Architecture and evolution of a minute plant genome.

It has been argued that the evolution of plant genome size is principally unidirectional and increasing owing to the varied action of whole-genome duplications (WGDs) and mobile element proliferation. However, extreme genome size reductions have been reported in the angiosperm family tree. Here we report the sequence of the 82-megabase genome of the carnivorous bladderwort plant Utricularia gibba. Despite its tiny size, the U. gibba genome accommodates a typical number of genes for a plant, with the main difference from other plant genomes arising from a drastic reduction in non-genic DNA. Unexpectedly, we identified at least three rounds of WGD in U. gibba since common ancestry with tomato (Solanum) and grape (Vitis). The compressed architecture of the U. gibba genome indicates that a small fraction of intergenic DNA, with few or no active retrotransposons, is sufficient to regulate and integrate all the processes required for the development and reproduction of a complex organism.

Functional analysis of the Arabidopsis PLDZ2 promoter reveals an evolutionarily conserved low-Pi-responsive transcriptional enhancer element.

Plants have evolved a plethora of responses to cope with phosphate (Pi) deficiency, including the transcriptional activation of a large set of genes. Among Pi-responsive genes, the expression of the Arabidopsis phospholipase DZ2 (PLDZ2) is activated to participate in the degradation of phospholipids in roots in order to release Pi to support other cellular activities. A deletion analysis was performed to identify the regions determining the strength, tissue-specific expression, and Pi responsiveness of this regulatory region. This study also reports the identification and characterization of a transcriptional enhancer element that is present in the PLDZ2 promoter and able to confer Pi responsiveness to a minimal, inactive 35S promoter. This enhancer also shares the cytokinin and sucrose responsive properties observed for the intact PLDZ2 promoter. The EZ2 element contains two P1BS motifs, each of which is the DNA binding site of transcription factor PHR1. Mutation analysis showed that the P1BS motifs present in EZ2 are necessary but not sufficient for the enhancer function, revealing the importance of adjacent sequences. The structural organization of EZ2 is conserved in the orthologous genes of at least eight families of rosids, suggesting that architectural features such as the distance between the two P1BS motifs are also important for the regulatory properties of this enhancer element.

Dry but Not Humid Thermal Processing of Aloe vera Gel Promotes Cytotoxicity on Human Intestinal Cells HT-29.

Aloe vera products, both in food and cosmetics, are becoming increasingly popular due to their claimed beneficial effects, which are mainly attributed to the active compound acemannan. Usually, these end products are based on powdered starting materials. High temperatures during the drying process to obtain the starting materials have several advantages, like shortening the drying time, eliminating toxic aloin and reducing bacterial contamination. Nevertheless, there are two major drawbacks: first, at temperatures of 80 °C or higher, structural changes in acemannan, especially its deacetylation (>46%), are triggered, which does not happen at lower temperatures (14% at 60 °C); secondly, a toxic principle is formed at higher temperatures, resulting in a higher cytotoxicity. Thus, two temperature-dependent but opposing effects cause with a median cytotoxic concentration of CC50 = 0.4× a peak of cytotoxicity at 80 °C; at 60 °C this cytotoxic substance is not formed and at 100 °C aloin is more readily eliminated, resulting in a CC50 = 1.1× and CC50 = 1.4×, respectively. The cytotoxic substance generated by dry heat at 80 °C is not a modified polysaccharide because its polysaccharide-enriched alcohol-insoluble fraction is with CC50 = 0.9× less cytotoxic. Moreover, this substance is polar enough to be washed away with ethanol. Additionally, when Aloe gel is heated at 80 °C under humid conditions (pasteurization), the cytotoxicity does not increase (CC50 = 1.6×). Finally, to produce powdered starting materials from Aloe gel, it is recommended to use temperatures of around 60 °C in order to preserve the acemannan structure (and thus biological activity) and the low cytotoxicity.

Activation of the phenylpropanoid biosynthesis pathway reveals a novel action mechanism of the elicitor effect of chitosan on avocado fruit epicarp.

