Edmundo Lozoya-Gloria

Recent developments in abscission: shedding light on the shedding process

Plants are able to shed organs that are diseased, stressed or at the terminal stage of their development. Shedding takes place at a morphologically distinct site, the abscission zone, in a process that is accelerated by ethylene and retarded by auxin. Associated with cell separation is an increase in the expression of a spectrum of gene products, including hydrolytic enzymes and peptides that protect the exposed fracture surface from pathogenic attack. Key research objectives are the identification of abscission-specific gene promoters and the elucidation of the morphogenetic events that regulate cell differentiation in the abscission zone. Manipulation of both the site and rate of shedding will then become feasible.

Symptom Remission and Specific Resistance of Pepper Plants After Infection by Pepper golden mosaic virus

ABSTRACT Pepper golden mosaic virus (PepGMV) is an important begomovirus infecting solanaceous crops in Mexico and Central America. Under controlled conditions for growth and inoculation with a low-pressure biolistic device, PepGMV-infected pepper plants consistently showed symptom remission or host recovery 12 to 15 days postinoculation (dpi). Inoculated plants initially developed the characteristic PepGMV symptoms; however, newer leaves presented a significant decrease or disappearance of symptoms. Younger asymptomatic, recovered leaves accumulated lower quantities of viral DNA and transcripts than the ones found in the symptomatic tissue. Nonetheless, viral DNA did not disappear during the evaluation period (up to 35 dpi), suggesting that a population of viral molecules escape from plant defensive mechanisms to maintain a subliminal, symptomless infection. Recovery was correlated with a specific resistance to PepGMV but not to Pepper huasteco yellow vein virus, a different gemi-nivirus commonly found in mixed infections with PepGMV. Virus-related small interfering RNAs were detected in practically all tissues (from symptomatic to recovered leaves) but it was not possible to establish a correlation between concentration and symptom severity. The participation of a posttranscriptional gene silencing mechanism in the recovery process and specific resistance is discussed.

Overexpression in Catharanthus roseus hairy roots of a truncated hamster 3-hydroxy-3-methylglutaryl-CoA reductase gene

Catharanthus roseus (L.) G. Don hairy roots harboring hamster 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) (EC 1.1.1.88) cDNA without membrane-binding domain were evaluated by quantifying the levels of sterols and some indol-alkaloids. Clone 236, with the highest hybridization signal, had the lowest soluble and microsomal HMGR activity and produced more ajmalicine and catharanthine than the control but had reduced campesterol concentration. Clone 19, with low hybridization signal, had high soluble HMGR activity and produced high levels of campesterol and five to seven times more serpentine than the control but a low level of ajmalicine and no accumulation of catharanthine. These results suggest a possible role for HMGR in indole alkaloid biosynthesis and a possible cosuppression of both the endogenous and foreign HMGR genes in clone 236.

Biosynthesis of the sesquiterpenic phytoalexin capsidiol in elicited root cultures of chili pepper (Capsicum annuum)

The biosynthesis of the sesquiterpenic phytoalexin capsidiol was investigated using in vitro root cultures of chili pepper (Capsicum annuum) elicited with cellulase. Optimal concentrations of cellulase and sucrose for capsidiol production were established. A simple spectrophotometric procedure to quantify capsidiol was improved. Monoclonal antibodies against a tobacco sesquiterpene cyclase were used to detect a similar protein in pepper root extracts. We found that capsidiol was secreted to the medium and the maximal production was achieved at 24 h after elicitation. In contrast, the maximal amount of the elicitor inducible sesquiterpene cyclase was found between 6 and 8 h. Addition of small amounts of polyvinylpyrrolidone was necessary for sesquiterpene cyclase enzyme activity assays.

The capsicum transcriptome DB: a "hot" tool for genomic research

Chili pepper (Capsicum annuum) is an economically important crop with no available public genome sequence. We describe a genomic resource to facilitate Capsicum annuum research. A collection of Expressed Sequence Tags (ESTs) derived from five C. annuum organs (root, stem, leaf, flower and fruit) were sequenced using the Sanger method and multiple leaf transcriptomes were deeply sampled using with GS-pyrosequencing. A hybrid assembly of 1,324,516 raw reads yielded 32,314 high quality contigs as validated by coverage and identity analysis with existing pepper sequences. Overall, 75.5% of the contigs had significant sequence similarity to entries in nucleic acid and protein databases; 23% of the sequences have not been previously reported for C. annuum and expand sequence resources for this species. A MySQL database and a user-friendly Web interface were constructed with search-tools that permit queries of the ESTs including sequence, functional annotation, Gene Ontology classification, metabolic pathways, and assembly information. The Capsicum Transcriptome DB is free available from http://www.bioingenios.ira.cinvestav.mx:81/Joomla/

Montanoa tomentosa glandular trichomes containing kaurenoic acids chemical profile and distribution

Montanoa tomentosa has been used in traditional medicine in Mexico to treat diverse female health disorders; it is particularly useful in inducing childbirth. Microscopic analysis of leaf surfaces of M. tomentosa revealed the presence of glandular trichomes. The chemical profile and distribution of glandular trichomes from different developmental stages of M. tomentosa leaves were investigated. Two diterpenic acids, kaurenoic and grandiflorenic were detected in glandular trichomes through the glandular microsampling technique and GC/MS analysis. In the glandular trichomes of the leaves also up to twenty-six volatile terpenes were identified, where beta-eudesmol and valencene were the most abundant terpenes.

