Effects of exogenous manganese on its plant growth, subcellular distribution, chemical forms, physiological and biochemical traits in Cleome viscosa L.

Cleome viscosa L. is a promising species for the phytoremediation of Mn-contaminanted soil. To reveal the adaptive mechanisms of species to Mn stress, plant growth, Mn subcellular distribution, Mn chemical forms, and plant physiological and biochemical traits were characterized in plants grown under different concentrations of Mn2+ (0, 1000, 5000, 10000, 15000 and 20000 µM). The results showed that C. viscosa plant biomass initially increased and then decreased with rising Mn treatment concentration. C. viscosa plants can accumulate high levels of Mn in roots and leaves, and both the bioconcentration factor (BCF) and the translocation factor (TF) exhibited values higher than one. Mn was primarily retained in the cell wall and soluble fractions. Predominant chemical forms of Mn were pectate and protein, phosphates, and oxalates-integrated Mn. The activities of antioxidant enzymes, including superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), and the contents of proline, soluble sugar, and soluble protein initially increased and then decreased with enhancing Mn treatment concentration, whereas the malondialdehyde (MDA) content simultaneously displayed a gradual increase. Combined, these results indicate that C. viscosa can tolerate Mn-stress conditions by increasing antioxidant enzyme activities and non-enzymatic metabolites contents. In addition, Mn immobilization in the cell wall and soluble fractions, alongside the storage of Mn in low-activity chemical forms are further important mechanisms to cope with high environmental Mn concentration. This study reveals the adaptive mechanisms of plants to Mn stress, and provides a theoretical basis for the use of C. viscosa as a candidate phytoremediation plant for Mn-contaminated soil.

Nutritional compound analysis and morphological characterization of spider plant (Cleome gynandra) - an African indigenous leafy vegetable.

Spider plant is among the important indigenous African leafy vegetables having the potential to contribute to food and nutritional security in sub-Saharan Africa. The main objective of this study was to quantify the mineral concentration, to identify and quantify glucosinolates and flavonoids in spider plant and further to characterize spider plant entries using important morphological traits. Thirty spider plant entries from different African countries, comprising of farmers' cultivars, gene bank accessions and advanced lines were grown in a field experiment and harvested for leaves, stems, flowers and siliques at different developmental stages. Five plant types based on the stem and petiole colorations were identified. Significant genotypic differences were shown for all the morphological traits except for 100 seed weight and silique weight. High mineral concentrations in the leaf tissue were observed especially for potassium, calcium, magnesium, phosphorus, iron, manganese and zinc. The aliphatic 3-hydroxypropyl glucosinolate was the main glucosinolate detected in all tissues with the highest concentrations in the reproductive organs. Glycosides of quercetin, kaempferol and isorhamnetin were the main flavonoids. Isorhamnetin glycosides were detected in trace amounts in both, leaves and inflorescences, while quercetin and kaempferol glycosides were the dominant flavonoids in the leaves and inflorescences, respectively. This knowledge of beneficial nutrient contents is an incentive for promoting spider plant consumption for improved human health while the morphological diversity analysis will be important for the further development of the spider plant germplasm.

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.

Intra-plant floral variation in Cleome viscosa L. and its possible significance in breeding system.

Cleome viscosa L., an annual rainy season weed, is cosmopolitan in distribution. Two naturally growing populations of C. viscosa from Jammu, J & K, India have been studied for floral variation at an intra-plant level and its possible role in its life cycle. Plants of both the populations bear flowers which exhibit tremendous intra-plant variation in size (large and small) and sex (hermaphrodite, staminate and pistillate). The average number of flowers per plant varied significantly and so did their structural and functional details. Greater propensity, however, was towards hermaphroditism at both plant and flower levels. The large and small sized flowers differed in their morphology and reproductive features; the former were significantly larger than the latter. Anthesis, anther dehiscence and stigma receptivity were coupled in all flower types. This functional aspect along with the structural proximity between stamens at two lengths and pistil further facilitated self-pollination. However, conspicuous floral display attracted diverse pollinator fauna (Apis dorsata, Halictus albescens, Nomia curvipes and N. elliotii) which in turn mediated cross pollination. Nevertheless, each floral type contributed towards plant's fitness in its own way. Hermaphrodite flowers exhibited both self and cross pollination and assured survival by setting fruits and seeds with the large sized counterparts more productive. All these floral variations seemed to impart flexibility to the pollination system and provide fitness over the short flowering season.

