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The Trichoderma harzianum fungus is one of the most widely used biological control agents in agriculture. A new T. harzianum THITR01 strain was isolated and their spores were mutagenized with ethyl methane sulfonate obtaining 174 mutants. M7, M14, M21 and M24 are mutant strains that showed 97.4%-100% antagonist effect against Sclerotium rolfsii, Rhizoctonia solani and Sclerotinia sclerotiorum compared to the wild strain. Under potato dextrose agar (PDA) medium with either NaCl, sorbitol or NaHCO3 there were nonsignificant growth rate differences between the mutants and the wild strain. M14 and M21 mutant strains were inoculated on ‘Jalapeño’ pepper plants (Capsicum annuum L. var. annuum) with unavailable P promoted a significant increase in root fresh weight (54% and 40%, respectively) and dry weight (28% in both strains), compared to plant inoculation with the wild strain. The M14 strain presented the highest P solubilization ability (13.4 μg g-1) in the substrate and promoted a change on root architecture. There was a higher relative water content (82.9%) in drought stressed plants inoculated with the M24 mutant than in plants that were inoculated with the wild type strain (76.3%), and also higher levels of proline in chili pepper plants inoculated with the M24 mutant (939.5 μg g-1 dry weight) than in plants inoculated with the wild type strain (419.8 μg g-1 dry weight). Therefore, M14, M21 and M24 mutant strains could potentially be used as biocontrol agents and plant protector from abiotic stress.

La variedad Drymifolia de aguacate (Persea americana Mill.) es usada generalmente como portainjerto por lo que es importante implementar las técnicas de embriogénesis somática y la organogénesis in vitro para su mejora genética, conservación y propagación clonal. En este trabajo se evaluaron embriogénesis somática directa, indirecta y organogénesis de la variedad Drymifolia. La germinación de los embriones somáticos maduros se indujo con 0.5 mg L-1 de 6-N-bencil amino purina (BAP) y 1 mg L-1 de ácido giberélico (GA3). De las seis accesiones evaluadas, Celaya 79, Comonfort 53, San Miguel, BG24, BG181 y Zutano, solo San Miguel respondió al proceso de embriogénesis. En la embriogénesis directa la eficiencia más alta de regeneración se obtuvo con 0.2 mg L-1 de picloram (46%) y 10 mg L-1 de ANA (40%). En la embriogénesis indirecta la accesión San Miguel formó callos con 0.2 mg L-1 de picloram y tuvo una eficiencia de regeneración de 45% conservando su potencial de regeneración hasta por seis meses. Con respecto a la organogénesis, se cultivaron embriones cigóticos inmaduros decapitados en medio con reguladores de crecimiento o sin ellos y las seis accesiones respondieron positivamente a ambas condiciones. La accesión Comonfort 53 tuvo mayor eficiencia de regeneración (54%) con reguladores de crecimiento. Este estudio realizado del 2016 a 2018, proporciona un enfoque nuevo y prometedor para la regeneración y multiplicación de plantas de P. americana var. Drymifolia a través de embriones cigóticos.

Las pudriciones de raíz, corona y tallo en trigo se consideran una amenaza seria para este cereal en diversas partes del mundo ya que las pérdidas que ocasionan en el rendimiento y calidad de grano llegan hasta 89% y pueden ser comparables con las causadas por las royas, además de que los hongos asociados a estas pudriciones sintetizan micotoxinas que pueden contaminar productos alimenticios. Esta problemática en México ha sido poco estudiada; sin embargo, en los últimos años, estas enfermedades en trigos de riego y temporal han sido más incidentes lo que está causando preocupación en los productores de este cereal en el país. Esta revisión resume aspectos actuales de la enfermedad, las fuentes de resistencia disponibles a nivel mundial, así como la forma de cómo opera dicha resistencia en el patosistema Fusarium-trigo. Las bases genéticas de la resistencia a pudriciones de raíz, tallo y corona de los trigos hexaploides se ha examinado; a través, del mapeo de Quantitative Trait Loci (QTL). Hasta la fecha, 44 QTL han sido identificados en 14 cromosomas con alelos que inducen la resistencia a esta enfermedad y que derivan de trigos hexaploides y de parientes cercanos. El mejoramiento genético para esta enfermedad mediante la introgresión de los alelos con estos QTL es la estrategia más factible y la piramidación de estos QTL parece ser la estrategia más viable en los programas de mejoramiento genético. Paso importante para el manejo de la problemática en las zonas productores de trigo en México.

In recent years, benefits of silicon in plants grown under stress conditions have been reported. The objective of the research was to evaluate the response at a physiological and biochemical level of millet (Panicum miliaceum L.) to fertilization with Si under controlled stress conditions during vegetative stage, drought, salinity and the control (without stress). After stress, shoot and root length, DM content, peroxidase (POD) and catalase (CAT) activity, proline, H2O2 and Si content, seed yield and germination percentage were measured. Drought and salinity significantly decreased shoot (24%, 21%) and root (30% in drought) development, weight (36%, 29%) and seed number (30%, 21%) per plant. Application of Si significantly increased seed number (289) and seed weight (1.20 g) in the control plants, increased seed weight (0.83 g) in plants under drought and germination percentage (99%) in plants under salinity. Silicon increased 2.1 times POD activity in drought, 1.4 times in salinity and control plants; CAT activity increased 10.6 times in salinity and 1.7 times in control plants. Silicon decreased 10 times proline levels in plants under drought; in salinity and control, proline content increased 1.3 times with Si and H2O2 levels decreased in these treatments. The Si content in plants fertilized with Si under drought, salinity and control was 6%, 3.54% and 5.45% respectively. In conclusion, Si can improve plant stress tolerance by stimulating POD and CAT activity, and regulating proline levels, allowing it to improve the production and physiological seed.

