Respiratory and C4-photosynthetic NAD-malic enzyme coexist in bundle sheath cell mitochondria and evolved via association of differentially adapted subunits.

In plant mitochondria, nicotinamide adenine dinucleotide-malic enzyme (NAD-ME) has a housekeeping function in malate respiration. In different plant lineages, NAD-ME was independently co-opted in C4 photosynthesis. In the C4 Cleome species, Gynandropsis gynandra and Cleome angustifolia, all NAD-ME genes (NAD-MEα, NAD-MEß1, and NAD-MEß2) were affected by C4 evolution and are expressed at higher levels than their orthologs in the C3 species Tarenaya hassleriana. In T. hassleriana, the NAD-ME housekeeping function is performed by two heteromers, NAD-MEα/ß1 and NAD-MEα/ß2, with similar biochemical properties. In both C4 species, this role is restricted to NAD-MEα/ß2. In the C4 species, NAD-MEα/ß1 is exclusively present in the leaves, where it accounts for most of the enzymatic activity. Gynandropsis gynandra NAD-MEα/ß1 (GgNAD-MEα/ß1) exhibits high catalytic efficiency and is differentially activated by the C4 intermediate aspartate, confirming its role as the C4-decarboxylase. During C4 evolution, NAD-MEß1 lost its catalytic activity; its contribution to the enzymatic activity results from a stabilizing effect on the associated α-subunit and the acquisition of regulatory properties. We conclude that in bundle sheath cell mitochondria of C4 species, the functions of NAD-ME as C4 photosynthetic decarboxylase and as a housekeeping enzyme coexist and are performed by isoforms that combine the same α-subunit with differentially adapted ß-subunits.

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.

Unravelling triterpene biosynthesis through functional characterization of an oxidosqualene cyclase (OSC) from Cleome arabica L.

Cleome arabica is a medicinal plant contains diverse bioactive compounds and terpenoids are the major components. However, the isolation and purification of the active triterpenes from this plant involve long and complicated procedures. The present work investigates the triterpenes profiles of different tissues, besides that, describes the isolation, heterologous expression and functional characterization of C. arabica gene coding for triterpenes synthases. The phytochemical investigation through GC-MS revealed significant accumulation of pentacyclic triterpenes in leaves and siliques at mature stage compared to the stems and roots of C. arabica. Among the pentacyclic triterpenes, the lupeol reached the highest level of 320 µg/g DW in leaves at maturity stage compared to the other tissues. The biosynthesis of a pentacyclic triterpene was investigated through isolation and cloning of a full-length oxidosqualene cyclase cDNA (CaOSC) from mature leaves of C. arabica. The bioinformatic analyses revealed that CaOSC was highly homologous with the characterized lupeol synthases and shared 79.3% identity to camelliol C synthase from A. thaliana. Heterologous expression of CaOSC gene in Saccharomyces cerevisiae synthesized lupeol as a single product. The lupeol biosynthesis was exponentially increased after induction through the fermentation process reaching the maximum of 2.33 µg/ml for 240 h. Furthermore, organ-specific expression of lupeol gene was exactly matched the accumulation pattern in different tissues of C. arabica during phenological cycle. Thus, the identified CaOSC will be useful in enhancing triterpene yield for industrial purposes.

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.

An assessment of the capacity for phosphoenolpyruvate carboxykinase to contribute to C4 photosynthesis.

Three C4 acid decarboxylases, phosphoenolpyruvate carboxykinase (PEPCK), NADP-malic enzyme (NADP-ME), and NAD-malic enzyme (NAD-ME) were recruited from C3 plants to support C4 photosynthesis. In Poaceae, there are established lineages having PEPCK type species, and some NADP-ME lineages in which PEPCK contributes to C4. Besides family Poaceae, recently PEPCK has been reported to function in C4 photosynthesis in eudicot species including Cleome gynandra (Cleomaceae), Trianthema portulacastrum and Zaleya pentandra (Aizoaceae). We evaluated PEPCK by enzyme assay and western blots in representatives of Poaceae, Aizoaceae, Cleomaceae, and Chenopodiaceae compared to that in the PEPCK type C4 grass Spartina anglica. Eragrostis nutans was identified as the first NAD-ME type C4 grass having substantial amounts of PEPCK. In the eudicots, including C. gynandra, Cleome angustifolia, T. portulacastrum, Z. pentandra, and nine C4 members of family Chenopodiaceae (which has the most C4 species and diversity in forms among eudicot families), amounts of PEPCK were generally very low (barely detectable up to 4% of that in S. anglica). Based on these results, C4 species can be classified biochemically according to the dominant decarboxylase recruited for C4 function; and, Poaceae remains the only family in which PEPCK is known to have a significant role in C4 photosynthesis.

Coexistence but independent biosynthesis of catechyl and guaiacyl/syringyl lignin polymers in seed coats.