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.

Differentiation of C4 photosynthesis along a leaf developmental gradient in two Cleome species having different forms of Kranz anatomy.

In family Cleomaceae there are NAD-malic enzyme-type C4 species having different forms of leaf anatomy. Leaves of Cleome angustifolia have Glossocardioid-type anatomy with a single complex Kranz unit which surrounds all the veins, while C. gynandra has Atriplicoid anatomy with multiple Kranz units, each surrounding an individual vein. Biochemical and ultrastructural differentiation of mesophyll (M) and bundle sheath (BS) cells were studied along a developmental gradient, from the leaf base (youngest) to the tip (mature). Initially, there is cell-specific expression of certain photosynthetic enzymes, which subsequently increase along with structural differentiation. At the base of the leaf, following division of ground tissue to form M and BS cells which are structurally similar, there is selective localization of Rubisco and glycine decarboxylase to BS cells. Thus, a biochemical C3 default stage, with Rubisco expression in both cell types, does not occur. Additionally, phosphoenolpyruvate carboxylase (PEPC) is selectively expressed in M cells near the base. Surprisingly, in both species, an additional layer of spongy M cells on the abaxial side of the leaf has the same differentiation with PEPC, even though it is not in contact with BS cells. During development along the longitudinal gradient there is structural differentiation of the cells, chloroplasts, and mitochondria, resulting in complete formation of Kranz anatomy. In both species, development of the C4 system occurs similarly, irrespective of having very different types of Kranz anatomy, different ontogenetic origins of BS and M, and independent evolutionary origins of C4 photosynthesis.

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.

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.

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.

Cleome, a genus closely related to Arabidopsis, contains species spanning a developmental progression from C(3) to C(4) photosynthesis.

C(4) photosynthesis involves alterations to leaf development, cell biology and biochemistry. Different lineages of C(4) plants use varying mechanisms to generate the C(4) pathway. Although the biochemistry of C(4) photosynthesis was described around 20 years ago, the phylogenetic distance between Arabidopsis and the traditional C(4) models has not facilitated the transfer of knowledge from Arabidopsis research to understanding C(4) systems. We show that Cleome, a genus closely related to Arabidopsis, contains species spanning a developmental progression from C(3) to C(4) photosynthesis. The majority of species we assessed are C(3) plants but have increased venation in leaves. Three C(3) species have both increased venation and enlarged bundle sheath cells, and there is also a tendency to accumulate proteins and transcripts needed for C(4) photosynthesis. Cleome gynandra shows all the characteristics needed for efficient C(4) photosynthesis, including alterations to leaf biochemistry, cell biology and development, and belongs to the NAD-dependent malic enzyme subtype. Combined with its phylogenetic proximity to Arabidopsis, the developmental progression from C(3) to C(4) photosynthesis within the genus provides a potentially excellent new model to increase our understanding of C(4) photosynthesis, and provide insights into its evolution.

Biological activities of an extract from Cleome viscosa L. (Capparaceae).

Electron micrograph examination of the leaf and stem surfaces of Cleome viscosa L (Family Capparaceae) revealed the presence of secretory glandular trichomes with club-cylinder and cylinder morphologies. In the present study, the leaves and stems of C. viscosa were extracted with hexane and the extract was evaluated for the following biological activities: anti-bacterial, anti-fungal, contact insecticidal and nematicidal. The extract was found to be a potent anti-bacterial agent according to the thin layer chromatography autobiographic assay. Activity-directed isolation studies of the anti-bacterially active compounds led to a 14-member ring cembranoid diterpene being identified as one of the effective agents. Minimum inhibitory concentration (MIC) values (microg/spot) of 5.0 microg/spot and 1.0 microg/spot were found for the diterpene on Bacillus subtilis (Gram-positive) and Pseudomonas fluorescens (Gram-negative), respectively. The diterpene did not inhibit the growth of the fungus Cladosporium cucumerinum. The extract demonstrated a pyrethroid type of contact insecticidal activity on adult Cylas formicarius elegantulus Summer (Coleoptera: Curculionidae). The extract also had high nematicidal activity with a percentage Abbott's value of 72.69 on the plant parasitic nematode Meloidogyne incognita Chitwood; however, the extract lost its potency upon subfractionation.