La chía es un recurso muy valioso ya que es la principal fuente de ácidos grasos insaturados, principalmente α-linolénico, además de proteína y fibra. En esta investigación se realizaron siete colectas en siete localidades para su estudio y comparación. Se midió tamaño de semilla, peso de mil semillas, peso volumétrico, color, porcentaje de germinación y extracción de aceite para análisis en cromatografía. La colecta de chía cultivada de Guanajuato presentó mayor variabilidad en tamaño de semilla y la de Roque la menor. Las colectas silvestres tuvieron el menor tamaño de semilla. La cultivada de Guanajuato tiene el peso mayor en semilla y las silvestres el menor, estas últimas no germinaron, pero sí lo hicieron las domesticadas. No hubo diferencias en peso volumétrico; sin embargo, si hubo diferencias en los cromatogramas (HPLC) y en las características físicas y variación de color en las domesticadas, incluidas semillas de color blanco. Esta investigación prospectiva muestra que la variabilidad presente en la chía silvestre y cultivada puede ser aprovechada para el fitomejoramiento.

The major challenges that agriculture is facing in the twenty-first century are increasing droughts, water scarcity, flooding, poorer soils, and extreme temperatures due to climate change. However, most crops are not tolerant to extreme climatic environments. The aim in the near future, in a world with hunger and an increasing population, is to breed and/or engineer crops to tolerate abiotic stress with a higher yield. Some crop varieties display a certain degree of tolerance, which has been exploited by plant breeders to develop varieties that thrive under stress conditions. Moreover, a long list of genes involved in abiotic stress tolerance have been identified and characterized by molecular techniques and overexpressed individually in plant transformation experiments. Nevertheless, stress tolerance phenotypes are polygenetic traits, which current genomic tools are dissecting to exploit their use by accelerating genetic introgression using molecular markers or site-directed mutagenesis such as CRISPR-Cas9. In this review, we describe plant mechanisms to sense and tolerate adverse climate conditions and examine and discuss classic and new molecular tools to select and improve abiotic stress tolerance in major crops.

For more than a decade molecular biology has offered the tools to isolate plant genes and to analyze their structures. In general, knowledge of the gene product is a prerequisite for gene isolation. In the first part of this chapter the methodologies available for plant gene isolation are discussed and the examples described. The strategy for gene isolation depends on the abundancy of the gene product as well as on the knowledge of the expression pattern or primary structure of the gene and encoded protein. The second part will deal with molecular techniques which can be applied to isolate genes of which the gene products are unknown. For many genes isolated by the methods described so far it is often difficult to prove that the clone contains the desired gene. A major interest of plant biology is on development, cell differentiation, and cell response to environmental factors at the molecular level.

La glicina betaína es uno de los solutos compatibles con el metabolismo más eficiente. En las plantas, la biosíntesis de la glicina betaína se lleva a cabo en dos pasos enzimáticos. Primero se oxida el sustrato colina a betaína aldehído por medio de la colina monooxigenasa (CMO); el segundo paso es catalizado por una betaína aldehído deshidrogenasa (BADH) que convierte la betaína aldehído a glicina betaína. Estudios previos mostraron que la transcripción del gen ahybadh17, que codifica para la BADH de Amaranthus hypochondriacus, es inducida por estrés hídrico y salinidad, en paralelo a un incremento en los niveles de glicina betaína. En el presente trabajo se analizó la expresión del promotor del gen ahybadh4 de A. hypochondriacus en plantas transgénicas de Arabidopsis thaliana. Para este propósito se construyó un gen quimérico con una secuencia de 1.5 kb correspondiente a la posible región promotora del gen ahybadh4 fusionada con el gen reportero uid A de la -glucuronidasa (GUS). Las semillas de las plantas transgénicas (T3) obtenidas, fueron crecidas durante 7, 14 y 21 días después de la germinación y fueron sometidas a tratamientos de estrés osmótico y ácido abscísico (ABA) por un periodo de 16 horas. Los análisis histoquímico y fluorimétrico de GUS mostraron una expresión constitutiva y específica en la raíz. Estos resultados permiten concluir que la secuencia 5¿ de 1.5 kb del gen ahybadh4 no se regula por estrés osmótico o ABA en A. thaliana. ispartof: Revista Fitotecnia Mexicana vol:26 pages:133-140 status: published

En este trabajo se describen las cinéticas de hidratación y deshidratación en plantas de Selaginella lepidophylla y su influencia en la actividad de la trehalosa 6-fosfato sintasa y en los niveles de trehalosa. Al hidratar plantas totalmente secas por 24 h, su contenido relativo de agua aumentó de 3.2 % a 92.7 %, mientras que su potencial hídrico se elevó de niveles indetectables en plantas secas a -0.3 MPa. Las plantas secas mostraron una actividad enzimática de 0.023 U mg-1 proteína, al aumentar a 0.135 U mg-1 proteína (581 %) a las 2 h de hidratación; a las 24 h de hidratación la actividad descendió a 0.081 U mg-1 proteína. La concentración de trehalosa en las plantas secas fue de 11.9 mg g–1 ps, que se incrementó a 53 mg g-1 ps durante las primeras 8 h de hidratación. En las plantas totalmente rehidratadas y sometidas a desecación por 24 h, la velocidad de pérdida de agua fue rápida y su contenido relativo de agua se redujo de 94.4 % a 4.8 %; su potencial hídrico descendió de –0.3 a –2.1 MPa durante las primeras 12 h del tratamiento. La actividad de trehalosa 6-fosfato sintasa disminuyó durante las primeras 2 h de deshidratación, y luego se incrementó hasta alcanzar una actividad máxima de 0.068 U mg-1 proteína a las 8 h; a las 24 h de desecación la planta conserva la actividad de trehalosa 6-fosfato sintasa aún con un contenido relativo de agua de 4.4 %. La concentración de trehalosa aumentó de 10.2 a 50.4 mg g-1 ps a las 8 h de deshidratación, y a las 24 h la concentración de trehalosa se mantiene alta (12 mg g-1 ps).

