4 photosynthesis is a complex trait that has a high degree of natural variation, involving anatomical and biochemical changes relative to the ancestral C3 state. It has evolved at least 66 times across a variety of lineages and the evolutionary route from C3 to C4 is likely conserved but not necessarily genetically identical. As such, a variety of C4 species are needed to identify what is fundamental to the C4 evolutionary process in a global context. In order to identify the genetic components of C4 form and function, a number of species are used as genetic models. These include Zea mays (maize), Sorghum bicolor (sorghum), Setaria viridis (Setaria), Flaveria bidentis, and Cleome gynandra. Each of these species has different benefits and challenges associated with its use as a model organism. Here, we propose that RNA profiling of a large sampling of C4, C3-C4, and C3 species, from as many lineages as possible, will allow identification of candidate genes necessary and sufficient to confer C4 anatomy and/or biochemistry. Furthermore, C4 model species will play a critical role in the functional characterization of these candidate genes and identification of their regulatory elements, by providing a platform for transformation and through the use of gene expression profiles in mesophyll and bundle sheath cells and along the leaf developmental gradient. Efforts should be made to sequence the genomes of F. bidentis and C. gynandra and to develop congeneric C3 species as genetic models for comparative studies. In combination, such resources would facilitate discovery of common and unique C4 regulatory mechanisms across genera.

Plant regeneration and stable transformation in the floricultural plant Cleome spinosa, a C3 plant closely related to the C4 plant C. gynandra.

Cleome spinosa is widely used as a garden ornamental in many countries. Here we determined the optimal conditions for plant regeneration from different tissue explants grown in vitro. Induction medium containing MS salts, MS vitamins, 3% sucrose, 1 mg l⁻¹ BA, 200 mg l⁻¹ timentin, and 0.8% agar was sufficient for shoot regeneration of all the tissue explants examined, including leaf, hypocotyl, and cotyledon. Subsequently, an Agrobacterium tumefaciens-mediated method was developed to transform the vector pCHS, which carries the transgenes Petunia chalcone synthase (chs) and selection marker neomycin phosphotransferase II (nptII), into C. spinosa. From a total of 368 cotyledon explants, 13 putative transgenic lines were regenerated from selection medium supplemented with 50 mg l⁻¹ kanamycin and 200 mg l⁻¹ timentin, and transferred to the greenhouse. Genomic PCR and Southern blot analyses revealed that the nptII transgene was present in all 13 transgenic plants. Similarly, when the Petunia chs transgene was used as a probe in Southern blot analysis, single or multiple hybridization bands were detected in 12 out of the 13 transgenic plants. In addition, T1 progeny assay from selected transformants showed that the nptII transgene can be transmitted in a Mendelian manner from transgenic parents into their progeny. This is the first report of stable transformation of the C3 dicotyledon C. spinosa, which will facilitate functional comparison of cell-type specific genes with counterpart C4 dicotyledon C. gynandra using transgenic approaches.

Comparison of ROS formation and antioxidant enzymes in Cleome gynandra (C4) and Cleome spinosa (C3) under drought stress.

Differences between antioxidant responses to drought in C(3) and C(4) plants are rather scanty. Even, we are not aware of any research on comparative ROS formation and antioxidant enzymes in C(3) and C(4) species differing in carboxylation pathway of same genus which would be useful to prevent other differences in plant metabolism. With this aim, relative shoot growth rate, relative water content and osmotic potential, hydrogen peroxide (H(2)O(2)) content and NADPH oxidase (NOX) activity, antioxidant defence system (superoxide dismutase (SOD), catalase (CAT), peroxidase (POX), ascorbate peroxidase (APX), glutathione reductase (GR) enzymes and their isoenzymes), CAT1 mRNA level, and lipid peroxidation in seedlings of Cleome spinosa (C(3)) and Cleome gynandra (C(4)) species of Cleome genus exposed to drought stress for 5 and 10 day (d) were comparatively investigated. Constitutive levels of antioxidant enzymes (except SOD) were consistently higher in C. spinosa than in C. gynandra under control conditions. CAT1 gene expression in C. spinosa was correlated with CAT activity but CAT1 gene expression in C. gynandra at 10 d did not show this correlation. Drought stress caused an increase in POX, CAT, APX and GR in both species. However, SOD activity was slightly decreased in C. gynandra while it was remained unchanged or increased on 5 and 10 d of stress in C. spinosa, respectively. Parallel to results of malon dialdehyde (MDA), H(2)O(2) content was also remarkably increased in C. spinosa as compared to C. gynandra under drought stress. These results suggest that in C. spinosa, antioxidant defence system was insufficient to suppress the increasing ROS production under stress condition. On the other hand, in C. gynandra, although its induction was lower as compared to C. spinosa, antioxidant system was able to cope with ROS formation under drought stress.

Multiple Arabidopsis genes primed for recruitment into C4 photosynthesis.

