Factors affecting fructosyltransferases and fructan exohydrolase activities in <Emphasis Type="Italic">Agave tequilana</Emphasis> Weber var. azul

There is great interest in the fructosyltransferases (FTFs) involved in fructan metabolism and agents affecting their activity. Agaves accumulate fructans, fructose polymers linked by glycosidic β(2–1) and β(2–6) bonds in linear or branched configurations. In plants, fructans provide protection under stress conditions. The sucrose:sucrose 1-fructosyltransferase (1-SST), fructan:fructan 1-fructosyltransferase (1-FFT), fructan:fructan 6G-fructosyltransferase (6G-FFT), and fructan exohydrolase (FEH) activities were analyzed in micropropagated Agave tequilana plants in the absence and presence of HgCl₂, AgNO₃, MgCl_2, sodium deoxycholate (DNa), and sodium dodecyl sulfate (SDS). Kestose, nystose and neokestose were synthesized by the respective FTFs. HgCl₂ and AgNO₃ inhibited all FTFs, mainly up to 90 % in 1-SST and 1-FFT. DNa increased 1-SST (32 %) and 1-FFT (45 %) activities, and SDS increased 6G-FFT activity by 96 %. Finally, AgNO₃ inhibited FEH activity by 78 %. Our results might be relevant on the regulation of FTFs in agave and other crops, for instance by the increment the fructans synthesis in stressed plants.     

Effect of light quality and culture medium on somatic embryogenesis of <Emphasis Type="Italic">Agave tequilana</Emphasis> Weber var. Azul

Somatic embryogenesis in Agave tequilana Weber var. Azul was affected by the interaction between the light regimes applied during the induction phase and the expression phase. When embryogenic calli was exposed to white or red light during the expression phase, an average of two germinated embryos per explant was obtained regardless of the light treatment used for callus induction. Conversely, the highest number of germinated embryos, an average of 18 per explant, was obtained when applying either white or red light during the induction phase and then wide-spectrum light during the expression phase. Culture medium had also a great influence in this process, with embryo germination being reduced by up to 70%, depending on the light treatment, when using Schenk and Hildebrandt (SH) medium instead of Murashige and Skoog (MS) medium.     

Overexpression of smORF YNR034W-A/<Emphasis Type="Italic">EGO4</Emphasis> in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> increases the fermentative efficiency of <Emphasis Type="Italic">Agave tequilana</Emphasis> Weber must

Fermentative processes are widely used to produce food, beverages and biofuels. Saccharomyces cerevisiae is an efficient ethanol-producing microorganism. However, a concentration of high ethanol and other metabolites can affect yeast viability and decrease the ethanol yield. Many studies have focused on improving the fermentative efficiency, mostly through the genetic engineering of genes that have a direct impact on specific metabolic pathways. In the present study, we characterized a small open reading frame encoding a protein with an unknown function and biological role termed YNR034W-A. We analyzed the expression profile of the YNR034W-A gene during growth and glucose treatment, finding that it is expressed during the diauxic shift and stationary phase and is negatively regulated by glucose. We overexpressed the YNR034W-A gene in the BY4741 laboratory strain and a wild-type yeast strain (AR5) isolated during the Tequila fermentation process. Transformant derivatives of the AR5 strain showed an improved fermentative efficiency during fermentation of Agave tequilana Weber juice. We suggest that the improved fermentative efficiency is the result of a higher stress tolerance response in the YNR034W-A overexpressing transformant.     

Production of Fermentable Sugars and Hydrogen-Rich Gas from <Emphasis Type="Italic">Agave tequilana</Emphasis> Biomass

The Mexican tequila industry annually processes approximately 1 × 10⁶ Agave tequilana plants, generating approximately 1.78 × 10⁸ kg of bagasse per year. This biomass is considered an attractive alternative to fossil fuels as an energy source and to produce biofuels and/or chemical products because it is produced and used without adversely affecting the environment. The first aim of the present work was to determine the effect of temperature, the concentration of H₂SO_4, and reaction time on the hydrolysis of agave bagasse to maximize the fermentable sugars using a steam explosion. This step process generated 71.11 g/L of reducible sugars in the supernatant (59.29 % glucose, 29.05 % xylose, and 11.66 % fructose) and unconverted organic matter of enzymatic hydrolysis bagasse (35.4 % α-cellulose, 7.33 % hemicellulose, 49.91 % lignin, and 7.31 % ashes). A mathematical surface response analysis of the hydrolysis was used for process optimization. The second aim involves the study of the thermodynamics of the reforming of unconverted organic matter from enzymatic hydrolysis of Agave tequilana bagasse (ATB) evaluated by the Gibbs free energy minimization method for hydrogen production. The effect of the parameters on the system performance measures, such as reaction temperature (T), Water/Biomass ratio (WBR), and pressure (P), were also investigated. The maximum H₂ production obtained was 23.2 mol of H₂/271.5 g ATB with a WBR ≥ 11 and a temperature of 740 °C. These findings indicate that the temperature and WBR are essential factors in the production of H₂, which was reflected in the efficiency of the process.     