Drought is one of the most limiting factors in agricultural production worldwide. The aim of this work was to evaluate the per se response to drought stress of three maize (Zea mays L.) semi-inbred lines (CHIH, COAH, and ARZM) during the anthesis and grain filling stages. These semi-inbred lines (BC1S1) are S1 offspring of crosses between drought-tolerant maize and a high-yield line bred by the Centro Internacional de Mejoramiento de Maíz y Trigo (CIMMYT). Materials were evaluated during a 15-d controlled drought and post-drought irrigation reestablishment period. The photosynthetic rate decreased by 50% in CHIH during drought and it recovered after stress was removed. Proline levels increased 2.3 times in COAH during stress and nonsignificant increases were observed in ARZM or CHIH. However, glycine betaine accumulated at 1061-1133 ng mg-1 fresh weight (FW) before stress, but decreased to 879-1000 ng mg-1 FW during drought stress. The trehalose concentration increased 1.8 times in ARZM (5.60 ng mg-1 FW) under drought stress and remained at similar levels in CHIH (6.48 ng mg-1 FW) and COAH (8.63 ng mg-1 FW) before and during drought stress. In contrast, trehalose-6-phosphate decreased 33% to 38% under drought stress in all three BC1S1 entries and recovered its initial levels after irrigation. Grain biomass loss under drought stress was 54% for ARZM, 48.2% for COAH, and only 26.5% for CHIH. These results showed that CHIH, COAH, and ARZM are drought-tolerant and suggest that osmoprotectant accumulation might play a key role in their physiological performance and grain biomass trait.

La semilla de melón chino se ha usado de manera tradicional para elaborar horchata; sin embargo, se ha perdido la costumbre de consumirla. A fin de ampliar el conocimiento sobre este recurso vegetal se extrajeron las proteínas de reserva solubles de la semilla. El contenido total de proteína fue de 27.34% para el melón chino y de 24.9% para el melón verde (liso). Las globulinas constituyen la fracción mayoritaria y las prolaminas, la minoritaria. En el patrón electroforético, la fracción globulina muestra bandas abundantes y bien definidas. Las proteínas precipitadas con ácido clorhídrico o frío mostraron un patrón electroforético semejante en ambas variedades. Las proteínas se extrajeron mejor a valores de pH entre 5 y 12, pero no a valores de 2 a 4. El tratamiento con tripsina y quimotripsina mostró acumulación de bandas en el frente del gel, lo cual demuestra la presencia de péptidos digeridos. La actividad inhibitoria contra tripsina es mayor en la fracción albúminas con 741 UI mL-1 y fracción globulina con 587 UI mL-1. El contenido mineral fue de 0.5% para fósforo y de 0.28 para magnesio en melón chino; para el verde fue de 0.36% para potasio y de 0.28% para fósforo. El contenido de aceite fue de 35.8% en semilla de melón chino y de 23.2% en el verde, del cual se sabe que 67.5% son ácidos grasos insaturados; el aceite fue de color ámbar, de olor agradable. El uso de la semilla de melón es un recurso vegetal que debe promoverse por su alto contenido de proteína y aceite, ya que es valioso para la alimentación humana. También puede usarse en la formulación de alimento para ganado o de larvas de insecto cultivadas en laboratorio.

En la presente investigación se osmoacondicionaron semillas de dos razas de maíz, con PEG o con nitrato de potasio más ácido giberélico, para estimar su efecto en las variables: germinación, peso y longitud de radícula y plántula, acidez titulable y patrón electroforético de proteínas; se les sometió a remojo (hipoxia, “soak test”) y se cuantifico la respuesta. El objetivo fue contribuir a la caracterización de los maíces nativos de México que se siembran bajo condiciones de temporal. Los maíces Bofo y Elotes Occidentales mostraron la mayor imbibición con KNO3 + ácido giberélico durante 12 h. El porcentaje de germinación después del remojo, fue mayor con KNO3 + ácido giberélico durante 6 h y fue superior al testigo. En cuanto a la longitud de radícula y plúmula, los tratamientos superaron al testigo, en ambos conteos. Con Elotes Occidentales, luego de la prueba de remojo, los tratamientos resultaron superiores al testigo en longitud de plúmula. La semilla osmoacondicionada tuvo una tendencia a presentar mayor longitud de plúmula y radícula y mayor porcentaje de germinación cuando la semilla se sometió posteriormente a remojo. Los patrones electroforéticos son muy semejantes, lo que indica gran parecido a nivel bioquímico entre las razas estudiadas; es deseable un fraccionamiento más fino al extraer las proteínas y técnicas más sensibles para la detección de diferencias.

El osmoacondicionamiento es usado para mejorar la germinación y el comportamiento de plántula en campo. Se usan diferentes sustancias, como ácido giberélico, nitrato de potasio y polietilenglicol (PEG). Los cambios que ocurren en la semilla osmoacondicionada, que le permiten responder mejor durante el proceso de la germinación y el establecimiento de plántula, no se conocen bien. En México contamos con gran diversidad de variedades de maíces que se siembran bajo condiciones de temporal. Los antecedentes muestran que las razas nativas presentan diferencias en las fases de la germinación. En este trabajo se investigó (año 2015) el efecto del osmoacondicionamiento sobre el maíz tipo palomero para dilucidar si tiene un efecto positivo. En la prueba de remojo previo de la semilla (soak test) no se observaron diferencias entre el testigo y los tratamientos de osmoacondicionamiento. El palomero resultó resistente a la inmersión en agua. No hubo efecto de los tratamientos sobre el peso total de plántula, comparados con el testigo; en la variable longitud de plúmula, todos los tratamientos fueron superiores al testigo; para longitud de radícula destacan los tratamientos con PEG a 6 y 12 h en ambos conteos y fueron superiores al testigo. Los tratamientos con KNO3 incrementaron la cantidad de proteína soluble de semilla completa. En el endospermo se observó un incremento en la proteína extraída. En acidez titulable hubo diferencias entre tratamientos. Se observaron algunas diferencias en los patrones electroforéticos de las proteínas de la semilla. El osmoacondicionamiento es aconsejable para esta raza por su efecto sobre longitud de plúmula y radícula.