Induced gene expression of 1-aminocyclopropane-1-carboxylic acid (ACC oxidase) in pepper (Capsicum annuum L.) by arachidonic acid ☆

Abstract Elicitation of pepper ( Capsicum annuum ) seedlings with arachidonic acid (AA) resulted in different defense responses. The transcriptional stimulation of the pepper phytoalexin biosynthesis related gene 5- epi -aristolochene synthase (PEAS) was demonstrated and a partial cDNA of the 1-aminocyclopropane-1-carboxylate (ACC) oxidase gene (CA-ACCO) was also isolated. Ethylene accumulation was subsequent to the CA-ACCO gene expression in AA treated fruits. The level of CA-ACCO transcript was assayed in pepper seedlings after treatment with salicylic acid (SA), methyl jasmonate (MeJa), cellulase, and Phytophthora capsici infection, as well as in wounded and different ripening stage fruits. The CA-ACCO gene was strongly expressed under stress conditions but hardly detected in breaker stage pepper fruits in contrast with tomato ripening fruits. Southern blot analysis revealed 3–4 members of the CA-ACCO gene family according to similar genes as reported in other plants.

Non-severe thermochemical hydrolysis of stover from white corn and sequential enzymatic saccharification and fermentation to ethanol.

A parametric study, with an initial load of 15%w/w of dry stover from white corn, was conducted to evaluate the sequential thermochemical hydrolysis (TH), enzymatic saccharification (ES) and fermentation of the whole slurry with ethanologenic Escherichia coli. The TH was designed to release the maximum amount of xylose with a concomitant formation of minimal amounts of furans. It was found that 29.0% or 93.2% of the xylan was recovered as free xylose at 130°C after 8 min in the presence of 1% or 2%w/w H2SO4 and produced only 0.06 or 0.44 g/L of total furans, respectively. After 24h of ES, 76.14-77.18 g/L of monosaccharides (pentoses and hexoses) were obtained. These slurries, which contained 0.03-0.26 g/L of total furans and 5.14-5.91 g/L of acetate, were fermented with 3.7 g/L of ethanologenic E. coli to produce 24.5-23.5 g/L of ethanol.

Biosynthesis of uterotonic diterpenes from Montanoa tomentosa (zoapatle)

Montanoa tomentosa (zoapatle) is a Central American plant used in Mexico in traditional herbal medicine to ease childbirth labor and to cure certain female disorders. Recently, crude extracts of M. tomentosa have been reported to have an aphrodisiacal effect on male rats. The bioactive molecules are the uterotonic diterpenes kaurenoic acid (KA), grandiflorenic acid (GF), and monoginoic acid (MO). Roots of M. tomentosa contain all three diterpenes, whereas in leaves only kaurenoic and GF are present. However, despite the pharmacological importance of these compounds, specific information about their biosynthesis and localization in the plant is not available. In this investigation, we followed the metabolic transformation of a tritium-labeled diterpene-precursor via geranylgeranyl diphosphate into each of the three diterpenes. Inhibitors of gibberellin biosynthesis were used to elucidate the sequence of conversion of the intermediates. Our results suggest the biosynthetic conversion of KA into GF by a putative cytochrome P450-like desaturase. Partial characterization of the enzyme revealed that it requires NADPH and O2 but is inhibited by 50 microM paclobutrazol, suggesting a cytochrome P450 desaturase like enzyme (EC 1.14.14.-). Optimal reaction conditions are 32 degrees C and a pH of 7.6, respectively. Apparent kinetics parameters for KA gave a K(m,app) of 36.31 microM, and a V(max, app) of 13.6 nmol KA mg(1)protein h(-1). Based on the data presented, a putative biosynthetic pathway is proposed for the uterotonic diterpenes of M. tomentosa.

Antisense expression of hmg1 from Arabidopsis thaliana encoding 3-hydroxy-3-methylglutaryl coenzyme A reductase, reduces isoprenoid production in transgenic tobacco plants

Summary With the aim of studying the production of chloroplast isoprenoid derivatives, chimeric gene constructs comprising partial and full-length forms of the Arabidopsis thaliana hmg1 gene, encoding 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR), in sense and antisense forms were fused to a chloroplast transit peptide (CTP) sequence from a pea ribulose bisphosphate carboxylase/oxygenase (Rubisco) small subunit gene. These plasmids were placed downstream from the constitutive CaMV 35S promoter and introduced into tobacco ( Nicotiana tabacum cv. Xanthi) by Agrobacterium -mediated transformation. Southern and northern blot analysis confirmed the transformation and transcription of the respective constructs within transgenic tobacco plants. In addition total and chloroplast specific HMGR enzyme activities, and the levels of chlorophyll, carotenoids and total sterols were analyzed. Results of these analyses suggest that neither partial nor full-length sense constructs had any effect on chloroplast isoprenoid production. However, the corresponding antisense constructs decreased general isoprenoid levels. The significance of these results is discussed.