Association between vitamin content, plant morphology and geographical origin in a worldwide collection of the orphan crop Gynandropsis gynandra (Cleomaceae).

MAIN CONCLUSION: The variability in nutrient content and morphology in Gynandropsis gynandra is associated with the geographic origin of the accessions and provides a basis for breeding for higher levels of vitamin C, carotenoids or tocopherols in higher-yielding cultivars. We examined the variation in carotenoids, tocopherols and ascorbic acid as well as morphological traits in a worldwide germplasm of 76 accessions of the orphan leafy vegetable Gynandropsis gynandra (Cleomaceae) using greenhouse experiments and high-performance liquid chromatography analysis. The levels of carotenoids and tocopherols accumulating in the leaves varied significantly across accessions and were linked with the geographical origin and morphological variation. The main carotenoids included lutein, ß-carotene, α-carotene and violaxanthin. A twofold to threefold variation was observed for these compounds. The main tocopherols detected were α-tocopherol and γ-tocopherol with a 20-fold variation. A ninefold variation in vitamin C concentration and independent of geographical origin was observed. Overall, the accessions were grouped into three clusters based on variation in nutrient content and morphology. West African accessions were short plants with small leaves and with high tocopherol contents and relatively low carotenoid contents, Asian accessions were short plants with broad leaves and with relatively low carotenoid and high tocopherol contents, while East-Southern African plants were tall with high contents of both carotenoids and chlorophylls and low tocopherol contents. Carotenoids were positively correlated with plant height as well as foliar and floral traits but negatively correlated with tocopherols. The absence of a significant correlation between vitamin C and other traits indicated that breeding for high carotenoids or tocopherols content may be coupled with improved leaf yield and vitamin C content. Our study provides baseline information on the natural variation available for traits of interest for breeding for enhanced crop yield and nutrient content in Gynandropsis gynandra.

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.

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.

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.

Rendimento e composição do óleo essencial de Piper aduncum L. cultivado em Manaus, AM, em função da densidade de plantas e épocas de corte / Yield and composition of the essential oil from Piper aduncum L, cultivated in Manaus, AM, regarding plant density and cutting period

A pimenta-de-macaco (Piper aduncum L. - Piperaceae) é uma espécie usada na medicina popular em quase todo o Brasil e apresenta alto teor de óleo essencial (2,5 a 4,0%) rico em dilapiol - éter fenílico com ação inseticida. O presente trabalho teve como objetivo contribuir para o sistema de cultivo da espécie P. aduncum nas condições de Manaus - AM. O experimento foi realizado na área experimental da Embrapa Amazônia Ocidental. O delineamento utilizado foi o de blocos casualizados com quatro repetições, cada repetição com quatro plantas na área útil. Sete densidades de plantas (0,5 m x 0,5 m; 1,0 m x 0,5 m; 1,0 m x 1,0 m; 1,5 m x 1,0 m; 1,5 m x 1,5 m; 2,0 m x 1,5 m e 2,0 m x 2,0 m, E1 a E7, respectivamente) e três épocas de corte (C1 - 6 meses após transplantio; C2 - rebroto de C1 aos 6 meses - plantas com idade de 12 meses, no campo; C3 - rebroto de C2 aos 6 meses - plantas com 18 meses, no campo), portanto em esquema fatorial. Foi observado que o segundo corte (C2) apresentou maior produção, no espaçamento E4. Não houve diferença estatística para o teor de óleo essencial em C1, independente da densidade de plantas. Com relação ao rendimento (produção) em óleo essencial, o melhor foi observado com o corte C2, nos espaçamentos E3 e E4, com 76,0 kg/ha e 88 kg/ha, respectivamente, contendo até 85,6% de dilapiol no óleo. Assim, a melhor condição de cultivo nas condições de teste foi observada para o período de corte C2 e espaçamentos de 1,0 m x 1,0 m, e de 1,0 m x 1,5 m.

Pimenta-de-macaco (monkey pepper - Piper aduncum L. - Piperaceae) is a species used in folk medicine throughout Brazil, containing an essential oil (2.5 to 4.0% yield) rich in dillapiole, an arylpropanoid with insecticide action. The objective of this research was to improve the cultivation system of P. aduncum in local conditions of Manaus, AM. The study was conducted in the experimental fields of Embrapa Western Amazon, and used randomized blocks with four replicates, each replicate with four plants in its testing area. Seven plant densities (0.5 m x 0.5 m; 1.0 m x 0.5 m; 1.0 m x 1.0 m; 1.5 m x 1.0 m; 1.5 m x 1.5 m; 1.5 m 2.0 m; 2.0 m x 2.0 m, E1 to E7 respectively) and three cutting periods (C1 - 6 months after transplanting, C2 - regrowth of C1 after 6 months (plants with 12 months in the field) and C3 - regrowth of C2 after 6 months (plants with 18 months in the field), therefore in a factorial scheme. A larger production was observed in the second cutting (C2) in the plant density E4. There were no statistical differences in the essential oil content in C1, regardless of densities. In relation to the essential oil yield, the best result was observed for the cutting period C2 in the E3 and E4 densities, 76.0 kg/ha and 88 kg/ha, respectively, with a dillapiole content of up to 85.6%. Therefore, the best cultivation condition according to the test was observed for the cutting after 12 months of transplantation and plant density of 1.0 m x 1.0 m or 1.5 m x 1.5 m.

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.

Anthocyanin production in callus cultures of Cleome rosea: modulation by culture conditions and characterization of pigments by means of HPLC-DAD/ESIMS.

Leaf and stem explants of Cleome rosea formed calluses when cultured on MS medium supplemented with different concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D) or 4-amino-3,5,6-trichloropicolinic acid (PIC). The highest biomass accumulation was obtained in the callus cultures initiated from stem explants on medium supplemented with 0.90 microM 2,4-D. Reddish-pink regions were observed on callus surface after 6-7 months in culture and these pigments were identified as anthocyanins. Anthocyanins production was enhanced by reducing temperature and increasing light irradiation. Pigmented calluses transferred to MS1/2 with a 1:4 ratio NH(4)(+)/NO(3)(-), 70 g L(-1) sucrose and supplementation with 0.90 microM 2,4-D maintained a high biomass accumulation and showed an increase of 150% on anthocyanin production as compared with the initial culture conditions. Qualitative analysis of calluses was performed by high performance liquid chromatography coupled to diode array detector and electrospray ionization mass spectrometry (HPLC-DAD/ESIMS). Eleven anthocyanins were characterized and the majority of them were identified as acylated cyanidins, although two peonidins were also detected. The major peak was composed by two anthocyanins, whose proposed identity were cyanidin 3-(p-coumaroyl) diglucoside-5-glucoside and cyanidin 3-(feruloyl) diglucoside-5-glucoside.

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.