Alejandro Calderón-Urrea

Ferrocenyl chalcones versus organic chalcones: a comparative study of their nematocidal activity.

A series of 30 organic chlacones and 33 ferrocenyl (Fc) chalcones were synthesized and characterized by melting point, elemental analysis, spectroscopy ((1)H NMR and FTIR) and, in two cases, by X-ray crystallography. The biological activity of each compound (10(-4)M in DMSO) against the model nematode Caenorhabditis elegans was examined in terms of % mortality (percent nematodes that died) and % fecundity (percent nematodes that reproduced) and compared to that obtained for the control medium (1% DMSO) over a 14-day period. Detailed conformational analyses for two Fc-chalcones (studied also by X-ray crystallography) were performed via molecular modeling studies. In general, the organic chalcones were found to be less polar than their Fc analogs. Some structure-activity relationships (SARs) were determined: (a) The nematocidal activities of the organic chalcones in this series were found to be much greater than those of their ferrocenyl analogs. (b) The position of the carbonyl group played a central role in the biological activity of both classes of chalcones studied. (c) For both classes of chalcones, lipophilicity of a compound seemed to play a significant role in its nematocidal activity. (d) The planarity of a ferrocenyl-chlacone seems to play a role in its activity.

Transcriptome Analysis of Pepper (Capsicum annuum) Revealed a Role of 24-Epibrassinolide in Response to Chilling

Brassinosteroids (BRs) have positive effects on many processes during plant growth, development, and various abiotic stress responses. However, little information is available regarding the global gene expression of BRs in response to chilling stress in pepper. In this study, we used RNA sequencing to determine the molecular roles of 24-epibrassinolide (EBR) during a chilling stress response. There were 39,829 transcripts, and, among them, 656 were differently-expressed genes (DEGs) following EBR treatment (Chill+EBR) compared with the control (Chill only), including 335 up-regulated and 321 down-regulated DEGs. We selected 20 genes out of the 656 DEGs for RT-qPCR analysis to confirm the RNA-Seq. Based on GO enrich and KEGG pathway analysis, we found that photosynthesis was significantly up-enriched in biological processes, accompanied by significant increases in the net photosynthetic rate (Pn), Fv/Fm, and chlorophyll content. Furthermore, the results indicate that EBR enhanced endogenous levels of salicylic acid (SA) and jasmonic acid (JA) while suppressing the ethylene (ETH) biosynthesis pathway, suggesting that BRs function via a synergistic cross-talk with SA, JA, and ETH signaling pathways in response to chilling stress. In addition, EBR induced cellulose synthase-like protein and UDP-glycosyltransferase, suggesting a contribution to the formation of cell wall and hormone metabolism. EBR also triggered the calcium signaling transduction in cytoplasm, and activated the expression of cellular redox homeostasis related genes, such as GSTX1, PER72, and CAT2. This work, therefor, identified the specific genes showed different expression patterns in EBR-treated pepper and associated with the processes of hormone metabolism, redox, signaling, transcription, and defense. Our study provides the first evidence of the potent roles of BRs, at the transcription level, to induce the tolerance to chilling stress in pepper as a function of the combination of the transcriptional activities, signaling transduction, and metabolic homeostasis.

Early development of the root-knot nematode Meloidogyne incognita.

Detailed descriptions of the early development of parasitic nematodes are seldom available. The embryonic development of the plant-parasitic nematode Meloidogyne incognita was studied, focusing on the early events. A fixed pattern of repeated cell cleavages was observed, resulting in the appearance of the six founder cells 3 days after the first cell division. Gastrulation, characterized by the translocation of cells from the ventral side to the center of the embryo, was seen 1 day later. Approximately 10 days after the first cell division a rapidly elongating two-fold stage was reached. The fully developed second stage juvenile hatched approximately 21 days after the first cell division. When compared to the development of the free-living nematode Caenorhabditis elegans, the development of M. incognita occurs approximately 35 times more slowly. Furthermore, M. incognita differs from C. elegans in the order of cell divisions, and the early cleavage patterns of the germ line cells. However, cytoplasmic ruffling and nuclear migration prior to the first cell division as well as the localization of microtubules are similar between C. elegans and M. incognita.

