Cloning of the Coffea canephora SERK1 promoter and its molecular analysis during the cell-to-embryo transition

Background: Somatic embryogenesis receptor-like kinase 1 (SERK1) is a cell membrane receptor active in different plant tissues and involved in cell differentiation activities including somatic embryogenesis. The identification of promoter elements responsible for SERK1 expression during the onset of somatic embryogenesis can be useful to understand the molecular regulation of the cell-to embryo transition, and these promoter elements represent biotechnological tools in plant organ tissue culture. Results: A −1,620 bp DNA sequence located upstream of the Coffea canephora SERK1 gene homologue (CcSERK1) was isolated, and then, different segments containing key response elements (REs) for somatic embryogenesis onset and development were fused to the uidA (encoding a ß-glucuronidase, GUS) reporter gene to evaluate its expression in transgenic leaf explants. DNA segments of −1,620 and −1048 bp in length directed uidA expression with patterns in leaf explants similar to those occurring during somatic embryogenesis. When a −792-bp fragment was used, uidA expression disappeared only in leaf explants and pro-embryogenic mass but persisted in developing embryos. No uidA expression was detected in any embryogenic stage when a −618-bp fragment was used. Conclusion: DNA deletions showed that a −1048-bp sequence located upstream of the CcSERK1 gene is sufficient to direct gene expression during the onset and the development of C. canephora somatic embryogenesis. The DNA segment located between −1048 and −792 bp (containing BBM and WUS REs) is needed for gene expression before embryogenesis onset but not during embryo development. The promoter segment between −792 and −618 bp (including GATA, ARR1AT, and ANT REs) regulates gene expression in developing embryos.

Genetic Transformation of Pentalinon andrieuxii Tissue Cultures.

Pentalinon andrieuxii is a species used in Mayan traditional medicine due to its biological properties. Recent studies indicate that it produces a pentacyclic triterpene-denominated betulinic acid, which presents various biological activities: antibacterial, antifungal, antiplasmodial, anti-inflammatory, antimalarial, anticancer, leishmanicidal, and antiviral, as well as steroids and sterols with leishmanicidal properties. A recent study also reported the presence of urechitol A and B in the roots; these are secondary metabolites whose biochemical function is as yet unknown. This plant therefore represents a natural source of metabolites with potential application in the pharmaceutical industry. In this chapter, a protocol is described for obtaining transgenic plants, at the reporter gene of the ß-glucuronidase (GUS) via Agrobacterium tumefaciens from hypocotyl and root explants. The protocol established herein could be employed for the manipulation of the genes involved in the biosynthesis of isoprenoids or secondary metabolites of interest. To our knowledge, this is the first report of stable transformation of Pentalinon andrieuxii via Agrobacterium tumefaciens.

Ectopic expression of the Coffea canephora SERK1 homolog-induced differential transcription of genes involved in auxin metabolism and in the developmental control of embryogenesis.

Somatic embryogenesis receptor-like kinase 1 (SERK1) is a membrane receptor that might serve as common co-regulator of plant cell differentiation processes by forming heterodimers with specific receptor-like kinases. The Coffea canephora SERK1 homolog (CcSERK1) was cloned in this work, and its early function in the transcription of embryogenesis master genes and of genes encoding proteins involved in auxin metabolism was investigated by externally manipulating its expression in embryogenic leaf explants, before the appearance of embryogenic structures. Overexpression of CcSERK1 early during embryogenesis caused an increase in the number of somatic embryos when the 55-day process was completed. Suppression of CcSERK1 expression by RNA interference almost abolished somatic embryogenesis. Real time-PCR experiments revealed that the transcription of the CcAGL15, CcWUS, CcBBM, CcPKL, CcYUC1, CcPIN1 and CcPIN4 homologs was modified in direct proportion to the expression of CcSERK1 and that only CcLEC1 was inversely affected by the expression levels of CcSERK1. The expression of the CcYUC4 homolog was induced to more than 80-fold under CcSERK1 overexpression conditions, but it was also induced when CcSERK1 expression was silenced. The level of CcTIR1 was not affected by CcSERK1 overexpression but was almost abolished during CcSERK1 silencing. These results suggest that CcSERK1 co-regulates the induction of somatic embryogenesis in Coffea canephora by early activation of YUC-dependent auxin biosynthesis, auxin transport mediated by PIN1 and PIN4, and probably auxin perception by the TIR1 receptor, leading to the induction of early-stage homeotic genes (CcAGL15, CcWUS, CcPKL and CcBBM) and repression of late-stage homeotic genes (CcLec1).

Achiote (Bixa orellana L.): a natural source of pigment and vitamin E.

Commercialization of agricultural products, including seeds and its derived products, represents an important economic source for developing countries. Natural colorants obtained from the seeds of achiote plant (annatto) have been used since pre-Hispanic times. Also, production of this crop has been important for Mayan cuisine. Annual world production of achiote seeds is approximately 14,500 tons (dry weight). Two thirds of the production is commercialized as dried seeds and the rest as colorant. Latin America produces 60% of the total world production, followed by Africa (27%) and Asia (12%). The main producers in Latin America are Peru, Brazil and Mexico. The purpose of the present paper is to review the most recent literature on Bixa orellana L. focusing on bixin, norbixin, tocotrienols and tocopherols biosynthesis, use and industrial applications of annatto extracts, as well as its nutraceutical potential and its benefits for human health.