Effect of <Emphasis Type="Italic">Agave tequilana</Emphasis> juice on cell wall polysaccharides of three <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> strains from different origins

In this study, a characterization of cell wall polysaccharide composition of three yeasts involved in the production of agave distilled beverages was performed. The three yeast strains were isolated from different media (tequila, mezcal and bakery) and were evaluated for the β(1,3)-glucanase lytic activity and the β-glucan/mannan ratio during the fermentation of Agave tequilana juice and in YPD media (control). Fermentations were performed in shake flasks with 30 g l⁻¹ sugar concentration of A. tequilana juice and with the control YPD using 30 g l⁻¹ of glucose. The three yeasts strains showed different levels of β-glucan and mannan when they were grown in A. tequilana juice in comparison to the YPD media. The maximum rate of cell wall lyses was 50% lower in fermentations with A. tequilana juice for yeasts isolated from tequila and mezcal than compared to the bakery yeast.     

Efficient chemical and enzymatic saccharification of the lignocellulosic residue from <Emphasis Type="Italic">Agave tequilana</Emphasis> bagasse to produce ethanol by <Emphasis Type="Italic">Pichia caribbica</Emphasis>

Bagasse of Agave tequilana (BAT) is the residual lignocellulosic waste that remains from tequila production. In this study we characterized the chemical composition of BAT, which was further saccharified and fermented to produce ethanol. BAT was constituted by cellulose (42%), hemicellulose (20%), lignin (15%), and other (23%). Saccharification of BAT was carried out at 147°C with 2% sulfuric acid for 15 min, yielding 25.8 g/l of fermentable sugars, corresponding to 36.1% of saccharificable material (cellulose and hemicellulose contents, w/w). The remaining lignocellulosic material was further hydrolyzed by commercial enzymes, ~8.2% of BAT load was incubated for 72 h at 40°C rendering 41 g/l of fermentable sugars corresponding to 73.6% of the saccharificable material (w/w). Mathematic surface response analysis of the acid and enzymatic BAT hydrolysis was used for process optimization. The results showed a satisfactory correlation (R ² = 0.90) between the obtained and predicted responses. The native yeast Pichia caribbica UM-5 was used to ferment sugar liquors from both acid and enzymatic hydrolysis to ethanol yielding 50 and 87%, respectively. The final optimized process generated 8.99 g ethanol/50 g of BAT, corresponding to an overall 56.75% of theoretical ethanol (w/w). Thus, BAT may be employed as a lignocellulosic raw material for bioethanol production and can contribute to BAT residue elimination from environment.     

Low-Input Fermentations of <Emphasis Type="Italic">Agave tequilana</Emphasis> Leaf Juice Generate High Returns on Ethanol Yields

During tequila production, up to 75 % w/w of the Agave plant is discarded when leaves are removed from the stem. The discarded leaves represent an extensive amount of unexploited biomass that was used here for bioethanol production in no-input fermentations, where no acid or enzymatic hydrolysis, supplementation of nutrients or standardization of carbohydrate content occur. Ethanol yield from Agave leaf juice is unaffected by sterilization but reduced if fermentation is reliant solely on endogenous microorganisms. Non-Saccharomyces yeasts, including Kluyveromyces marxianus and Candida akabanensis, proved to be more robust than standard Saccharomyces spp. and yielded up to 88 % of the theoretical maximum ethanol from leaf juice. Combining leaf and stem juice, as from a whole plant, was predicted to maximize yield at up to 19,439 L/ha of ethanol from mature plants.     

