Impact of altered cell wall composition on saccharification efficiency in stem tissue of Arabidopsis RABA GTPase-deficient knockout mutants

Use of biomass for second-generation biofuel production is severely hindered by the inherent recalcitrance of the plant cell wall to digestion. Trafficking is crucial for compartmentalisation within the cell. This process is partly regulated by small Rab GTPase proteins. In particular, control of trafficking to the cell wall is regulated through the RABA clade. Manipulation of this regulatory system offers tantalising opportunities for manipulation of cell wall composition and hence recalcitrance. Trafficking-defective rabA mutants have already been shown to impact cell wall composition. To study the impacts of these mutants on cell wall digestion, we developed a saccharification process for Arabidopsis based on the hot water method. We then showed that following pre-treatment, stems from the T-DNA knockouts of the three RABA4 genes expressed in Arabidopsis stem show an increased sugar release on saccharification. These rabA4 mutants have been shown to impact the “hemicellulose-rich” fraction during cell wall fractionation. Furthermore, we go on to show that these mutant lines also show increased sugar release when subjected to saccharification without pre-treatment. Finally, we used X-ray diffraction to show that rabA4 mutants had no impact on cellulose crystallinity, thus supporting the hypothesis that the increases in saccharification were not due to alterations of the cellulose microfibrils but were a direct effect of reduced hemicellulose levels. We also present data to show that the growth characteristics of these plants were unaffected. The data obtained from these lines are most easily explained by the reported alteration in hemicellulose increasing pre-treatment efficiency

Concertacion y seguridad social : de la legitimidad social a la legitimidad tecnocrática

El estudio aborda la concertación, un análisis de las políticas de seguridad social constatando su evolución desde una legitimidad social a una legitimidad tecnocrática en la crisis del welfare state y en la crisis económica con particular referencia al sistema español. Desde la normalización de la acción política de los sindicatos y agentes sociales en sus diversas manifestaciones, como la participación institucional y la "legislación negociada" en la búsqueda del consenso y el negociado de intercambios en el marco político frente a la imposición unilateral. La frustración de la concertación social se produce cuando el gobierno interpreta la democracia como algo puramente aritmético de mayorías que ejercen sin más el poder, en épocas de debilidad sindical, de debilidad del estado nación, de dificultades para las políticas redistributivas, de reforzamiento de los planteamientos neoliberales. La búsqueda de legitimidad en el pacto social no se ve en estas ocasiones como necesaria sino que la legitimidad se traslada al discurso economicista y tecnocrático, no político. Se vuelve a la búsqueda de una legitimidad a través del mercado y del intercambio entre particulares.L'estudi aborda la concertació, una anàlisi de les polítiques de seguretat social constatant la seva evolució des d'una legitimitat social a una legitimitat tecnocràtica en la crisi del welfare state i en la crisi econòmica amb particular referència al sistema espanyol. Des de la normalització de l'acció política dels sindicats i agents socials en les seves diverses manifestacions, com la participació institucional i la "legislació negociada" en la cerca del consens i el negociat d'intercanvis en el marc polític enfront de la imposició unilateral. La frustració de la concertació social es produeix quan el govern interpreta la democràcia com alguna cosa purament aritmètic de majories que exerceixen sense més el poder, en èpoques de feblesa sindical, de feblesa de l'estat nació, de dificultats per a les polítiques redistributives, de reforçament dels plantejaments neoliberals. La cerca de legitimitat en el pacte social no es veu en aquestes ocasions com a necessària sinó que la legitimitat es trasllada al discurs economicista i tecnocràtic, no polític. Es torna a la cerca d'una legitimitat a través del mercat i de l'intercanvi entre particulars.The study addresses the conclusion an analysis of policies of social security noting exposing its evolution from a social legitimacy to a technocratic legitimacy in the crisis of the welfare state and the economic crisis with particular reference to the Spanish system. Since the normalization of the political action of the trade unions and social partners in its various manifestations, such as institutional participation and negotiated "legislation" in the search for consensus and the Bureau of exchanges in the political framework against the unilateral imposition. The frustration of the social agreement occurs when the Government interprets the democracy as something purely arithmetic of majorities that exercise without more power, in times of Union weakness, weakness of the State nation, of difficulties for redistributive policies, strengthening of neoliberal approaches. The search for legitimacy in the social pact is not on these occasions as necessary but that legitimacy is moved to the economistic and technocratic, non-political speech. Turns to the search for legitimacy through the market and the exchange between individuals

Development of a direct transformation method by GFP screening and in vitro whole plant regeneration of Capsicum frutescens L.

