Publications by authors named "Jarunya Narangajavana"

32 Publications

Integrative omics approaches revealed a crosstalk among phytohormones during tuberous root development in cassava.

Plant Mol Biol 2020 Aug 5. Epub 2020 Aug 5.

RIKEN Center for Sustainable Resource Science, 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama, Kanagawa, 230-0045, Japan.

Key Message: Integrative omics approaches revealed a crosstalk among phytohormones during tuberous root development in cassava. Tuberous root formation is a complex process consisting of phase changes as well as cell division and elongation for radial growth. We performed an integrated analysis to clarify the relationships among metabolites, phytohormones, and gene transcription during tuberous root formation in cassava (Manihot esculenta Crantz). We also confirmed the effects of the auxin (AUX), cytokinin (CK), abscisic acid (ABA), jasmonic acid (JA), gibberellin (GA), brassinosteroid (BR), salicylic acid, and indole-3-acetic acid conjugated with aspartic acid on tuberous root development. An integrated analysis of metabolites and gene expression indicated the expression levels of several genes encoding enzymes involved in starch biosynthesis and sucrose metabolism are up-regulated during tuberous root development, which is consistent with the accumulation of starch, sugar phosphates, and nucleotides. An integrated analysis of phytohormones and gene transcripts revealed a relationship among AUX signaling, CK signaling, and BR signaling, with AUX, CK, and BR inducing tuberous root development. In contrast, ABA and JA inhibited tuberous root development. These phenomena might represent the differences between stem tubers (e.g., potato) and root tubers (e.g., cassava). On the basis of these results, a phytohormonal regulatory model for tuberous root development was constructed. This model may be useful for future phytohormonal studies involving cassava.
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http://dx.doi.org/10.1007/s11103-020-01033-8DOI Listing
August 2020

Enhancing of anthracnose disease resistance indicates a potential role of antimicrobial peptide genes in cassava.

Genetica 2020 Aug 11;148(3-4):135-148. Epub 2020 Jul 11.

Department of Biotechnology, Faculty of Science, Mahidol University, Bangkok, Thailand.

Cassava (Manihot esculenta Crantz.) is an important economic crop in tropical countries. Demands for using cassava in food, feed and biofuel industries have been increasing worldwide. Cassava anthracnose disease, caused by Colletotrichum gloeosporioides f.sp. manihotis (CAD), is considered a major problem in cassava production. To minimize the effects of such disease, this study investigated the response of cassava to attack by CAD and how the plants defend themselves against this threat. Genome-wide identification of antimicrobial peptide genes (AMPs) and their expression in response to fungal infection was performed in the resistant cassava cultivar (Huay Bong 60; HB60) in comparison with the highly susceptible cultivar (Hanatee; HN). A total of 114 gene members of AMP were identified in the cassava genome database. Fifty-six gene members were selected for phylogenetic tree construction and analysis of putative cis-acting elements in their promoter regions. Differential expression profiles of six candidate genes were observed in response to CAD infection of both cassava cultivars. Upregulation of snakins, MeSN1 and MeSN2 was found in HB60, whereas MeHEL, Me-AMP-D2 and MeLTP2 were highly induced in HN. The MeLTP1 gene was not expressed in either cultivar. HB60 showed a reduced severity rating in comparison to HN after CAD infection. The biomembrane permeability test of fungal CAD was strongly affected after treatment with protein extract derived from CAD-infected HB60. Altogether, these findings suggest that snakins have a potential function in the CAD defense response in cassava. These results could be useful for cassava improvement programs to fight fungal pathogen.
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http://dx.doi.org/10.1007/s10709-020-00097-0DOI Listing
August 2020

Opposite physiological effects upon jasmonic acid and brassinosteroid treatment on laticifer proliferation and co-occurrence of differential expression of genes involved in vascular development in rubber tree.

Physiol Mol Biol Plants 2019 Sep 18;25(5):1283-1299. Epub 2019 Jul 18.

1Department of Biotechnology, Faculty of Science, Mahidol University, Rama 6 Rd., Rajthewee, Bangkok, 10400 Thailand.