<Emphasis Type="Italic">Ty</Emphasis>1<Emphasis Type="Italic">/copia</Emphasis>- and <Emphasis Type="Italic">Ty</Emphasis>3<Emphasis Type="Italic">/gypsy</Emphasis>-like DNA sequences in <Emphasis Type="Italic">Helianthus </Emphasis>species

Two repeated DNA sequences isolated from a partial genomic DNA library of Helianthus annuus, p HaS13 and p HaS211, were shown to represent portions of the int gene of a Ty3 /gypsy retroelement and of the RNase-Hgene of a Ty1 /copia retroelement, respectively. Southern blotting patterns obtained by hybridizing the two probes to BglII- or DraI-digested genomic DNA from different Helianthus species showed p HaS13 and p HaS211 were parts of dispersed repeats at least 8 and 7 kb in length, respectively, that were conserved in all species studied. Comparable hybridization patterns were obtained in all species with p HaS13. By contrast, the patterns obtained by hybridizing p HaS211 clearly differentiated annual species from perennials. The frequencies of p HaS13- and p HaS211-related sequences in different species were 4.3x10(4)-1.3x10(5) copies and 9.9x10(2)-8.1x10(3) copies per picogram of DNA, respectively. The frequency of p HaS13-related sequences varied widely within annual species, while no significant difference was observed among perennial species. Conversely, the frequency variation of p HaS211-related sequences was as large within annual species as within perennials. Sequences of both families were found to be dispersed along the length of all chromosomes in all species studied. However, Ty3 /gypsy-like sequences were localized preferentially at the centromeric regions, whereas Ty1/ copia-like sequences were less represented or absent around the centromeres and plentiful at the chromosome ends. These findings suggest that the two sequence families played a role in Helianthusgenome evolution and species divergence, evolved independently in the same genomic backgrounds and in annual or perennial species, and acquired different possible functions in the host genomes.     

Complete chloroplast genome sequence of <Emphasis Type="Italic">Capsicum</Emphasis><Emphasis Type="Italic">baccatum</Emphasis> var. <Emphasis Type="Italic">baccatum</Emphasis>

Molecular markers derived from the complete chloroplast genome can provide effective tools for species identification and phylogenetic resolution. Complete chloroplast (cp) genome sequences of Capsicum species have been reported. We herein report the complete chloroplast genome sequence of Capsicum baccatum var. baccatum, a wild Capsicum species. The total length of the chloroplast genome is 157,145 bp with 37.7 % overall GC content. One pair of inverted repeats, 25,910 bp in length, was separated by a small single-copy region (17,974 bp) and large single-copy region (87,351 bp). This region contains 86 protein-coding genes, 30 tRNA genes, 4 rRNA genes, and 11 genes contain one or two introns. Pair-wise alignments of chloroplast genome were performed for genome-wide comparison. Analysis revealed a total of 134 simple sequence repeat (SSR) motifs and 282 insertions or deletions variants in the C. baccatum var. baccatum cp genome. The types and abundances of repeat units in Capsicum species were relatively conserved, and these loci could be used in future studies to investigate and conserve the genetic diversity of the Capsicum species.     

Life cycle of <Emphasis Type="Italic">Uromyces appendiculatus</Emphasis> var. <Emphasis Type="Italic">azukicola</Emphasis> on <Emphasis Type="Italic">Vigna angularis</Emphasis>

Field observations and inoculation experiments revealed that Uromyces appendiculatus var. azukicola has an autoecious and macrocyclic life cycle and produces spermogonia, aecia, uredinia, and telia on Vigna angularis var. angularis and V. angularis var. nipponensis. From inoculation experiments, it was suggested that this rust fungus has different host relationships from other varieties. Morphological examinations revealed that the characteristics of urediniospores and teliospores are different among varieties, although aeciospores are morphologically similar to each other.Contribution no. 182, Laboratory of Plant Parasitic Mycology, Institute of Agriculture and Forestry, University of Tsukuba, Japan     

Impact of precursors and plant growth regulators on <Emphasis Type="Italic">in vitro</Emphasis> growth,bioactive lignans,and antioxidant content of <Emphasis Type="Italic">Phyllanthus</Emphasis> species

The production of specific secondary metabolites in vitro can be improved through medium supplementation with secondary metabolite precursors, plant growth regulators (PGRs), and abiotic and biotic elicitors. In the present study, node and internode explants of Phyllanthus amarus and P. urinaria collected from Karkala region, Udupi District, Karnataka, India, were inoculated aseptically onto Murashige and Skoog (MS) medium for callus induction. Uniform calluses were inoculated onto MS medium fortified with one of two precursor’s cinnamic acid (CA) or phenylalanine (PA), or with naphthalene acetic acid (NAA). After 30 d of treatment, calluses from treatment and control groups were harvested and quantitatively analyzed for three lignans (phyllanthin, hypophyllanthin and niranthin) and an antioxidant (ellagic acid). Increased amounts of the lignans and ellagic acid were obtained through supplementation with CA, PA, and NAA, and higher ellagic acid was present at higher amounts than the three lignans. These results demonstrated that the Phyllanthus species collected from Karkala region (designated “Accessions3”) show substantial response to CA, PA, and NAA treatment and represent a potential source of donor plants with higher amounts of lignans and antioxidants. These plants can be cultivated on a large scale both in vitro and in vivo for production of important bioactive compounds. Production of these compounds can be further enhanced through induction of somaclonal variant plants with higher amounts of bioactive molecule production and through production of transgenic plants overexpressing genes related to lignan- and phenolic-compound biosynthesis.     