Lignins are phenylpropanoid polymers, derived from monolignols, commonly found in terrestrial plant secondary cell walls. We recently reported evidence of an unanticipated catechyl lignin homopolymer (C lignin) derived solely from caffeyl alcohol in the seed coats of several monocot and dicot plants. We previously identified plant seeds that possessed either C lignin or traditional guaiacyl/syringyl (G/S) lignins, but not both. Here, we identified several dicot plants (Euphorbiaceae and Cleomaceae) that produce C lignin together with traditional G/S lignins in their seed coats. Solution-state NMR analyses, along with an in vitro lignin polymerization study, determined that there is, however, no copolymerization detectable (i.e., that the synthesis and polymerization of caffeyl alcohol and conventional monolignols in vivo is spatially and/or temporally separated). In particular, the deposition of G and C lignins in Cleome hassleriana seed coats is developmentally regulated during seed maturation; C lignin appears successively after G lignin within the same testa layers, concurrently with apparent loss of the functionality of O-methyltransferases, which are key enzymes for the conversion of C to G lignin precursors. This study exemplifies the flexible biosynthesis of different types of lignin polymers in plants dictated by substantial, but poorly understood, control of monomer supply by the cells.

The dicotyledonous NAD malic enzyme C4 plant Cleome gynandra displays age-dependent plasticity of C4 decarboxylation biochemistry.

The C(4) photosynthetic pathway enriches carbon dioxide in the vicinity of Rubisco, thereby enabling plants to assimilate carbon more efficiently. Three canonical subtypes of C(4) exist, named after their main decarboxylating enzymes: NAD-dependent malic enzyme type, NADP-dependent malic enzyme type and phosphoenolpyruvate carboxykinase type. Cleome gynandra is known to perform NAD-ME type C(4) photosynthesis. To further assess the mode of C(4) in C. gynandra and its manifestation in leaves of different age, total enzyme activities of eight C(4) -related enzymes and the relative abundance of 31 metabolites were measured. C. spinosa was used as a C(3) control. C. gynandra was confirmed as an NAD-ME type C(4) plant in mid-aged leaves, whereas a mixed NAD-ME and PEPCK type was observed in older leaves. Young leaves showed a C(3) -C(4) intermediate state with respect to enzyme activities and metabolite abundances. Comparative transcriptome analysis of mid-aged leaves of C. gynandra and C. spinosa showed that the transcript of only one aspartate aminotransferase (AspAT) isoform is highly abundant in C. gynandra. However, the canonical model of the NAD-ME pathway requires two AspATs, a mitochondrial and a cytosolic isoform. Surprisingly, our results indicate the existence of only one highly abundant AspAT isoform. Using GFP-fusion, this isozyme was localised exclusively to mitochondria. We propose a revised model of NAD-ME type C(4) photosynthesis in C. gynandra, in which both AspAT catalysed reactions take place in mitochondria and PEPCK catalyses an alternative decarboxylating pathway.

Diversity in forms of C4 in the genus Cleome (Cleomaceae).

BACKGROUND AND AIMS: Cleomaceae is one of 19 angiosperm families in which C(4) photosynthesis has been reported. The aim of the study was to determine the type, and diversity, of structural and functional forms of C(4) in genus Cleome. Methods Plants of Cleome species were grown from seeds, and leaves were subjected to carbon isotope analysis, light and scanning electron microscopy, western blot analysis of proteins, and in situ immunolocalization for ribulose bisphosphate carboxylase oxygenase (Rubisco) and phosphoenolpyruvate carboxylase (PEPC). KEY RESULTS: Three species with C(4)-type carbon isotope values occurring in separate lineages in the genus (Cleome angustifolia, C. gynandra and C. oxalidea) were shown to have features of C(4) photosynthesis in leaves and cotyledons. Immunolocalization studies show that PEPC is localized in mesophyll (M) cells and Rubisco is selectively localized in bundle sheath (BS) cells in leaves and cotyledons, characteristic of species with Kranz anatomy. Analyses of leaves for key photosynthetic enzymes show they have high expression of markers for the C(4) cycle (compared with the C(3)-C(4) intermediate C. paradoxa and the C(3) species C. africana). All three are biochemically NAD-malic enzyme sub-type, with higher granal development in BS than in M chloroplasts, characteristic of this biochemical sub-type. Cleome gynandra and C. oxalidea have atriplicoid-type Kranz anatomy with multiple simple Kranz units around individual veins. However, C. angustifolia anatomy is represented by a double layer of concentric chlorenchyma forming a single compound Kranz unit by surrounding all the vascular bundles and water storage cells. CONCLUSIONS: NAD-malic enzyme-type C(4) photosynthesis evolved multiple times in the family Cleomaceae, twice with atriplicoid-type anatomy in compound leaves having flat, broad leaflets in the pantropical species C. gynandra and the Australian species C. oxalidea, and once by forming a single Kranz unit in compound leaves with semi-terete leaflets in the African species C. angustifolia. The leaf morphology of C. angustifolia, which is similar to that of the sister, C(3)-C(4) intermediate African species C. paradoxa, suggests adaptation of this lineage to arid environments, which is supported by biogeographical information.