During growth of woody plant-trunk, the secondary meristem functions in giving rise the xylem and phloem. Rubber tree ( Muell. Arg.), in addition, contains laticifers (latex producing vessels) in the vicinity of phloem. Insights into regulatory mechanisms of gene networks underlying laticifer proliferation in rubber tree has remained very limited. The candidate vascular development-related genes were selected to investigate for expression profile in phloem and xylem tissues of high latex yield- and high wood yield-clones of rubber tree. The differential gene expression between the mature branch-xylem and -phloem tissues was clearly observed. The -regulatory motif analysis revealed the existent of putative jasmonic acid (JA)- and brassinosteroid (BR)-responsive regulatory motifs in promoter regions of these genes, and consequently the effect of exogenous application of JA, BR or their respective signaling inhibitors, on the formation of laticifers in rubber tree was demonstrated. Interestingly, the laticifer numbers were significantly increased in JA-treatment, correlated with up-regulation of phloem development-related genes in both rubber tree clones. On the contrary, the laticifers were decreased in BR-treatment accompanying by up-regulation of xylem development-related genes, especially in high wood yield-rubber tree clone. BR-inhibitor treatment also enhanced laticifer numbers, while JA-inhibitor suppressed laticifer differentiation. Taken together, this study unveils the molecular interplay between JA/BR on vascular development in rubber tree and how this impacts the appearance of laticifers in this plant. This process is vital for a better understanding on laticifer differentiation and its impact in the manipulation of wood and latex yield in rubber tree improvement program.
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http://dx.doi.org/10.1007/s12298-019-00686-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6745593PMC
September 2019

Phytohormone priming elevates the accumulation of defense-related gene transcripts and enhances bacterial blight disease resistance in cassava.

Plant Physiol Biochem 2018 Jan 27;122:65-77. Epub 2017 Nov 27.

Department of Biotechnology, Faculty of Science, Mahidol University, Phayathai, Bangkok, Thailand; Center of Excellence on Agricultural Biotechnology: (AG-BIO/PERDO-CHE), Bangkok, Thailand. Electronic address:

Cassava bacterial blight (CBB) disease caused by Xanthomonas axonopodis pv. manihotis (Xam) is a severe disease in cassava worldwide. In addition to causing significant cassava yield loss, CBB disease has not been extensively studied, especially in terms of CBB resistance genes. The present research demonstrated the molecular mechanisms underlining the defense response during Xam infection in two cassava cultivars exhibiting different degrees of disease resistance, Huay Bong60 (HB60) and Hanatee (HN). Based on gene expression analysis, ten of twelve putative defense-related genes including, leucine-rich repeat receptor-like kinases (LRR-RLKs), resistance (R), WRKY and pathogenesis-related (PR) genes, were differentially expressed between these two cassava cultivars during Xam infection. The up-regulation of defense-related genes observed in HB60 may be the mechanism required for the reduction of disease severity in the resistant cultivar. Interestingly, priming with salicylic acid (SA) or methyl jasmonate (MeJA) for 24 h before Xam inoculation could enhance the defense response in both cassava cultivars. The disease severity was decreased 10% in the resistant cultivar (HB60) and was remarkably reduced 21% in the susceptible cultivar (HN) by SA/MeJA priming. Priming with Xam inoculation modulated cassava4.1_013417, cassava4.1_030866 and cassava4.1_020555 (highest similarity to MeWRKY59, MePR1 and AtPDF2.2, respectively) expression and led to enhanced resistance of the susceptible cultivar in the second infection. The putative cis-regulatory elements were predicted in an upstream region of these three defense-related genes. The different gene expression levels in these genes between the two cultivars were due to the differences in cis-regulatory elements in their promoter regions. Taken together, our study strongly suggested that the induction of defense-related genes correlated with defense resistance against Xam infection, and exogenous application of SA or MeJA could elevate the defense response in both cultivars of cassava. This finding should pave the way for management to reduce yield loss from disease and genetic improvement in cassava.
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http://dx.doi.org/10.1016/j.plaphy.2017.11.016DOI Listing
January 2018

Genome-wide analysis of aquaporin gene family and their responses to water-deficit stress conditions in cassava.

Plant Physiol Biochem 2017 Dec 27;121:118-127. Epub 2017 Oct 27.

Department of Biotechnology, Faculty of Science, Mahidol University, Bangkok, Thailand; Center of Excellence on Agricultural Biotechnology (AG-BIO/PERDO-CHE), Bangkok, Thailand. Electronic address:

Cassava (Manihot esculenta Crantz) is an important economic crop in tropical countries. Although cassava is considered a drought-tolerant crop that can grow in arid areas, the impact of drought can significantly reduce the growth and yield of cassava storage roots. The discovery of aquaporin molecules (AQPs) in plants has resulted in a paradigm shift in the understanding of plant-water relationships, whereas the relationship between aquaporin and drought resistance in cassava still remains elusive. To investigate the potential role of aquaporin in cassava under water-deficit conditions, 45 putative MeAQPs were identified in the cassava genome. Six members of MeAQPs, containing high numbers of water stress-responsive motifs in their promoter regions, were selected for a gene expression study. Two cassava cultivars, which showed different degrees of responses to water-deficit stress, were used to test in in vitro and potted plant systems. The differential expression of all candidate MeAQPs were found in only leaves from the potted plant system were consistent with the relative water content and with the stomatal closure profile of the two cultivars. MePIP2-1 and MePIP2-10 were up-regulated and this change in their expression might regulate a special signal for water efflux out of guard cells, thus inducing stomatal closure under water-deficit conditions. In addition, the expression profiles of genes in the ABA-dependent pathway revealed an essential correlation with stomatal closure. The potential functions of MeAQPs and candidate ABA-dependent pathway genes in response to water deficit in the more tolerant cassava cultivar were discussed.
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http://dx.doi.org/10.1016/j.plaphy.2017.10.025DOI Listing
December 2017

The Histone Deacetylase Inhibitor Suberoylanilide Hydroxamic Acid Alleviates Salinity Stress in Cassava.

