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author:("Firon, norit")
1.  Survey of Genes Involved in Biosynthesis, Transport, and Signaling of Phytohormones with Focus on Solanum lycopersicum 
Phytohormones control the development and growth of plants, as well as their response to biotic and abiotic stress. The seven most well-studied phytohormone classes defined today are as follows: auxins, ethylene, cytokinin, abscisic acid, jasmonic acid, gibberellins, and brassinosteroids. The basic principle of hormone regulation is conserved in all plants, but recent results suggest adaptations of synthesis, transport, or signaling pathways to the architecture and growth environment of different plant species. Thus, we aimed to define the extent to which information from the model plant Arabidopsis thaliana is transferable to other plants such as Solanum lycopersicum. We extracted the co-orthologues of genes coding for major pathway enzymes in A. thaliana from the translated genomes of 12 species from the clade Viridiplantae. Based on predicted domain architecture and localization of the identified proteins from all 13 species, we inspected the conservation of phytohormone pathways. The comparison was complemented by expression analysis of (co-) orthologous genes in S. lycopersicum. Altogether, this information allowed the assignment of putative functional equivalents between A. thaliana and S. lycopersicum but also pointed to some variations between the pathways in eudicots, monocots, mosses, and green algae. These results provide first insights into the conservation of the various phytohormone pathways between the model system A. thaliana and crop plants such as tomato. We conclude that orthologue prediction in combination with analysis of functional domain architecture and intracellular localization and expression studies are sufficient tools to transfer information from model plants to other plant species. Our results support the notion that hormone synthesis, transport, and response for most part of the pathways are conserved, and species-specific variations can be found.
PMCID: PMC5038615  PMID: 27695302
phytohormone biosynthesis; transport; signaling; orthologue search; domain analysis; expression profiling; pathway conservation
2.  Altered Phenylpropanoid Metabolism in the Maize Lc-Expressed Sweet Potato (Ipomoea batatas) Affects Storage Root Development 
Scientific Reports  2016;6:18645.
There is no direct evidence of the effect of lignin metabolism on early storage root development in sweet potato. In this study, we found that heterologous expression of the maize leaf color (Lc) gene in sweet potato increased anthocyanin pigment accumulation in the whole plant and resulted in reduced size with an increased length/width ratio, low yield and less starch content in the early storage roots. RT-PCR analysis revealed dramatic up-regulation of the genes involved in the lignin biosynthesis pathway in developing storage roots, leading to greater lignin content in the Lc transgenic lines, compared to the wild type. This was also evidenced by the enhanced lignification of vascular cells in the early storage roots. Furthermore, increased expression of the β-amylase gene in leaves and storage roots also accelerated starch degradation and increased the sugar use efficiency, providing more energy and carbohydrate sources for lignin biosynthesis in the Lc transgenic sweet potato. Lesser starch accumulation was observed in the developing storage roots at the initiation stage in the Lc plants. Our study provides experimental evidence of the basic carbohydrate metabolism underlying the development of storage roots, which is the transformation of lignin biosynthesis to starch biosynthesis.
PMCID: PMC4698713  PMID: 26727353
3.  Analysis and visualization of RNA-Seq expression data using RStudio, Bioconductor, and Integrated Genome Browser 
Sequencing costs are falling, but the cost of data analysis remains high, often because unforeseen problems arise, such as insufficient depth of sequencing or batch effects. Experimenting with data analysis methods during the planning phase of an experiment can reveal unanticipated problems and build valuable bioinformatics expertise in the organism or process being studied. This protocol describes using R Markdown and RStudio, user-friendly tools for statistical analysis and reproducible research in bioinformatics, to analyze and document analysis of an example RNA-Seq data set from tomato pollen undergoing chronic heat stress. Also, we show how to use Integrated Genome Browser to visualize read coverage graphs for differentially expressed genes. Applying the protocol described here and using the provided data sets represent a useful first step toward building RNA-Seq data analysis expertise in a research group.
PMCID: PMC4387895  PMID: 25757788
Integrated Genome Browser; tomato; pollen; visualization; RNA-Seq; R; differential gene expression; edgeR
4.  Transcriptional profiling of sweetpotato (Ipomoea batatas) roots indicates down-regulation of lignin biosynthesis and up-regulation of starch biosynthesis at an early stage of storage root formation 
BMC Genomics  2013;14:460.