Less than 0.5% of total water in the world is available for human consumption and agriculture. The major part of the world's water is saline and salinity in soils interferes in germination of seeds and the posterior development of the plant. In order to increase the osmotolerance of tomato, seedlings were associated with Azospirillum brasilense Cd, Azospirillum brasilense Cd transformed bacteria with a plasmid harboring a trehalose biosynthesis gene-fusion or Chlorella vulgaris. Two plant culture media: Hydroponic and Murashige and Skoog were tested. In the first set of studies seedlings were associated to single free cells meanwhile in a second set single and combined free cells were studied. A positive interaction between transformed Azospirillum and Chlorella vulagris and tomato plants was observed. Seedlings showed a salt concentration tolerance, as sodium chloride, up to 200 mM. According to our results, the association of plants with A. brasilense Cd-BIF and C. vulgaris is a viable approach to increase their salt tolerance and biomass, as consequence the possible use of sea water to irrigate horticultural plants.

Trehalose is a non-reducing disaccharide formed by two glucose molecules. It is widely distributed in Nature and has been isolated from certain species of bacteria, fungi, invertebrates and plants, which are capable of surviving in a dehydrated state for months or years and subsequently being revived after a few hours of being in contact with water. This disaccharide has many biotechnological applications, as its physicochemical properties allow it to be used to preserve foods, enzymes, vaccines, cells etc., in a dehydrated state at room temperature. One of the most striking findings a decade ago was the discovery of the genes involved in trehalose biosynthesis, present in a great number of organisms that do not accumulate trehalose to significant levels. In plants, this disaccharide has diverse functions and plays an essential role in various stages of development, for example in the formation of the embryo and in flowering. Trehalose also appears to be involved in the regulation of carbon metabolism and photosynthesis. Recently it has been discovered that this sugar plays an important role in plant-microorganism interactions.

Alfalfa (Medicago sativa L.) is an important forage crop in many countries with high biomass production and the third-most-cultivated crop in the United States, with high protein content and potential use as biofuel. Here we describe the use of a chimeric translational fusion of yeast trehalose-6-phosphate synthase (EC 2.4.1.15) and trehalose-6-phosphate phosphatase (EC 3.1.3.12) genes to engineer stress tolerance in alfalfa. The gene fusion was overexpressed using either the constitutive 35S promoter or the stress-regulated rd29A promoter. Transgene insertion in the genome was checked by polymerase chain reaction and transcript expression by reverse transcription polymerase chain reaction. Several independent transgenic plants were selected in the presence of kanamycin and further analyzed. Yeast gene encoding trehalose-6-phosphate synthase (TPS1)-yeast gene encoding trehalose-6-phosphate phosphatase (TPS2) gene expression under the 35S promoter led to plants with stunted growth and less biomass, whereas TPS1-TPS2 expression driven by the rd29A promoter improved growth and provoked a significant increase in plant biomass at the foliage level in the different transgenic lines. Trehalose accumulated in all the different lines at similar levels under stress conditions. Transgenic plants displayed a significant increase in drought, freezing, salt, and heat tolerance. This is the first time that genetic engineering of trehalose metabolism is reported in alfalfa and demonstrates that it can improve multiple stress protection in this crop. Thus, the yeast TPS-TPP fusion protein represents a great potential for generating stress-tolerant crop plants for agriculture.

Bacteria of the genus Azospirillum increase the grain yield of several grass crops. In this work the effect of inoculating maize plants with genetically engineered Azospirillum brasilense for trehalose biosynthesis was determined. Transformed bacteria with a plasmid harboring a trehalose biosynthesis gene-fusion from Saccharomyces cerevisiae were able to grow up to 0.5 M NaCl and to accumulate trehalose, whereas wild-type A. brasilense did not tolerate osmotic stress or accumulate significant levels of the disaccharide. Moreover, 85% of maize plants inoculated with transformed A. brasilense survived drought stress, in contrast with only 55% of plants inoculated with the wild-type strain. A 73% increase in biomass of maize plants inoculated with transformed A. brasilense compared with inoculation with the wild-type strain was found. In addition, there was a significant increase of leaf and root length in maize plants inoculated with transformed A. brasilense. Therefore, inoculation of maize plants with A. brasilense containing higher levels of trehalose confers drought tolerance and a significant increase in leaf and root biomass. This work opens the possibility that A. brasilense modified with a chimeric trehalose biosynthetic gene from yeast could increase the biomass, grain yield and stress tolerance in other relevant crops.

SummaryThere are no reports on the possible protective role of trehalose in tropical monocot plants such as banana, when exposed to salt stress. In the present work, banana plants (Musa acuminata ‘Grand Nain’) were transformed with a fusion of two genes from Saccharomyces cerevisiae involved in the biosynthetic pathway of trehalose [trehalose-6-phosphate synthase (tps); and trehalose-6-phosphate phosphatase (tpp)] under the control of the Arabidopsis rd29A drought-stress inducible promoter. Transformed (rd29A::tps-tpp; TF) and wild-type (WT) plants were subjected to three different concentrations of NaCl (100, 200, or 300 mM) for 9 d. The trehalose concentration in the leaves of WT plants was approx. 3.5 µg g−1 fresh weight (FW) when not exposed to salinity, and increased only slightly (to 4.0 µg g−1 FW) when exposed to 100 or 200 mM NaCl. In TF plants, however, the trehalose concentration was approx. 4.0 µg g−1 FW when not exposed to salinity, but increased with salinity, reaching values of ≥ 8.0 µg g−1 ...

Improving stress tolerance is a major goal for agriculture. Trehalose is a key molecule involved in drought tolerance in anhydrobiotic organisms. Here we describe the construction of a chimeric translational fusion of yeast trehalose-6-phosphate synthase and trehalose-6-phosphate phosphatase. This construct was overexpressed in yeast cells displaying both TPS and TPP enzyme activities and trehalose biosynthesis capacity. In Arabidopsis thaliana, the gene fusion was overexpressed using either the 35S promoter or the stress-regulated rd29A promoter. Transgene insertion in the genome was checked by PCR and transcript expression by RT-PCR. Several independent homozygous lines were selected in the presence of kanamycin and further analyzed. Trehalose was accumulated in all these lines at low levels. No morphological or growth alterations were observed in lines overexpressing the TPS1-TPS2 construct, whereas plants overexpressing the TPS1 alone under the control of the 35S promoter had aberrant growth, color and shape. TPS1-TPS2 overexpressor lines were glucose insensitive, consistent with a suggested role of trehalose/T6P in modulating sugar sensing and carbohydrate metabolism. Moreover, TPS1-TPS2 lines displayed a significant increase in drought, freezing, salt and heat tolerance. This is the first time that trehalose accumulation in plants is shown to protect against freezing and heat stress. Therefore, these results demonstrate that engineering trehalose metabolism with a yeast TPS-TPP bifunctional enzyme confers multiple stress protection in plants, comprising a potential tool to improve stress-tolerance in crops. ispartof: Planta vol:226 issue:6 pages:1411-1421 ispartof: location:Germany status: published