Secondary metabolites play an important role in the avocado fruit defense system. Phenolic compounds are the main biosynthesized metabolites of this system response. Our objective in this investigation was to evaluate the induction of specific metabolic pathways using chitosan as an elicitor. Extracts obtained from avocado in intermediate and consumption maturity stages treated with chitosan exhibited an increase in antifungal activity, which caused inhibition of mycelial growth and a decrease in sporulation as well as spore germination of Colletotrichum gloeosporioides. Additionally, RNA from epicarp of the fruits treated and untreated with chitosan was obtained in order to evaluate the expression of genes related to phenylpropanoids and the antifungal compound 1-acetoxy-2-hydroxy-4-oxo-heneicosa-12,15-diene biosynthesis. An increased in gene expression of genes that participates in the phenylpropanoids route was observed during the stage of physiological fruit maturity, others genes such as Flavonol synthase (Fls), increased only in samples obtained from fruit treated with chitosan at consumption maturity. Our results reveal a new molecular mechanism where chitosan induces a specific accumulation of phenylpropanoids and antifungal diene; this partially explains avocado's resistance against fungal pathogens. Finally, we discuss the molecular connections between chitosan induction and gene expression to explain the biological events that orchestrate the resistance pathways in fruits.

Novel signals for plant development.

Classic signal molecules such as auxin, cytokinin, gibberellins, abscisic acid and more recently brassinosteroids have been extensively studied in the context of their role in morphogenetic processes in plants. In the past five years, it has become apparent that there are novel signaling molecules, such as N-acylethanolamides, alkamides, glutamate and nitric oxide, that might play important roles in the regulation of morphogenetic and adaptive processes. There is information pointing out that these molecules might be involved in diverse processes, including seed germination, pathogenesis, modulation of plant architecture and response to abiotic factors. In animals, alkamides and N-acylethanolamides act as endogenous signaling molecules that activate cannabinoid receptors, which are coupled to signal transduction cascades involving glutamate and nitric oxide. Hence, there is a possibility that cannabinoid signaling represents an evolutionary conserved pathway that modulates cellular and physiological processes in eukaryotes.

In Vitro Bioactivity of Methanolic Extracts from Amphipterygium adstringens (Schltdl.) Schiede ex Standl., Chenopodium ambrosioides L., Cirsium mexicanum DC., Eryngium carlinae F. Delaroche, and Pithecellobium dulce (Roxb.) Benth. Used in Traditional Medicine in Mexico.

Seven out of eight methanolic extracts from five plants native to Mexico were inactive against ten bacterial strains of clinical interest. The fruit extract of Chenopodium ambrosioides inhibited the bacteria Enterococcus faecalis (MIC = 4375 µg/ml), Escherichia coli (MIC = 1094 µg/ml), and Salmonella typhimurium (MIC = 137 µg/ml). The fruit extract of C. ambrosioides was with CC50 = 45 µg/ml most cytotoxic against the cell-line Caco-2, followed by the leaf extract from Pithecellobium dulce (CC50 = 126 µg/ml); interestingly, leaves of C. ambrosioides (CC50 = 563 µg/ml) and bark of P. dulce (CC50 = 347 µg/ml) extracts were much less cytotoxic. We describe for the first time the cytotoxic effect from extracts of the aerial parts and the flowers of Cirsium mexicanum (CC50 = 323 µg/ml and CC50 = 250 µg/ml, resp.). Phytochemical analysis demonstrated for both extracts high tannin and saponin and low flavonoid content, while terpenoids were found in the flowers. For the first time we report a cytotoxicological study on an extract of Eryngium carlinae (CC50 = 356 µg/ml) and likewise the bark extract from Amphipterygium adstringens (CC50 = 342 µg/ml). In conclusion the fruit extract of C. ambrosioides is a potential candidate for further biological studies.

Use of inductors in the control of Colletotrichum gloeosporioides and Rhizopus stolonifer isolated from soursop fruits: in vitro tests.

Soursop (Annona muricata) is a tropical fruit that can be infected by Colletotrichum gloeosporioides and Rhizopus stolonifer. Traditional methods used for postharvest disease control include the application of fungicides, however due to their excessive use, as well as their persistence in the environment, the development of new strategies that control pathogens are required. The application of chitosan (Chi), salicylic acid (SA) and methyl jasmonate (MJ) is an environmentally-friendly alternative with antimicrobial properties and also induces defense mechanisms in plant tissues. In this study, Colletotrichum was reactivated and Rhizopus was identified using morphological features and molecular tools. In vitro, the application of 0.5 and 1.0% of Chi alone or in combination with SA and MJ decreased mycelial growth and sporulation, a complete inhibition of spore germination was obtained. Thus, the application of Chi in combination with SA and MJ could be a smart strategy to inhibit the development of pathogens that attack soursop fruit.