Isotopologue profiling of triterpene formation under physiological conditions. Biosynthesis of lupeol-3-(3'-R-hydroxy)-stearate in Pentalinon andrieuxii.

The biosynthesis of lupeol-3-(3'R-hydroxy)-stearate (procrim b, 1) was investigated in the Mexican medicinal plant Pentalinon andrieuxii by (13)CO2 pulse-chase experiments. NMR analyses revealed positional enrichments of (13)C2-isotopologues in both the triterpenoid and the hydroxystearate moieties of 1. Five of the six isoprene units reflected a pattern with [1,2-(13)C2]- and [3,5-(13)C2]-isotopologues from the respective C5-precursors, IPP and DMAPP, whereas one isoprene unit in the ring E of 1 showed only the [3,5-(13)C2]-connectivity of the original C5-precursor, due to rearrangement of the dammarenyl cation intermediate during the cyclization process. The presence of (13)C2-isotopologues was indicative of [(13)C2]acetyl-CoA being the precursor units in the formation of the fatty acid moiety and of the triterpene via the mevalonate route. The observed labeling pattern was in agreement with a chair-chair-chair-boat conformation of the (S)-2,3-oxidosqualene precursor during the cyclization process, suggesting that the lupeol synthase from P. andrieuxii is of the same type as that from Olea europea and Taraxacum officinale, but different from that of Arabidopsis thaliana. The study shows that (13)CO2 pulse-chase experiments are powerful in elucidating, under in vivo conditions and in a single experiment, the biosynthesis of complex plant products including higher terpenes.

Growth measurements: estimation of cell division and cell expansion.

The main parameters for the estimation of growth within in vitro cultures are reviewed. Procedures to measure these parameters are described, emphasizing in each case their convenience of use, depending on the features of the culture evaluated.

Endogenous GUS-like activity in Capsicum chinense Jacq

The gene uidA, codes for beta-glucuronidase, which is one of the reporters more frequently utilized in transgenic plants. However, this can only be use if the selected organism does not present endogenous GUS-like activity. In tissues of C. chinense we found a GUS-like activity showing different levels of intensity. Histochemical screening showed that endogenous GUS-like activity decreased, or reduced significantly, in almost all tissues with exception of stament, when phosphate buffer was adjusted to pH 8. Subsequently, C. chinense zygotic embryo explants were transient transformed with Agrobacterium tumefaciens LBA4404 (pCAMBIA2301) and plantlets regenerated were histochemically stained in phosphate buffer pH 8. Observations of incubated tissues of C. chinense regenerants showed blue staining, suggesting expression of uidA. Incubated tissues of non-transformed regenerants did not show blue staining in phosphate buffer pH 8. The results show that for transformation experiments of C. chinense with uidA gene, pH 8 is recommended for histochemical staining.

Agrobacterium tumefaciens-transient genetic transformation of Habanero pepper (Capsicum chinense Jacq. ) leaf explants

Most of the pepper species of the genus Capsicum have been recalcitrant to efficient Agrobacterium tumefaciens-mediated stable or transient, genetic transformation. In the present work, we optimized a protocol for transient transformation of the Habanero pepper (Capsicum chinense Jacq.) through the standardization of several experimental factors. These included the age of the plants, the temperature, the length of co-cultivation, the application of a negative (vacuum) and/or a positive (infiltration) pressure, along with micro injection, the use of acetosyringone during the bacterial culturing, and modification of the pH during the GUS assay to eliminate the endogenous beta-glucuronidase activity. The standardized protocol, which yielded nearly 55 percent fully transformed leaf explants, was used to successfully mobilize two empty binary vectors (pCAMBIA2301 and pCAMex), as well as the C. chinense cDNAs encoding the pathogenesis-related protein 10 and esterase, respectively.

Agrobacterium-mediated transient transformation of Mexican prickly poppy (Argemone mexicana L)

Shoot apex, leaf primordia, leaf sections and roots from Mexican prickly poppy seedlings, were inoculated with Agrobacterium tumefaciens harboring the binary vector pCAMBIA2301, which contained the beta-glucuronidase (uid A) gene. Histochemical beta-glucuronidase (GUS) assay in infected explants showed transient gus gene expression between 3 and 12 days after inoculation. To our knowledge, this is the first report of A. mexicana susceptibility to A. tumefaciens-mediated genetic transformation.

Growth measurements: estimation of cell division and cell expansion.

The main parameters for the estimation of growth within in vitro cultures are reviewed. Procedures to measure these parameters are described, emphasizing in each case their convenience of use, depending on the features of the culture to evaluate.

Effect of acetylsalicylic acid on secondary metabolism ofCatharanthus roseus tumor suspension cultures.

Addition of various concentrations (0.5-20 mM) of acetylsalicylic acid (ASA) to tumor lines ofCatharanthus roseus cultivatedin vitro and requiring corn starch as carbon source, produced remarkable effects on secondary metabolite production. An increase of 505% total alkaloids per culture (cells plus liquid medium), 1587% total phenolics (liquid medium), 612% total furanocoumarins (liquid medium) and 1476% total anthocyanins (liquid medium) was detected. 1 mM ASA in combination with other elicitors, such as homogenates ofAspergillus fumigatus or trans-cinnamic acid, did not further increase the metabolite content substantially. The results suggest that ASA could act as a new biotic elicitor of metabolite production inC. roseus cell suspension culture.