Selection and validation of reference genes for RT-qPCR analysis in potato under abiotic stress

BackgroundReal-time quantitative PCR (RT-qPCR) is the most commonly used method for accurately detecting gene expression patterns. As part of RT-qPCR analysis, normalization of the data requires internal control gene(s) that display uniform expression under different biological conditions. However, no invariable internal control gene exists, and therefore more than one reference gene is needed to normalize RT-qPCR results. Identification of stable reference genes in potato will improve assay accuracy for selecting stress-tolerance genes and identifying molecular mechanisms conferring stress tolerance in this species.ResultsIn the experiment, we assessed the expression of eight candidate internal control genes, namely elongation factor-1alpha (EF1α), actin, tubulin, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), adenine phosphoribosyl transferase (APRT), 60S ribosomal protein L8 (L8), Cullin 3A (CUL3A), and exocyst complex component sec3 (sec3), in a diverse set of potato samples representing drought stress and osmotic stress challenges, and using geNorm, NormFinder, BestKeeper and RefFinder softwares.ConclusionsThe results indicated that EF1α and sec3 were the most stably expressed genes in the potato under drought and osmotic stress conditions. This work will facilitate future work on gene expression studies in potato and also benefit other species of the Solanaceae, such as tomato.

Nontemplate Polymerization of Free Nucleotides Into Genetic Elements by Thermophilic DNA Polymerase in vitro

DNA synthesis is the cornerstone of all life forms and is required to replicate and restore the genetic information. Usually, DNA synthesis is carried out only by DNA polymerases semiconservatively to copy preexisting DNA templates. We report here that DNA strands were synthesized ab initio in the absence of any DNA or RNA template by thermophilic DNA polymerases at (a) a constant high temperature (74°C), (b) alternating temperatures (94°C/60°C/74°C), or (c) physiological temperatures (37°C). The majority of the ab initio synthesized DNA represented short sequence blocks, repeated sequences, intergenic spacers, and other unknown genetic elements. These results suggest that novel DNA elements could be synthesized in the absence of a nucleic acid template by thermophilic DNA polymerases in vitro. Biogenesis of genetic information by thermophilic DNA polymerase-mediated nontemplate DNA synthesis may explain the origin of genetic information and could serve as a new way of biosynthesis of genetic information that may have facilitated the evolution of life. Supplemental materials are available for this article. Go to the publishers online edition of Nucleosides, Nucleotides, and Nucleic Acids to view the free supplemental file.

Autotoxicity in cucumber (Cucumis sativus L.) seedlings is alleviated by silicon through an increase in the activity of antioxidant enzymes and by mitigating lipid peroxidation

Autotoxicity in plants limits their growth and that of nearby plants of the same species, which has obvious implications in crop yield and quality. Silicon (Si) has been shown to increase plant tolerance to autotoxic stress. However, the physiological mechanisms underlying the effects of Si in alleviating autotoxicity during germination in cucumber (Cucumis sativus L.) are unknown. Cinnamic acid derivatives, such as 3-phenylpropionic acid (PA), are a class of autotoxins present in cucumber root exudates. Our objective was to investigate Si-induced autotoxic stress tolerance in cucumber seedlings by focusing on the effects of Si on the induction of antioxidant defense pathways. We found that PA treatment significantly reduced seed germination, radicle length, lateral root number, fresh weight, AsA and GSH contents, and the activities of SOD, CAT, and APX in cucumber seedlings, while it increased membrane permeability and levels of MDA, proline, O2-, and H2O2. Application of Si enhanced growth of PA-treated plants and significantly increased germination rate, radicle length, lateral root number, fresh weight, AsA and GSH levels, and SOD, CAT, POD, and APX activities. These results suggest that exogenous Si alleviates autotoxicity caused by PA during seed germination by increasing antioxidant enzyme activities and mitigating lipid peroxidation.