Common bean crops are staple food of great economic importance in developing countries and the most important source of plant protein around the world. Nevertheless, an efficient and reliable regeneration method is still lacking for this crop. Here it is described two economically important bean varieties, Bayomex (BAY) and Flower of May 199 (FM199), that yielded 84 and 78% explants with shoots and developed 3.6 and 2.61 shoots per explant respectively, in MS culture media with B5 vitamins (MSB5) and 10 µM benzylaminopurine (BAP). Multiple shoots of BAY and FM199 were promoted in MSB5 with BAP or thidiazuron (TDZ). Using 4.54 µM TDZ induced 2.5 and 2.9 shoots per explant and at 4.44 µM BAP 1.7 and 2.2 shoots per explant were formed in BAY and FM199, respectively. In the hypocotyl zone, rooting was induced with TDZ between 0.41 and 1.73 µM and with BAP between 0.44 and 1.68 µM. Callus was induced with 2.72 and 4.54 µM TDZ, adding 2.66 and 4.44 µM BAP. The FM199 shoots obtained with 2.72 and 4.54 µM TDZ and subsequently cultured in media with 2.72 µM TDZ promoted plant development and regeneration. There was an increase a 4.44 shoots per explant using the BAY variety with TDZ and subsequently grown with 4.44 µM BAP. Rooting of shoots was promoted in the media with 0.44 µM BAP or without phytoregulators. Regenerated plants were acclimatized in soil, grew normally and developed seeds.

Bacteria of the genus Arthrobacter are commonly found in the soil and plant rhizosphere . In this study we report the draft genome of Arthrobacter chlorophenolicus strain Mor30.16 that was isolated from rhizosphere of beans grown in Cuernavaca Morelos, Mexico. This strain promotes growth and ameliorates drought stress in bean plants.

The resurrection plant Selaginella lepidophylla (Division Lycophyta) belongs to an ancient lineage of vascular plants that can withstand complete desiccation for years and be revived after only a few hours of rehydration. To better understand the molecular basis of dehydration tolerance, a complementary DNA library was constructed from S. lepidophylla microphyll fronds dehydrated for 2.5 h and used to generate an expressed sequence tag (EST) database. ESTs were obtained for 1046 clones representing 874 unique transcripts. Putative functions were assigned to 653 (62.4%) of these clones after comparison with protein databases, whereas 212 (20.2%) sequences having significant similarity to known sequences whose functions are unclear and 181 (17.3%) sequences having no similarity to known sequences. The S. lepidophylla ESTs were compared to the Selaginella moellendorffii EST database using the tBLASTX algorithm. Approximately 36–38% of the S. lepidophylla ESTs exhibited similarity to the S. moellendorffii ESTs, whereas 62–63% were unique to S. lepidophylla and S. moellendorffii , respectively. For those S. lepidophylla ESTs for which functional assignments could be made, the largest functional categories included photosynthesis (17%), followed by primary metabolism (14%) and disease/defense-related functions (11%). S. lepidophylla had a higher relative percentage of ESTs within the transporter, cell structure, secondary metabolism, molecular chaperones (i.e., heat shock proteins), and abiotic stress-related (i.e., late embryogenesis abundance proteins) functional categories than did S. moellendorffii . Thus, S. lepidophylla may serve as a rich genetic resource for the identification of novel genes associated with environmental stress and dehydration tolerance.

Insertion of foreign DNA into plant genomes occurs randomly and with low frequency. Hence, a selectable marker is generally required to identify transgenic plants. Until now, all selection systems have been based on the use of non-plant genes, derived from microorganisms and usually conferring antibiotic or herbicide resistance. The use of microorganism-derived genes however has raised biosafety concerns.We have developed a novel selection system based on enhancing the expression of a plant-intrinsic gene and the use of a harmless selection agent. Selection takes advantage of the reduced glucose sensitivity of seedlings with enhanced expression of AtTPS1, a gene encoding trehalose-6-P synthase. As a result, transformants can be identified as developing green seedlings amongst the background of small, pale non-transformed plantlets on high glucose medium. In addition, vegetative regeneration of tobacco leaf explants is very sensitive to high external glucose. Overexpression of AtTPS1 in tobacco allows selecting glucose insensitive transgenic shoots. ispartof: Journal of Biotechnology vol:121 issue:3 pages:309-317 ispartof: location:Netherlands status: published

Trehalose (a-D-glucopyranosil-(1,1)-a-D-glucopyranoside) is a non-reducing disaccharide, formed by two molecules of glucose. It is present in a broad spectrum of different organisms playing roles as reserve carbohydrate and conferring tolerance to abiotic stress. It posses broad biotechnological applications, since it is able to protect proteins and biological membranes, which allows its use for preserving foods, enzymes, vaccines, cells, tissues and organs, and can also substitute sucrose in confitery and bakery. The presence of this disaccharide in plants, has several functions, where besides of being an osmoprotectant it plays an essential role in several developmental stages, such as embryo maturation and flowering. Trehalose also is involved in regulating carbon metabolism and photosynthesis, and its accumulation is related with tolerance to abiotic stresses. Finally, this sugar seems to perform an essential role in the plant-microorganism interaction. ispartof: Revista Chapingo. Serie Horticultura vol:11 issue:2 pages:193-202 status: published