Role of Arabidopsis <Emphasis Type="Italic">ABF1</Emphasis>/<Emphasis Type="Italic">3</Emphasis>/<Emphasis Type="Italic">4</Emphasis> during <Emphasis Type="Italic">det1</Emphasis> germination in salt and osmotic stress conditions

Key message

Arabidopsis det1 mutants exhibit salt and osmotic stress resistant germination. This phenotype requires HY5, ABF1, ABF3, and ABF4.

Abstract

While DE-ETIOLATED 1 (DET1) is well known as a negative regulator of light development, here we describe how det1 mutants also exhibit altered responses to salt and osmotic stress, specifically salt and mannitol resistant germination. LONG HYPOCOTYL 5 (HY5) positively regulates both light and abscisic acid (ABA) signalling. We found that hy5 suppressed the det1 salt and mannitol resistant germination phenotype, thus, det1 stress resistant germination requires HY5. We then queried publically available microarray datasets to identify genes downstream of HY5 that were differentially expressed in det1 mutants. Our analysis revealed that ABA regulated genes, including ABA RESPONSIVE ELEMENT BINDING FACTOR 3 (ABF3), are downregulated in det1 seedlings. We found that ABF3 is induced by salt in wildtype seeds, while homologues ABF4 and ABF1 are repressed, and all three genes are underexpressed in det1 seeds. We then investigated the role of ABF3, ABF4, and ABF1 in det1 phenotypes. Double mutant analysis showed that abf3, abf4, and abf1 all suppress the det1 salt/osmotic stress resistant germination phenotype. In addition, abf1 suppressed det1 rapid water loss and open stomata phenotypes. Thus interactions between ABF genes contribute to det1 salt/osmotic stress response phenotypes.

     

Green roof <Emphasis Type="Italic">Petunia</Emphasis>, <Emphasis Type="Italic">Ageratum</Emphasis>, and <Emphasis Type="Italic">Mentha</Emphasis> responses to water stress,seaweeds, and trinexapac-ethyl treatments

The availability of sufficient irrigation water and the development of drought-tolerant species are challenging factors in the design and maintenance of green roofs in modern cities. Green roof plants of Petunia hybrid Headliner^® Red Star, Ageratum hybrid Artist^® blue, and Mentha spicata L. grown in a simulated green roof pot system under controlled greenhouse conditions. The plants were watered every 2 or 6 days (2DWI/6DWI) for 8 weeks accompanied by either a 6-day treatment of seaweed extracts of Ascophyllum nodosum as a soil drench or foliar spray, or two concentrations of trinexapac-ethyl (TE) biweekly sprays. Following treatments, leaf number, leaf area, dry weights, plant height, stomatal conductanse, photosynthetic and transpiration rates and leaf water potential and relative water content were determined in two seasons during 2016 and 2017. The prolonged irrigation intervals reduced plant growth as revealed by morphological and physiological parameters. The application of SWE as drench treatment improved Petunia and Ageratum plant vegetative growth, stomatal conductance, photosynthetic and transpiration rates and leaf water potential and relative water content during prolonged irrigation intervals. TE increased the vegetative growth as well as the physiological performance of Ageratum plants. However, neither SWE nor TE treatments improved the performance of Mentha plants under prolonged irrigation intervals. It was suggested that improved photosynthetic rates were stimulated by enhanced stomatal conductance leading to improved leaf water potential as well as increased relative water content during prolonged irrigation conditions.     

<Emphasis Type="Italic">Galleria</Emphasis><Emphasis Type="Italic">mellonella</Emphasis> are Resistant to <Emphasis Type="Italic">Pneumocystis</Emphasis><Emphasis Type="Italic">murina</Emphasis> Infection

Studying Pneumocystis has proven to be a challenge from the perspective of propagating a significant amount of the pathogen in a facile manner. The study of several fungal pathogens has been aided by the use of invertebrate model hosts. Our efforts to infect the invertebrate larvae Galleria mellonella with Pneumocystis proved futile since P. murina neither caused disease nor was able to proliferate within G. mellonella. It did, however, show that the pathogen could be rapidly cleared from the host.     