Lateral Root Development in Potato Is Mediated by Stu-mi164 Regulation of NAC Transcription Factor

The NAC designation is derived from petunia (Petunia hybrida) gene NO APICAL MERISTEM (NAM) and Arabidopsis genes ATAF1/ATAF2 and CUP-SHAPED COTYLEDON2 (CUC2), which belongs to the family of plant-specific transcription factors (TFs), and plays important role in plant development processes, such as response to biotic and abiotic stress, and hormone signaling. MicroRNAs (miRNAs) are a class of small, non-coding endogenous RNAs which play versatile and significant role in plant stress response and development via negatively affecting gene expression at a post-transcriptional level. Here, we showed that Stu-mi164 had a complementary sequence in the CDS sequence of potato NAC TFs, and that NAC expression exhibited significant differences under osmotic stress. We measured expression levels of the Stu-mi164 target gene StNAC262 between control and PEG-treated plants using real-time PCR, and the results demonstrated that they had inverse relationship. We suggested that Stu-miR164 might drive overexpression of NAC gene under osmotic stress in potato. To confirm the regulation of NAC TFs by Stu-mi164, we developed transgenic plants, using Agrobacterium tumefaciens–mediated transformation, of the potato cultivars “Gannongshu 2” and “Kexin 3” overexpressing the Stu-mi164 or the TF StNAC262. Real-time PCR analysis of transgenic potato plants under osmotic (PEG) stress, showed that potato plants overexpressing Stu-mi164 had reduced expression of StNAC262 and their osmotic resistance decreased. Furthermore, these plants had low number of lateral roots although the same length as the control. Our findings support the regulatory role of Stu-miRNAs in controlling plant response to osmotic stress via StNAC262.

Significant and unique changes in phosphorylation levels of four phosphoproteins in two apple rootstock genotypes under drought stress

Drought stress is a major problem around the world and there is still little molecular mechanism about how fruit crops deal with moderate drought stress. Here, the physiological and phosphoproteomic responses of drought-sensitive genotype (M26) and drought-tolerant genotype (MBB) under moderate drought stress were investigated. Our results of the physiology analysis indicated that the MBB genotype could produce more osmosis-regulating substances. Furthermore, phosphoproteins from leaves of both genotypes under moderate drought stress were analyzed using the isobaric tags for relative and absolute quantification technology. A total of 595 unique phosphopeptides, 682 phosphorylated sites, and 446 phosphoproteins were quantitatively analyzed in the two genotypes. Five and thirty-five phosphoproteins with the phosphorylation levels significantly changed (PLSC) were identified in M26 and MBB, respectively. Among these, four PLSC phosphoproteins were common to both genotypes, perhaps indicating a partial overlap of the mechanisms to moderate drought stress. Gene ontology analyses revealed that the PLSC phosphoproteins represent a unique combination of metabolism, transcription, translation, and protein processing, suggesting that the response in apple to moderate drought stress encompasses a new and unique homeostasis of major cellular processes. The basic trend was an increase in protein and organic molecules abundance related to drought. These increases were higher in MBB than in M26. Our study is the first to address the phosphoproteome of apple rootstocks in response to moderate drought stress, and provide insights into the molecular regulation mechanisms of apple rootstock under moderate drought stress.

Overexpression of sense and antisense ced-9 in tobacco plants confers resistance to Meloidogyne incognita

Transgenic tobacco plants expressing the Caenorhabditis elegans programmed cell death gene ced-9, in both sense and antisense orientations, were produced using Agrobacteriumtumefaciens-mediated transformation. The generated transgenic tobacco plants were tested for resistance to the root-knot nematode Meloidogyne incognita by measuring gall formation, size of galls generated, and the ability of juvenile-2 (J2) to hatch. Results showed that expression of ced-9 gene in either sense (ced-9F) or antisense (ced-9R) orientation in hemizygous transgenic tobacco plants induced prevention of M. incognita proliferation (as measured by gall number reduction) and J2 hatching. Furthermore, the results also showed that ced-9R in homozygous transgenic tobacco plants prevented J2 hatching, whereas ced-9F homozygous transgenic tobacco plants lost nematicidal function. Although our study demonstrates that expression of either ced-9R or ced-9F genes in tobacco plants significantly reduces infection by M. incognita, further investigation is required to understand the specific mechanisms involved for this control. It is possible that the nematode resistance seen with both sense (ced-9F) and antisense (ced-9R) sequences is the result of two independent mechanisms, one acting on invading nematodes and the other acting during embryogenesis of M. incognita, ultimately resulting in plant protection.

Expression of the cell death protein CED-4 of Caenorhabditis elegans in transgenic tobacco plants confers resistance to Meloidogyne incognita

Plant parasitic nematodes are devastating to agricultural production. Recent estimates indicate that losses due to nematode infestation can reach US