In the human pathogen Pseudomonas aeruginosa, betaine aldehyde dehydrogenase (BADH) may play a dual role assimilating carbon and nitrogen from choline or choline precursors--abundant at infection sites--and producing glycine betaine, which protects the bacteria against the high-osmolarity stress prevalent in the infected tissues. We cloned the P. aeruginosa BADH gene and expressed the BADH protein in Escherichia coli. The recombinant protein appears identical to its native counterpart, as judged by Western blot, N-terminal amino acid sequence, tryptophan-fluorescence emission spectra, circular-dichroism spectroscopy, size-exclusion chromatography, and kinetic properties. Computational analysis indicated that the promoter sequence of the putative operon that includes the BADH gene has a consensus-binding site for the choline-sensing transcription repressor BetI, and putative boxes for ArcA and Lrp transcription factors but no known elements of response to osmotic stress. This is consistent with the strong induction of BADH expression by choline and with the lack of effect of NaCl. As there were significant amounts of BADH protein and activity in P. aeruginosa cells grown on glucose plus choline, as well as the BADH activity exhibiting tolerance to salt, it is likely that glycine betaine is synthesized in vivo and could play an important osmoprotectant role under conditions of infection.

Transformed root cultures of Galphimia glauca (Malpighiaceae) were established by infecting cotyledons and hypocotyls with Agrobacterium rhizogenes ATCC 15 834. Cotyledon-derived cell lines were grown in liquid B5 nutrient medium without phytohormones and have shown the typical hairy roots phenotype over two years of continuous subculturing. PCR analysis was used to confirm the integration of rol A and rol C genes into the plant genome. The transformed cultures synthesized three major norfriedelanes, the new glaucacetalins A-C (1-3), which were secreted into the nutrient medium. The structural elucidation of these in vitro produced metabolites was performed by the application of high resolution NMR techniques that proved them to be triterpenoids related to the known galphimines, the sedative principles of this plant species. These results suggest the possibility of further biotechnological exploration of sedative friedelane biosynthesis by in vitro plant organ cultures.

In Arabidopsis (Arabidopsis thaliana), trehalose is present at almost undetectable levels, excluding its role as an osmoprotectant. Here, we report that overexpression of AtTPS1 in Arabidopsis using the 35S promoter led to a small increase in trehalose and trehalose-6-P levels. In spite of this, transgenic plants displayed a dehydration tolerance phenotype without any visible morphological alterations, except for delayed flowering. Moreover, seedlings overexpressing AtTPS1 exhibited glucose (Glc)- and abscisic acid (ABA)-insensitive phenotypes. Transgenic seedlings germinated on Glc were visibly larger with green well expandedcotyledonary leaves and fully developed roots, in contrast with wild-type seedlings showing growth retardation and absence of photosynthetic tissue. An ABA dose-response experiment revealed a higher germination rate for transgenic plants overexpressing AtTPS1 showing insensitive germination kinetics at 2.5 mM ABA. Interestingly, germination in the presence of Glc did not trigger an increase in ABA content in plants overexpressing AtTPS1. Expression analysis by quantitative reverse transcription-PCR in transgenic plants showed up-regulation of the ABI4 and CAB1 genes. In the presence of Glc, CAB1 expression remained high, whereas ABI4, HXK1, and ApL3 levels were down-regulated in the AtTPS1-overexpressing lines. Analysis of AtTPS1 expression in HXK1-antisense or HXK1-sense transgenic lines suggests the possible involvement of AtTPS1 in the hexokinase-dependent Glc-signaling pathway. These data strongly suggest that AtTPS1 has a pivotal role in the regulation of Glc and ABA signaling during vegetative development. ispartof: Plant physiology vol:136 issue:3 pages:3649-3659 ispartof: location:United States status: published

The resurrection plant Craterostigma plantagineum has the ability to survive complete dehydration. In an attempt to further understand desiccation tolerance in this plant, the CpMYB10 transcription factor gene was functionally characterized. CpMYB10 is rapidly induced by dehydration and abscisic acid (ABA) treatments in leaves and roots, but no expression was detected in fully hydrated tissues. Electrophoretic mobility shift assay experiments showed binding of rCpMYB10 to specific mybRE elements within the LEA Cp11-24 and CpMYB10 promoters. Localization of CpMYB10 transcript by in situ reverse transcription-PCR reactions showed expression in vascular tissues, parenchyma, and epidermis both in leaves and roots in response to ABA. Transgenic Arabidopsis plants transformed with CpMYB10 promoter fused to GUS gene showed reporter expression under ABA and stress conditions in several organs. Overexpression of CpMYB10 cDNA in Arabidopsis led to desiccation and salt tolerance of transgenics lines. Interestingly, it was found that plants overexpressing CpMYB10 exhibited Glc-insensitive and ABA hypersensitive phenotypes. Therefore, our results indicate that CpMYB10 in Arabidopsis is mediating stress tolerance and altering ABA and Glc signaling responses.

Plants, such as Arabidopsis thaliana and Selaginella lepidophylla, contain genes homologous with the trehalose-6-phosphate synthase (TPS) genes of bacteria and fungi. Most plants do not accumulate trehalose with the desert resurrection plant S. lepidophylla, being a notable exception. Overexpression of the plant genes in a Saccharomyces cerevisiae tps1 mutant results in very low TPS-catalytic activity and trehalose accumulation. We show that truncation of the plant-specific N-terminal extension in the A. thalianaAtTPS1 and S. lepidophyllaSlTPS1 homologues results in 10–40-fold higher TPS activity and 20–40-fold higher trehalose accumulation on expression in yeast. These results show that the plant TPS enzymes possess a high-potential catalytic activity. The growth defect of the tps1 strain on glucose was restored, however, the proper homoeostasis of glycolytic flux was not restored, indicating that the plant enzymes were unable to substitute for the yeast enzyme in the regulation of hexokinase activity. Further analysis of the N-terminus led to the identification of two conserved residues, which after mutagenesis result in strongly enhanced trehalose accumulation upon expression in yeast. The plant-specific N-terminal region may act as an inhibitory domain allowing modulation of TPS activity.