BackgroundCapsicum is a genus of important spice crop that belongs to the chili lineage. However, many Capsicum species (family Solanaceae) are known to be recalcitrant to genetic transformation and in vitro regeneration, thus hampering the effort in using Capsicum species for detailed biological investigation. In this study, we have developed an optimized protocol for the direct transformation of Capsicum frutescens L. cv. Hot Lava via a biolistic particle delivery system. In addition, in vitro whole plant regeneration from the hypocotyl explants of C. frutescens was established.ResultsIn this biolistic system study, explant target distance, bombardment helium (He) pressure and the size of microcarrier were the key parameters to be investigated. The optimized parameters based on screening of GFP expression were determined to be 6 cm target distance, 1350 psi of helium pressure and 1.6 ?m of gold particle (microcarrier) size. The greatest number of shoots were obtained from hypocotyl as explant using Murashige and Skoog medium supplemented with 5.0 mg/L BAP and 0.1 mg/L NAA. An average of 5 shoots per explant were formed. Out of which, one shoot managed to form root and developed into whole plant.ConclusionsWe have obtained an optimized protocol for the biolistic transformation of chili and in vitro regeneration of chili plantlets. The establishment of the protocols will provide a platform for molecular breeding and biological studies of the chili plants

Transcriptome-wide identification and characterization of the Rab GTPase family in mango

© 2020, Springer Nature B.V. The Rab GTPase family plays a vital role in several plant physiological processes including fruit ripening. Fruit softening during ripening involves trafficking of cell wall polymers and enzymes between cellular compartments. Mango, an economically important fruit crop, is known for its delicious taste, exotic flavour and nutritional value. So far, there is a paucity of information on the mango Rab GTPase family. In this study, 23 genes encoding Rab proteins were identified in mango by a comprehensive in silico approach. Sequence alignment and similarity tree analysis with the model plant Arabidopsis as a reference enabled the bona fide assignment of the deduced mango proteins to classify into eight subfamilies. Expression analysis by RNA-Sequencing (RNA-Seq) showed that the Rab genes were differentially expressed in ripe and unripe mangoes suggesting the involvement of vesicle trafficking during ripening. Interaction analysis showed that the proteins involved in vesicle trafficking and cell wall softening were interconnected providing further evidence of the involvement of the Rab GTPases in fruit softening. Correlation analyses showed a significant relationship between the expression level of the RabA3 and RabA4 genes and fruit firmness at the unripe stage of the mango varieties suggesting that the differences in gene expression level might be associated with the contrasting firmness of these varieties. This study will not only provide new insights into the complexity of the ripening-regulated molecular mechanism but also facilitate the identification of potential Rab GTPases to address excessive fruit softening

Tomato Rab11a Characterization Evidenced a Difference Between SYP121-Dependent and SYP122-Dependent exocytosis

The regulatory functions of Rab proteins in membrane trafficking lie in their ability to perform as molecular switches that oscillate between a GTP- and a GDP-bound conformation. The role of tomato LeRab11a in secretion was analyzed in tobacco protoplasts. Green fluorescent protein (GFP)/red fluorescent protein (RFP)-tagged LeRab11a was localized at the trans-Golgi network (TGN) in vivo. Two serines in the GTP-binding site of the protein were mutagenized, giving rise to the three mutants Rab11S22N, Rab11S27N and Rab11S22/27N. The double mutation reduced secretion of a marker protein, secRGUS (secreted rat β-glucuronidase), by half, whereas each of the single mutations alone had a much smaller effect, showing that both serines have to be mutated to obtain a dominant negative effect on LeRab11a function. The dominant negative mutant was used to determine whether Rab11 is involved in the pathway(s) regulated by the plasma membrane syntaxins SYP121 and SYP122. Co-expression of either of these GFP-tagged syntaxins with the dominant negative Rab11S22/27N mutant led to the appearance of endosomes, but co-expression of GFP-tagged SYP122 also labeled the endoplasmic reticulum and dotted structures. However, co-expression of Rab11S22/27N with SYP121 dominant negative mutants decreased secretion of secRGUS further compared with the expression of Rab11S22/27N alone, whereas co-expression of Rab11S22/27N with SYP122 had no synergistic effect. With the same essay, the difference between SYP121- and SYP122-dependent secretion was then evidenced. The results suggest that Rab11 regulates anterograde transport from the TGN to the plasma membrane and strongly implicate SYP122, rather than SYP121. The differential effect of LeRab11a supports the possibility that SYP121 and SYP122 drive independent secretory event

Bioengineering of the plant culture of Capsicum frutescens with vanillin synthase gene for the production of vanillin