The number of fibrous roots that develop into storage roots determines sweetpotato yield. The aim of the present study was to identify the molecular mechanisms involved in the initiation of storage root formation, by performing a detailed transcriptomic analysis of initiating storage roots using next-generation sequencing platforms. A two-step approach was undertaken: (1) generating a database for the sweetpotato root transcriptome using 454-Roche sequencing of a cDNA library created from pooled samples of two root types: fibrous and initiating storage roots; (2) comparing the expression profiles of initiating storage roots and fibrous roots, using the Illumina Genome Analyzer to sequence cDNA libraries of the two root types and map the data onto the root transcriptome database.
Use of the 454-Roche platform generated a total of 524,607 reads, 85.6% of which were clustered into 55,296 contigs that matched 40,278 known genes. The reads, generated by the Illumina Genome Analyzer, were found to map to 31,284 contigs out of the 55,296 contigs serving as the database. A total of 8,353 contigs were found to exhibit differential expression between the two root types (at least 2.5-fold change). The Illumina-based differential expression results were validated for nine putative genes using quantitative real-time PCR. The differential expression profiles indicated down-regulation of classical root functions, such as transport, as well as down-regulation of lignin biosynthesis in initiating storage roots, and up-regulation of carbohydrate metabolism and starch biosynthesis. In addition, data indicated delicate control of regulators of meristematic tissue identity and maintenance, associated with the initiation of storage root formation.
This study adds a valuable resource of sweetpotato root transcript sequences to available data, facilitating the identification of genes of interest. This resource enabled us to identify genes that are involved in the earliest stage of storage root formation, highlighting the reduction in carbon flow toward phenylpropanoid biosynthesis and its delivery into carbohydrate metabolism and starch biosynthesis, as major events involved in storage root initiation. The novel transcripts related to storage root initiation identified in this study provide a starting point for further investigation into the molecular mechanisms underlying this process.
PMCID: PMC3716973  PMID: 23834507
Fibrous root; Ipomoea batatas; Lignin biosynthesis; Starch biosynthesis; Storage-root initiation; Transcription profiling
5.  Water status and associated processes mark critical stages in pollen development and functioning 
Annals of Botany  2012;109(7):1201-1214.
The male gametophyte developmental programme can be divided into five phases which differ in relation to the environment and pollen hydration state: (1) pollen develops inside the anther immersed in locular fluid, which conveys substances from the mother plant – the microsporogenesis phase; (2) locular fluid disappears by reabsorption and/or evaporation before the anther opens and the maturing pollen grains undergo dehydration – the dehydration phase; (3) the anther opens and pollen may be dispersed immediately, or be held by, for example, pollenkitt (as occurs in almost all entomophilous species) for later dispersion – the presentation phase; (4) pollen is dispersed by different agents, remaining exposed to the environment for different periods – the dispersal phase; and (5) pollen lands on a stigma and, in the case of a compatible stigma and suitable conditions, undergoes rehydration and starts germination – the pollen–stigma interaction phase.
This review highlights the issue of pollen water status and indicates the various mechanisms used by pollen grains during their five developmental phases to adjust to changes in water content and maintain internal stability.
Pollen water status is co-ordinated through structural, physiological and molecular mechanisms. The structural components participating in regulation of the pollen water level, during both dehydration and rehydration, include the exine (the outer wall of the pollen grain) and the vacuole. Recent data suggest the involvement of water channels in pollen water transport and the existence of several molecular mechanisms for pollen osmoregulation and to protect cellular components (proteins and membranes) under water stress. It is suggested that pollen grains will use these mechanisms, which have a developmental role, to cope with environmental stress conditions.
PMCID: PMC3359924  PMID: 22523424
Pollen; water status; dehydration; rehydration; angiosperm pollen; pollination
6.  Ethylene is involved in maintaining tomato (Solanum lycopersicum) pollen quality under heat-stress conditions 
AoB Plants  2012;2012:pls024.
The paper supports the view that ethylene plays a significant role in maintaining tomato pollen thermotolerance. Interfering with the ethylene signalling pathway or reducing ethylene levels and increased tomato pollen sensitivity to heat stress. On the other hand, increasing ethylene levels before heat-stress improved pollen quality.