Opuntia spp. is easily adaptable to arid and semiarid climates, and represents a valuable alternative for the agricultural development in dry regions. Twelve accessions of Opuntia spp. fruits were characterized to know the evolution of their bioactive compounds concentration such as phenolics, flavonoids and pigments (Isobetanins, betanins, indicaxanthins and total betalains) during their ripening process. DPPH and ABTS radical scavenging capacity were determined and correlated with pigments and bioactive compounds concentration. Bioactive compounds and pigments concentration increased along ripening. Higher concentration of total betalains were found in ripe ꞌBondaꞌ (O. guerrana Griffiths) and ꞌRojo Tolucaꞌ (Opuntia robusta var. Larreyi) accessions, being of 78.96 ± 0.54 mg 100 g-1 DW and 69.87 ± 0.29 mg 100 g-1 DW respectively. Findings in this work can help to select Opuntia spp. accessions with high potential to be established as a valuable crop in the arid zones of Mexico.

Availability of water is the most limiting factor for crop productivity and yield. Therefore, there is a strong need to understand plant adaptation against adverse envirommental conditions to impreve stress tolerance. Plant stress responses insolve the exprsotion of a plethora of genes with an adaptive role. The encoded products comprise enzymes catalyzing the synthesis of osmoprotectants or antioxidants, lipid desaturases, water channels, and the transporters among others. Here we show the characterization of 2 different genes. First, the CpMYB10 transcription factor gene from the resurrection plant Craterostigma plantagineum is rapidly induced by dehydration and ABA treatment in leaves and roots. Transsgenic Arabidops thaliana plants, transformed with CpMYB10 premeter fased to GUS gene, showed reporter expression under ABA and stress conditions in several organs. Overeapression of CpMYB10 cDNA in Arabidopais led to desiccation and salt tolerances of transgenic lines, which also exhibited glucose-insensitive and ABA-hypersensitive phenotyes. Also, the trehalose 6-phosphate synthase (AtTPS1) gene overxpressed in Arabidopsis conferred dehydration tolerance, and glucose- and ABA- insensitive phenotypes. Transgenic seedlings germinated on high concentrations of glucose developednormallt, in contrast to wild type seedlings. Germination in the presence of ABA revealed higher germination rate for transgenic plants overexpressing AtTPs1. Gene-expression analysis in transgenic plants overexpressing AtTPS1 showed up-or down-regulation of several genes involved in glucose sensing and ABA signaling. Therefore, our results provide evidence that CpMYB10 and AtTPS1, when overexpressed in Arabidopsis, led to a stress-tolerance phenotype through a cross-talk to ABA and glucose signaling pathways.

Spider venom contains a mixture of peptide toxins, some able to kill insects specifically to those considered as important pest. In this study, a peptide toxin produced by the Macrothele gigas spider, Magi 6, was cloned and expressed in tobacco plants, as this toxin has been shown to constitute an effective insecticide. For this purpose, a genetic construction for the cDNA that codifies for Magi 6 was subcloned in a plant expression vector using the 35S promoter and the 5′-end leader from tobacco mosaic virus, in order to transform tobacco leaf disks. The resulting plants demonstrated the presence of Magi 6 gene in the tobacco genome using PCR, and transcription of the cDNA was verified by means of RT-PCR. The expression of the Magi 6 peptide in tobacco was demonstrated by Western blot, which exhibited the expected size, thus suggesting a correct processing of the signal peptide. No morphological alterations in the different transgenic lines were observed, nor any change in plant growth. Subsequently, experiments were carried out challenging detached leaves or whole plants with the herbivorous insect Spodoptera frugiperda. The bioassays indicated that the transgenic lines were significantly more resistant than the wild type plants. This work demonstrated that the expression of Magi 6 peptide in transgenic plants conferred resistance to insect attack and opens the possibility of employing this peptide to improve the resistance of diverse plants.

Improving stress tolerance and yield in crops are major goals for agriculture. Here, we show a new strategy to increase drought tolerance and yield in legumes by overexpressing trehalose-6-phosphate synthase in the symbiotic bacterium Rhizobium etli. Phaseolus vulgaris (common beans) plants inoculated with R. etli overexpressing trehalose-6-phosphate synthase gene had more nodules with increased nitrogenase activity and higher biomass compared with plants inoculated with wild-type R. etli. In contrast, plants inoculated with an R. etli mutant in trehalose-6-phosphate synthase gene had fewer nodules and less nitrogenase activity and biomass. Three-week-old plants subjected to drought stress fully recovered whereas plants inoculated with a wild-type or mutant strain wilted and died. The yield of bean plants inoculated with R. etli overexpressing trehalose-6-phosphate synthase gene and grown with constant irrigation increased more than 50%. Macroarray analysis of 7,200 expressed sequence tags from nodules of plants inoculated with the strain overexpressing trehalose-6-phosphate synthase gene revealed upregulation of genes involved in stress tolerance and carbon and nitrogen metabolism, suggesting a signaling mechanism for trehalose. Thus, trehalose metabolism in rhizobia is key for signaling plant growth, yield, and adaptation to abiotic stress, and its manipulation has a major agronomical impact on leguminous plants.

Adaptation to desiccation tolerance or anhydrobiosis has puzzled scientists for more than 300 years. Over the last few decades, considerable emphasis has been placed on understanding the role of two key molecules involved in anhydrobiosis: a peculiar disaccharide named trehalose and the hydrophilic LEA (Late Embryogenesis Abundant) proteins. In an article published in the Biochemical Journal in 2005, Alan Tunnacliffe and colleagues found that LEA proteins (alone, or more so in combination with trehalose) can protect stress-sensitive enzymes, such as citrate synthase and lactate dehydrogenase, from aggregation due to desiccation and freezing. Upon heat-stress, however, LEA proteins alone cannot prevent these enzymes from aggregating unless trehalose is present. This is the first report that LEA proteins can act as ‘molecular shields’ to prevent aggregation-induced cell damage due to water loss.