Revision of the <Emphasis Type="Italic">Serrulati</Emphasis> species of <Emphasis Type="Italic">Penstemon</Emphasis> section <Emphasis Type="Italic">Saccanthera</Emphasis> subsection <Emphasis Type="Italic">Serrulati</Emphasis>

A revision of Penstemon sect. Saccanthera subsect. Serrulati includes a new species (P. salmonensis), a new variety (P. triphyllus var. infernalis), and the elevation of a subspecies to species (P. curtiflorus), bringing the total number of species to eight, which are keyed and described, complete with nomenclature and type citations.     

<Emphasis Type="Italic">Agrobacterium</Emphasis><Emphasis Type="Italic">tumefaciens</Emphasis>-mediated transformation of callus cells of <Emphasis Type="Italic">Crataegus</Emphasis><Emphasis Type="Italic">aronia</Emphasis>

A genetic transformation system has been developed for callus cells of Crataegus aronia using Agrobacterium tumefaciens. Callus culture was established from internodal stem segments incubated on Murashige and Skoog (MS) medium supplemented with 5 mg l⁻¹ Indole-3-butyric acid (IBA) and 0.5 mg l⁻¹ 6-benzyladenine (BA). In order to optimize the callus culture system with respect to callus growth and coloration, different types and concentrations of plant growth regulators were tested. Results indicated that the best average fresh weight of red colored callus was obtained on MS medium supplemented with 2 mg l⁻¹ 2,4-dichlorophenoxyacetic acid (2,4-D) and 1.5 mg l⁻¹ kinetin (Kin) (callus maintenance medium). Callus cells were co-cultivated with Agrobacterium harboring the binary plasmid pCAMBIA1302 carrying the mgfp5 and hygromycin phosphotransferase (hptII) genes conferring green fluorescent protein (GFP) activity and hygromycin resistance, respectively. Putative transgenic calli were obtained 4 weeks after incubation of the co-cultivated explants onto maintenance medium supplemented with 50 mg l⁻¹ hygromycin. Molecular analysis confirmed the integration of the transgenes in transformed callus. To our knowledge, this is the first time to report an Agrobacterium-mediated transformation system in Crataegus aronia.     

The tyrosine <Emphasis Type="Italic">O</Emphasis>-prenyltransferase SirD catalyzes <Emphasis Type="Italic">O</Emphasis>-, <Emphasis Type="Italic">N</Emphasis>-, and <Emphasis Type="Italic">C</Emphasis>-prenylations

Recently, the prenyltransferase SirD was found to be responsible for the O-prenylation of tyrosine in the biosynthesis of sirodesmin PL in Leptosphaeria maculans. In this study, the behavior of SirD towards phenylalanine/tyrosine and tryptophan derivatives was investigated. Product formation has been observed with 12 of 19 phenylalanine/tyrosine derivatives. It was shown that the alanine structure attached to the benzene ring and an electron donor, e.g., OH or NH₂, at its para-position are essential for the enzyme activity. Modifications were possible both at the side chain and the benzene ring. Enzyme products from seven phenylalanine/tyrosine derivatives were isolated and characterized by MS and NMR analyses including HSQC and HMBC and proven to be O- or N-prenylated derivatives at position C4 of the benzene rings. K _M values of six selected derivatives were found in the range of 0.10–0.68 mM. Catalytic efficiencies (K _cat/K _M ) were determined in the range of 430–1,110 s⁻¹·M⁻¹ with l-tyrosine as the best substrate. In addition, 7 of 14 tested tryptophan analogs were also accepted by SirD and converted to C7-prenylated derivatives, which was confirmed by comparison with products obtained from enzyme assays using a 7-dimethylallyltryptophan synthase 7-DMATS from Aspergillus fumigatus.     

Effect of <Emphasis Type="Italic">Agave tequilana</Emphasis> age,cultivation field location and yeast strain on tequila fermentation process

The effect of yeast strain, the agave age and the cultivation field location of agave were evaluated using kinetic parameters and volatile compound production in the tequila fermentation process. Fermentations were carried out with Agave juice obtained from two cultivation fields (CF1 and CF2), as well as two ages (4 and 8 years) and two Saccharomyces cerevisiae yeast strains (GU3 and AR5) isolated from tequila fermentation must. Sugar consumption and ethanol production varied as a function of cultivation field and agave age. The production of ethyl acetate, 1-propanol, isobutanol and amyl alcohols were influenced in varying degrees by yeast strain, agave age and cultivation field. Methanol production was only affected by the agave age and 2-phenylethanol was influenced only by yeast strain. This work showed that the use of younger Agave tequilana for tequila fermentation resulted in differences in sugar consumption, ethanol and volatile compounds production at the end of fermentation, which could affect the sensory quality of the final product.