Production of vanillin by bioengineering has gained popularity due to consumer demand towards vanillin produced by biological systems. Natural vanillin from vanilla beans is very expensive to produce compared to its synthetic counterpart. Current bioengineering works mainly involve microbial biotechnology. Therefore, alternative means to the current approaches are constantly being explored. This work describes the use of vanillin synthase (VpVAN), to bioconvert ferulic acid to vanillin in a plant system. The VpVAN enzyme had been shown to directly convert ferulic acid and its glucoside into vanillin and its glucoside, respectively. As the ferulic acid precursor and vanillin were found to be the intermediates in the phenylpropanoid biosynthetic pathway of Capsicum species, this work serves as a proof-of-concept for vanillin production using Capsicum frutescens (C. frutescens or hot chili pepper). The cells of C. frutescens were genetically transformed with a codon optimized VpVAN gene via biolistics. Transformed explants were selected and regenerated into callus. Successful integration of the gene cassette into the plant genome was confirmed by polymerase chain reaction. High performance liquid chromatography was used to quantify the phenolic compounds detected in the callus tissues. The vanillin content of transformed calli was 0.057% compared to 0.0003% in untransformed calli

Development of a direct transformation method by GFP screening and in vitro whole plant regeneration of Capsicum frutescens L.

Background Capsicum is a genus of important spice crop that belongs to the chili lineage. However, many Capsicum species (family Solanaceae) are known to be recalcitrant to genetic transformation and in vitro regeneration, thus hampering the effort in using Capsicum species for detailed biological investigation. In this study, we have developed an optimized protocol for the direct transformation of Capsicum frutescens L. cv. Hot Lava via a biolistic particle delivery system. In addition, in vitro whole plant regeneration from the hypocotyl explants of C. frutescens was established. Results In this biolistic system study, explant target distance, bombardment helium (He) pressure and the size of microcarrier were the key parameters to be investigated. The optimized parameters based on screening of GFP expression were determined to be 6 cm target distance, 1350 psi of helium pressure and 1.6 μm of gold particle (microcarrier) size. The greatest number of shoots were obtained from hypocotyl as explant using Murashige and Skoog medium supplemented with 5.0 mg/L BAP and 0.1 mg/L NAA. An average of 5 shoots per explant were formed. Out of which, one shoot managed to form root and developed into whole plant. Conclusions We have obtained an optimized protocol for the biolistic transformation of chili and in vitro regeneration of chili plantlets. The establishment of the protocols will provide a platform for molecular breeding and biological studies of the chili plants

Characterization of Southeast Asia mangoes (Mangifera indica L) according to their physicochemical attributes

Mango (Mangifera indica L.) is an economically important fruit crop grown in the tropics. One of the important traits of mango for successful commercial production is the storage quality of the fruit. This study was conducted to evaluate the postharvest qualities of three mango (Mangifera indica) varieties namely ‘Chokanan’, ‘Golden phoenix’ and ‘Water lily’ grown in Southeast Asia regions. The study found that variety and ripening stage had an impact on the postharvest qualities. In general, an increase in weight loss, L* value and soluble solids concentration (SSC) along with a reduction in titratable acidity (TA), firmness and hue value as ripening progressed were observed irrespective of the variety. Analysis of variance and multivariate analysis were used to characterize the ripening process. This study provides useful information for devising strategies in postharvest handling and implementation of breeding programs for mango crop improvement

The control of initiation of DNA replication in Escherichia coli.

A new type of DNA-BNA hybridisation system was used to investigate two aspects of the initiation of replication in E.coli. A colony bank of recombinant plasmids carrying various fragments of the E.coli chromosome was constructed by manipulation in vitro. A number of plasmids carrying specific E.coli genes were selected by direct complementation analysis or by F'lac mediated mobilisation. These plasmids together with other similar ones were used as hybridisation probes. The conditions for hybridisation and the labelling of LNA.;vitrowere refined. DNA-DNA hybridisation and chemical DNA measurements were used to demonstrate that chloramphenicol induces a burst of extra initiation at the origin of a dnaA46 strain growing at a semi-permissive temperature. The temporal pattern of the initiation is consistent with the hypothesis that the stimulation of.Jiiitiation is the result of the stimulation of the. synthesis of an RNA species. It was shown that there is a disturbance in the pattern of replication of Hfr strain KL99 during exponential growth compared with that in a closely related F" strain. The pattern is consistent with initiation occuxring at the F origin. The LNA/mass ratio of Hfr AB313 is not changed relative to that of the isogenic F∼ strain, indicating that the F plasmid origin of replication is not active in this strain during exponential growth. As both Hfr strains are capable of giving rise to F' plasmids it is argued that the difference in the point of insertion of F in the chromosome is the cause of the difference in the initiation pattern between the two strains. These results, are interpreted as being a reflection of a copy number titration control mechanism for F replication. Possible mechanisms for the primary control and for the subsequent steps in the initiation process are discussed