Structural relationships between diverse cis-acting elements are critical for the functional properties of a rbcS minimal light regulatory unit.

Light-regulation of photosynthesis-associated nuclear genes is mediated by multipartite cis-regulatory units located in their promoter regions. The combination, spacing, and relative orientation of transcription factor binding sites in these units influences the assembly of specific multiprotein complexes that control gene expression. In this work, the functional architecture of the conserved modular array 5 (CMA5), a previously characterized minimal light-regulatory unit of rbcS gene promoters, has been analysed. With the aim of defining the sequences of CMA5 that, besides the I- and G-box elements, are essential for CMA5 responsiveness to light and chloroplast-derived signals, a series of mutations affecting the sequences flanking these elements was performed. It was found that an I-box associated module, named IbAM5, is essential for CMA5 functionality and is able to bind nuclear proteins in vitro. The spacing requirements of the I- and G-box elements in achieving adequate combinatorial interaction were also studied as well as the effect of interchanging the relative position of these elements in the native rbcS promoter arrangement. The results show that helical phasing and distance between the I- and G-box motifs are critical to determine the functionality and transcriptional strength of CMA5. Furthermore, it is shown that the relative position of the IbAM5/I-box composite element and the G-box element is not critical for the enhancer activity of CMA5, as long as the proper distance between them is maintained. Taken together, these results suggest that the light-responsive, plastid-dependent activity of CMA5 requires the synergistic interaction of several DNA-binding transcription factors assembled in a higher-order nucleoprotein complex.

A specific variant of the PHR1 binding site is highly enriched in the Arabidopsis phosphate-responsive phospholipase DZ2 coexpression network.

PLDZ2 is a member of the Arabidopsis phospholipase D gene family that is induced in both shoot and root in response to phosphate (Pi) starvation. Recently, through deletion and gain-of-function analyses of the PLDZ2 promoter, we identified a 65 bp region (denominated enhancer EZ2) capable of conferring tissue-specific and low-Pi responses to a minimal inactive promoter. The EZ2 element contains two P1BS motifs, each of which is the binding site for PHR1 and related transcription factors. This structural organization is evolutionarily conserved in orthologous promoters within the rosid clade. To determine whether EZ2 is significantly over-represented in Arabidopsis genes coexpressed with PLDZ2, we constructed a PLDZ2 coexpression network containing 26 genes, almost half of them encoding enzymes or regulatory proteins involved in Pi recycling. A variant of the P1BS motif was found to be highly enriched in the promoter regions of these coexpressed genes, showing an EZ2-like arrangement in seven of them. No other motifs were significantly enriched. The over-representation of the EZ2 arrangement of P1BS motifs in the promoters of genes coexpressed with PLDZ2, suggests this unit has a particularly important role as a regulatory element in a coexpression network involved in the release of Pi from phospholipids and other molecules under Pi-limiting conditions.

Sugar and ABA responsiveness of a minimal RBCS light-responsive unit is mediated by direct binding of ABI4.

Photosynthesis-associated nuclear genes (PhANGs) are able to respond to multiple environmental and developmental signals, including light, sugars and abscisic acid (ABA). PhANGs have been extensively studied at the level of transcriptional regulation and several cis-acting elements important for light responsiveness have been identified in their promoter sequences. However, the regulatory elements involved in sugar and ABA regulation of PhANGs have not been completely characterized. Using conserved modular arrangement 5 (CMA5), a previously characterized minimal light-responsive unit, we show that in Arabidopsis thaliana this unit responds not only positively to light signals, but also negatively to sugars and ABA. The latter responses were found to be impaired in the abi4 mutant, indicating that ABSCISIC ACID INSENSITIVE-4 (ABI4) is a regulator involved in sugar and ABA repression of this minimal regulatory unit. Furthermore, we report a new sequence element conserved in several rbcS promoters, herewith named S-box, which is important for the sugar and ABA responsiveness of CMA5. This sequence corresponds to a putative ABI4-binding site, which is in fact bound by the Arabidopsis ABI4 protein in vitro. The S-box is closely associated with the G-box present in CMA5, and this association is conserved in the promoters of several RBCS genes. This phylogenetically conserved promoter feature probably reflects a common regulatory mechanism and identifies a point of convergence between light- and sugar-signaling pathways.