Background The compatible solute trehalose is a non-reducing disaccharide, which accumulates upon heat, cold or osmotic stress. It was commonly accepted that trehalose is only present in extremophiles or cryptobiotic organisms. However, in recent years it has been shown that although higher plants do not accumulate trehalose at significant levels they have actively transcribed genes encoding the corresponding biosynthetic enzymes. Results In this study we show that trehalose biosynthesis ability is present in eubacteria, archaea, plants, fungi and animals. In bacteria there are five different biosynthetic routes, whereas in fungi, plants and animals there is only one. We present phylogenetic analyses of the trehalose-6-phosphate synthase (TPS) and trehalose-phosphatase (TPP) domains and show that there is a close evolutionary relationship between these domains in proteins from diverse organisms. In bacteria TPS and TPP genes are clustered, whereas in eukaryotes these domains are fused in a single protein. Conclusion We have demonstrated that trehalose biosynthesis pathways are widely distributed in nature. Interestingly, several eubacterial species have multiple pathways, while eukaryotes have only the TPS/TPP pathway. Vertebrates lack trehalose biosynthetic capacity but can catabolise it. TPS and TPP domains have evolved mainly in parallel and it is likely that they have experienced several instances of gene duplication and lateral gene transfer.

Plants sense and respond to changes in carbon and nitrogen metabolites during development and growth according to the internal needs of their metabolism. Sugar-sensing allows plants to switch off photosynthesis when carbohydrates are abundant. These processes involve regulation of gene and protein activity to allow plants the efficient use of energy storage. Besides being a key element in carbon metabolism, glucose (Glc) has unravelled as a primary messenger in signal transduction. It has been proved that hexokinase (HXK) is a Glc sensor. An unusual disaccharide named trehalose is present in very low levels in most plants except for the desiccation-tolerant plants known as ‘resurrection’ plants where trehalose functions as an osmoprotectant. We have shown that overexpression of the Arabidopsis trehalose-6-phosphate synthase gene (AtTPS1) in Arabidopsis promotes trehalose and trehalose-6-phosphate (T6P) accumulation. Seedlings expressing AtTPS1 displayed a Glc-insensitive phenotype. Transgenic lines germinated normally on Glc, in contrast to wild-type seedlings showing growth retardation and absence of chlorophyll and root elongation. Gene-expression analysis in transgenic plants showed up-regulation of several genes involved in sugar signalling and metabolism. These data suggest that AtTPS1 and accordingly T6P and trehalose play an important role in the regulation of Glc sensing and signalling genes during plant development. ispartof: Biochemical Society Transactions vol:33 issue:Pt 1 pages:276-279 ispartof: location:Cirencester, UK status: published

Laccases are important enzymes for bioremediation and the best-characterised are from the fungus Trametes versicolor. Here, we describe the cloning and characterisation of a new variant of laccase from T. versicolor and its expression in Saccharomyces cerevisiae. We have performed a sequence-based analysis of Trametes laccases that leads to a proposal for a new nomenclature of this fungus laccases according to their phylogenetic relationships since their nomenclature based on IPs is ambiguous. We also describe the kinetic properties of the recombinant enzyme.

A number of systems to insert foreign DNA into a plant genome have been developed so far. However, only a small percentage of transgenic plants are obtained using any of these methods. Stable transgenic plants are selected by co-introduction of a selectable marker gene, which in most cases are genes that confer resistance against antibiotics or herbicides. In this chapter we describe a new method for selection of transgenic plants after transformation. The selection agent used is the nontoxic and common sugar glucose. Wild-type Arabidopsis thaliana plantlets that have been germinated on glucose have small white cotyledons and remain petite because the external sugar switches off the photosynthetic mechanism. The selectable marker gene encodes the essential trehalose-6-phophate synthase, AtTPS1, that catalyzes the first reaction of the two-step trehalose synthesis. Upon ectopic expression of AtTPS1 driven by the 35S promoter, transformed Arabidopsis thaliana plants became insensitive to glucose in comparison to wild-type plants. After transformation using AtTPS1 as a selection marker and 6% glucose as selection agent it is possible to single out the green and normal sized transgenic plants amid the nontransformed plantlets.

A number of systems to insert foreign DNA into a plant genome have been developed so far. However, only a small percentage of transgenic plants are obtained using any of these methods. Stable transgenic plants are selected by co-introduction of a selectable marker gene, which in most cases are genes that confer resistance against antibiotics or herbicides. In this chapter we describe a new method for selection of transgenic plants after transformation. The selection agent used is the nontoxic and common sugar glucose. Wild-type Arabidopsis thaliana plantlets that have been germinated on glucose have small white cotyledons and remain petite because the external sugar switches off the photosynthetic mechanism. The selectable marker gene encodes the essential trehalose-6-phophate synthase, AtTPS1, that catalyzes the first reaction of the two-step trehalose synthesis. Upon ectopic expression of AtTPS1 driven by the 35S promoter, transformed Arabidopsis thaliana plants became insensitive to glucose in comparison to wild-type plants. After transformation using AtTPS1 as a selection marker and 6% glucose as selection agent it is possible to single out the green and normal sized transgenic plants amid the nontransformed plantlets.

The accumulation of the disaccharide trehalose in anhydrobiotic organisms allows them to survive severe environmental stress. A plant cDNA,SlTPS1, encoding a 109-kD protein, was isolated from the resurrection plant Selaginella lepidophylla, which accumulates high levels of trehalose. Protein-sequence comparison showed that SlTPS1 shares high similarity to trehalose-6-phosphate synthase genes from prokaryotes and eukaryotes. SlTPS1mRNA was constitutively expressed in S. lepidophylla. DNA gel-blot analysis indicated that SlTPS1 is present as a single-copy gene. Transformation of a Saccharomyces cerevisiae tps1Δ mutant disrupted in theScTPS1 gene with S. lepidophylla SlTPS1restored growth on fermentable sugars and the synthesis of trehalose at high levels. Moreover, the SlTPS1 gene introduced into the tps1Δ mutant was able to complement both deficiencies: sensitivity to sublethal heat treatment at 39°C and induced thermotolerance at 50°C. The osmosensitive phenotype of the yeast tps1Δ mutant grown in NaCl and sorbitol was also restored by the SlTPS1 gene. Thus, SlTPS1 protein is a functional plant homolog capable of sustaining trehalose biosynthesis and could play a major role in stress tolerance in S. lepidophylla.