Background and aims
Exposure to higher-than-optimal temperatures reduces crop yield and quality, mainly due to sensitivity of developing pollen grains. The mechanisms maintaining high pollen quality under heat-stress conditions are poorly understood. Our recently published data indicate high heat-stress-induced expression of ethylene-responsive genes in tomato pollen, indicating ethylene involvement in the pollen heat-stress response. Here we elucidated ethylene's involvement in pollen heat-stress response and thermotolerance by assessing the effects of interfering with the ethylene signalling pathway and altering ethylene levels on tomato pollen functioning under heat stress.
Plants of the ethylene-insensitive mutant Never ripe (Nr)—defective in an ethylene response sensor (ERS)-like ethylene receptor—and the corresponding wild type were exposed to control or heat-stress growing conditions, and pollen quality was determined. Starch and carbohydrates were measured in isolated pollen grains from these plants. The effect of pretreating cv. Micro-Tom tomato plants, prior to heat-stress exposure, with an ethylene releaser or inhibitor of ethylene biosynthesis on pollen quality was assessed.
Principal results
Never ripe pollen grains exhibited higher heat-stress sensitivity, manifested by a significant reduction in the total number of pollen grains, reduction in the number of viable pollen and elevation of the number of non-viable pollen, compared with wild-type plants. Mature Nr pollen grains accumulated only 40 % of the sucrose level accumulated by the wild type. Pretreatment of tomato plants with an ethylene releaser increased pollen quality under heat stress, with an over 5-fold increase in the number of germinating pollen grains per flower. Pretreatment with an ethylene biosynthesis inhibitor reduced the number of germinating pollen grains following heat-stress exposure over 5-fold compared with non-treated controls.
Ethylene plays a significant role in tomato pollen thermotolerance. Interfering with the ethylene signalling pathway or reducing ethylene levels increased tomato pollen sensitivity to heat stress, whereas increasing ethylene levels prior to heat-stress exposure increased pollen quality.
PMCID: PMC3461890  PMID: 23050072
7.  Transcriptional profiling of maturing tomato (Solanum lycopersicum L.) microspores reveals the involvement of heat shock proteins, ROS scavengers, hormones, and sugars in the heat stress response 
Journal of Experimental Botany  2009;60(13):3891-3908.
Above-optimal temperatures reduce yield in tomato largely because of the high heat stress (HS) sensitivity of the developing pollen grains. The high temperature response, especially at this most HS-sensitive stage of the plant, is poorly understood. To obtain an overview of molecular mechanisms underlying the HS response (HSR) of microspores, a detailed transcriptomic analysis of heat-stressed maturing tomato microspores was carried out using a combination of Affymetrix Tomato Genome Array and cDNA-amplified fragment length polymorphism (AFLP) techniques. The results were corroborated by reverse transcription-PCR (RT-PCR) and immunoblot analyses. The data obtained reveal the involvement of specific members of the small heat shock protein (HSP) gene family, HSP70 and HSP90, in addition to the HS transcription factors A2 (HSFA2) and HSFA3, as well as factors other than the classical HS-responsive genes. The results also indicate HS regulation of reactive oxygen species (ROS) scavengers, sugars, plant hormones, and regulatory genes that were previously implicated in other types of stress. The use of cDNA-AFLP enabled the detection of genes representing pollen-specific functions that are missing from the tomato Affymetrix chip, such as those involved in vesicle-mediated transport and a pollen-specific, calcium-dependent protein kinase (CDPK2). For several genes, including LeHSFA2, LeHSP17.4-CII, as well as homologues of LeHSP90 and AtVAMP725, higher basal expression levels were detected in microspores of cv. Hazera 3042 (a heat-tolerant cultivar) compared with microspores of cv. Hazera 3017 (a heat-sensitive cultivar), marking these genes as candidates for taking part in microspore thermotolerance. This work provides a comprehensive analysis of the molecular events underlying the HSR of maturing microspores of a crop plant, tomato.
PMCID: PMC2736902  PMID: 19628571
cDNA-AFLP; gene expression; heat stress response; microarray; microspore maturation; tomato

Results 1-7 (7)