Virus-induced gene silencing is based on the sequence-specific degradation of RNA. Here, a gene silencing vector derived from EuMV-YP, named pEuMV-YP:ΔAV1, was used to silence ChlI and NPR1 genes in Nicotiana benthamiana. The silencing of the ChlI transcripts was efficient in the stems, petioles and leaves as reflected in tissue bleaching and reduced transcript levels. The silencing was stable, reaching the flowers and fruits, and was observed throughout the life cycle of the plants. Additionally, the silencing of the NPR1 gene was efficient in both N. benthamiana and Capsicum annuum. After silencing, the plants' viral symptoms increased to levels similar to those seen in wild-type plants. These results suggest that NPR1 plays a role in the compatible interactions of EuMV-YP N. benthamiana and EuMV-C. annum var. anaheim.

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).

The type-h thioredoxins (TRXs) play a fundamental role in oxidative stress tolerance and defense responses against pathogens. In pepper plants, type-h TRXs participate in the defense mechanism against Cucumber mosaic virus. The goal of this study was to analyze the role of the CaTRXh1-cicy gene in pepper plants during compatible interaction with a DNA virus, the Euphorbia mosaic virus-Yucatan Peninsula (EuMV-YP). The effects of a transient silencing of the CaTRXh1-cicy gene in pepper plants wëre evaluated by observing the accumulation of viral DNA and the visible symptoms of pepper plants under different treatments. The accumulation of salicylic acid (SA) and the relative expression of the defense genes NPR1 and PR10 were also evaluated. Results showed that viral DNA accumulation was higher in transiently CaTRXh1-cicy silenced plants that were also infected with EuMV-YP. Symptoms in these plants were more severe compared to the non-silenced plants infected with EuMV-YP. The SA levels in the EuMV-YP-infected plants were rapidly induced at 1 h post infection (hpi) in comparison to the non-silenced plants inoculated with EuMV-YP. Additionally, in pepper plants infected with EuMV-YP, the expression of NPR1 decreased by up to 41 and 58 % at 28 days post infection (dpi) compared to the non-silenced pepper plants infected with only EuMV-YP and healthy non-inoculated pepper plants, respectively. PR10 gene expression decreased by up to 70 % at 28 dpi. Overall, the results indicate that the CaTRXh1-cicy gene participates in defense mechanisms during the compatible interaction of pepper plants with the EuMV-YP DNA virus.

Introducción: Las bromelias son fuente de proteasas que no se han estudiado totalmente. Objetivos: Evaluar la actividad proteolítica de frutos silvestres de Bromelia karatas L. bajo distintas condiciones de temperatura, pH y NaCl, y estimar la estabilidad térmica de las proteasas. Materiales y métodos: Se comparó el efecto del pH (6, 7, 8, 9, 10 y 12), temperaturas de incubación (30, 40, 50, 60 y 70 °C) y concentraciones de NaCl (5, 10 y 20 %) sobre la actividad proteolítica de los frutos. La estabilidad térmica de las proteasas se evaluó a 30, 50 y 70 °C durante 240 minutos. Los pesos moleculares y los puntos isoeléctricos de las proteasas se estimaron por zimografía bidimensional no reductora. Los datos se analizaron mediante un ANOVA de un factor y la prueba de comparaciones múltiples de medias de Tukey (α = 0.05). Resultados y discusión: La actividad proteolítica del extracto de frutos de B. karatas (8.59 U·mg-1) fue mayor que la de Ananas comosus variedad piña miel (3.42 U·mg-1). La actividad fue mayor en valores de pH 6 y 7 y en concentraciones menores de 5 % de NaCl. Las proteasas se mantuvieron estables a 30 y 50 °C durante 210 minutos. El zimograma bidimensional, bajo condiciones no reductoras, mostró por lo menos 40 zonas claras con pesos moleculares aparentes entre 27.3 y 290 kDa, que representan potencialmente proteasas. Conclusión: Las proteasas de B. karatas tienen potencial de aplicación en la industria alimentaria.

Microalgae have been positioned as excellent models for producing new sources of energy (biofuels and biohydrogen). Some investigations in these biological models have been directed to know if the enzymes ferredoxin (FDX) and hydrogenase (HYD) are involved in the algae producing different concentrations of molecular hydrogen (H 2 ). To date, little is known about the concomitant transcriptional regulation of both enzymes during H 2 evolution in algae. In this research, we evaluated the relative expression of hdy and fdx genes during the evolution of H 2 in three microalgae (Chlorella vulgaris, Scenedesmus obliquus, and Chlamydomonas reinhardtii) in N-deprived anaerobic cultures in the presence of Fe, and 12:24 and 24:24 h dark:light cycles. We also detected structural differences in the enzymes. The 3D modeling indicated that the 3D structure of HYD and FDX are conserved in most algal genera, and the results of our grouping according to the aa characteristics of the proteins showed two grouping trends: One, according to the algae's phylogenetic classification, and another one according to the species-specific enzyme's characteristics, and the grouping could perhaps be more influenced by the algae's ability to produce H 2 . The three microalgae species reached maximum H 2 accumulation values in 24h:24 h dark:light conditions in Fe-supplemented media (4.2 AE 0.12 mL L À1 in C. vulgaris, 3.9 AE 0.10 mL L À1 in S. obliquus, and 4.5 AE 0.10 mL L À1 in C. reinhardtii), and the highest global relative expression of hyd and fdx genes was reached during the first hour of exposure to light, which suggests concomitant expression of both enzymes at the beginning of H 2 production. The behavior of the expression of the hyd and fdx genes in these algal species proved to be similar between species. A better understanding of the concomitant regulation of both enzymes could lay the groundwork for the future use of both enzymes to improve H 2 yields in microalgae.