C(4) photosynthesis occurs in the most productive crops and vegetation on the planet, and has become widespread because it allows increased rates of photosynthesis compared with the ancestral C(3) pathway. Leaves of C(4) plants typically possess complicated alterations to photosynthesis, such that its reactions are compartmented between mesophyll and bundle sheath cells. Despite its complexity, the C(4) pathway has arisen independently in 62 separate lineages of land plants, and so represents one of the most striking examples of convergent evolution known. We demonstrate that elements in untranslated regions (UTRs) of multiple genes important for C(4) photosynthesis contribute to the metabolic compartmentalization characteristic of a C(4) leaf. Either the 5' or the 3' UTR is sufficient for cell specificity, indicating that functional redundancy underlies this key aspect of C(4) gene expression. Furthermore, we show that orthologous PPDK and CA genes from the C(3) plant Arabidopsis thaliana are primed for recruitment into the C(4) pathway. Elements sufficient for M-cell specificity in C(4) leaves are also present in both the 5' and 3' UTRs of these C(3) A. thaliana genes. These data indicate functional latency within the UTRs of genes from C(3) species that have been recruited into the C(4) pathway. The repeated recruitment of pre-existing cis-elements in C(3) genes may have facilitated the evolution of C(4) photosynthesis. These data also highlight the importance of alterations in trans in producing a functional C(4) leaf, and so provide insight into both the evolution and molecular basis of this important type of photosynthesis.

Carbonic anhydrase and the molecular evolution of C4 photosynthesis.

C(4) photosynthesis, a biochemical CO(2)-concentrating mechanism (CCM), evolved more than 60 times within the angiosperms from C(3) ancestors. The genus Flaveria, which contains species demonstrating C(3), C(3)-C(4), C(4)-like or C(4) photosynthesis, is a model for examining the molecular evolution of the C(4) pathway. Work with carbonic anhydrase (CA), and C(3) and C(4) Flaveria congeners has added significantly to the understanding of this process. The C(4) form of CA3, a ß-CA, which catalyses the first reaction in the C(4) pathway by hydrating atmospheric CO(2) to bicarbonate in the cytosol of mesophyll cells (mcs), evolved from a chloroplastic C(3) ancestor. The molecular modifications to the ancestral CA3 gene included the loss of the sequence encoding the chloroplast transit peptide, and mutations in regulatory regions that resulted in high levels of expression in the C(4) mesophyll. Analyses of the CA3 proteins and regulatory elements from Flaveria photosynthetic intermediates indicated C(4) biochemistry very likely evolved in a specific, stepwise manner in this genus. The details of the mechanisms involved in the molecular evolution of other C(4) plant ß-CAs are unknown; however, comparative genetics indicate gene duplication and neofunctionalization played significant roles as they did in Flaveria.

Critical assessment of assembly strategies for non-model species mRNA-Seq data and application of next-generation sequencing to the comparison of C(3) and C(4) species.

Next-generation sequencing enables the study of species without a sequenced genome at the 'omics' level. Custom transcriptome databases are generated and global expression profiles can be compared. However, the assembly of transcriptome sequence reads into contigs remains a daunting task. In this study, five different assembly programs, both traditional overlap-based, 'read-centric' assemblers and de Bruijn graph data structure-based assemblers, were compared. To this end, artificial read libraries with and without simulated sequencing errors were constructed from Arabidopsis thaliana, based on quantitative profiles of mature leaf tissue. The open source TGICL pipeline and the commercial CLC bio genomics workbench produced the best assemblies in terms of contig length, hybrid assemblies, redundancy reduction, and error tolerance. The mature leaf transcriptomes of the C(3) species Cleome spinosa and the C(4) species Cleome gynandra were assembled and analysed. The pathways and cellular processes tagged in the transcriptome assemblies reflect processes of a mature leaf. The databases are useful for extracting transcripts related to C(4) processes as full-length or nearly full-length sequences.

Independent and parallel recruitment of preexisting mechanisms underlying C4 photosynthesis.

C4 photosynthesis allows increased photosynthetic efficiency because carbon dioxide (CO2) is concentrated around the key enzyme RuBisCO. Leaves of C4 plants exhibit modified biochemistry, cell biology, and leaf development, but despite this complexity, C4 photosynthesis has evolved independently in at least 45 lineages of plants. We found that two independent lineages of C4 plant, whose last common ancestor predates the divergence of monocotyledons and dicotyledons about 180 million years ago, show conserved mechanisms controlling the expression of genes important for release of CO(2) around RuBisCO in bundle sheath (BS) cells. Orthologous genes from monocotyledonous and dicotyledonous C3 species also contained conserved regulatory elements that conferred BS specificity when placed into C4 species. We conclude that these conserved functional genetic elements likely facilitated the repeated evolution of C4 photosynthesis.

An mRNA blueprint for C4 photosynthesis derived from comparative transcriptomics of closely related C3 and C4 species.

C(4) photosynthesis involves alterations to the biochemistry, cell biology, and development of leaves. Together, these modifications increase the efficiency of photosynthesis, and despite the apparent complexity of the pathway, it has evolved at least 45 times independently within the angiosperms. To provide insight into the extent to which gene expression is altered between C(3) and C(4) leaves, and to identify candidates associated with the C(4) pathway, we used massively parallel mRNA sequencing of closely related C(3) (Cleome spinosa) and C(4) (Cleome gynandra) species. Gene annotation was facilitated by the phylogenetic proximity of Cleome and Arabidopsis (Arabidopsis thaliana). Up to 603 transcripts differ in abundance between these C(3) and C(4) leaves. These include 17 transcription factors, putative transport proteins, as well as genes that in Arabidopsis are implicated in chloroplast movement and expansion, plasmodesmatal connectivity, and cell wall modification. These are all characteristics known to alter in a C(4) leaf but that previously had remained undefined at the molecular level. We also document large shifts in overall transcription profiles for selected functional classes. Our approach defines the extent to which transcript abundance in these C(3) and C(4) leaves differs, provides a blueprint for the NAD-malic enzyme C(4) pathway operating in a dicotyledon, and furthermore identifies potential regulators. We anticipate that comparative transcriptomics of closely related species will provide deep insight into the evolution of other complex traits.