Front Plant Sci 2016 9;7:2039. Epub 2017 Jan 9.

Plant Genomic Network Research Team, RIKEN Center for Sustainable Resource ScienceYokohama, Japan; CREST, Japan Science and Technology AgencySaitama, Japan; Plant Genomic Network Science Division, Kihara Institute for Biological Research, Yokohama City UniversityYokohama, Japan.

Cassava ( Crantz) demand has been rising because of its various applications. High salinity stress is a major environmental factor that interferes with normal plant growth and limits crop productivity. As well as genetic engineering to enhance stress tolerance, the use of small molecules is considered as an alternative methodology to modify plants with desired traits. The effectiveness of histone deacetylase (HDAC) inhibitors for increasing tolerance to salinity stress has recently been reported. Here we use the HDAC inhibitor, suberoylanilide hydroxamic acid (SAHA), to enhance tolerance to high salinity in cassava. Immunoblotting analysis reveals that SAHA treatment induces strong hyper-acetylation of histones H3 and H4 in roots, suggesting that SAHA functions as the HDAC inhibitor in cassava. Consistent with increased tolerance to salt stress under SAHA treatment, reduced Na content and increased K/Na ratio were detected in SAHA-treated plants. Transcriptome analysis to discover mechanisms underlying salinity stress tolerance mediated through SAHA treatment reveals that SAHA enhances the expression of 421 genes in roots under normal condition, and 745 genes at 2 h and 268 genes at 24 h under both SAHA and NaCl treatment. The mRNA expression of genes, involved in phytohormone [abscisic acid (ABA), jasmonic acid (JA), ethylene, and gibberellin] biosynthesis pathways, is up-regulated after high salinity treatment in SAHA-pretreated roots. Among them, an () involved in a crucial step of JA biosynthesis is strongly up-regulated by SAHA treatment under salinity stress conditions, implying that JA pathway might contribute to increasing salinity tolerance by SAHA treatment. Our results suggest that epigenetic manipulation might enhance tolerance to high salinity stress in cassava.
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http://dx.doi.org/10.3389/fpls.2016.02039DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5220070PMC
January 2017

Cassava (Manihot esculenta) transcriptome analysis in response to infection by the fungus Colletotrichum gloeosporioides using an oligonucleotide-DNA microarray.

J Plant Res 2016 Jul 2;129(4):711-726. Epub 2016 May 2.

Plant Genomic Network Research Team, RIKEN Center for Sustainable Resource Science, 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama, Kanagawa, 230-0045, Japan.

Cassava anthracnose disease (CAD), caused by the fungus Colletotrichum gloeosporioides f. sp. Manihotis, is a serious disease of cassava (Manihot esculenta) worldwide. In this study, we established a cassava oligonucleotide-DNA microarray representing 59,079 probes corresponding to approximately 30,000 genes based on original expressed sequence tags and RNA-seq information from cassava, and applied it to investigate the molecular mechanisms of resistance to fungal infection using two cassava cultivars, Huay Bong 60 (HB60, resistant to CAD) and Hanatee (HN, sensitive to CAD). Based on quantitative real-time reverse transcription PCR and expression profiling by the microarray, we showed that the expressions of various plant defense-related genes, such as pathogenesis-related (PR) genes, cell wall-related genes, detoxification enzyme, genes related to the response to bacterium, mitogen-activated protein kinase (MAPK), genes related to salicylic acid, jasmonic acid and ethylene pathways were higher in HB60 compared with HN. Our results indicated that the induction of PR genes in HB60 by fungal infection and the higher expressions of defense response-related genes in HB60 compared with HN are likely responsible for the fungal resistance in HB60. We also showed that the use of our cassava oligo microarray could improve our understanding of cassava molecular mechanisms related to environmental responses and development, and advance the molecular breeding of useful cassava plants.
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http://dx.doi.org/10.1007/s10265-016-0828-xDOI Listing
July 2016

Cassava root membrane proteome reveals activities during storage root maturation.

J Plant Res 2016 Jan 7;129(1):51-65. Epub 2015 Nov 7.

Deparment of Biotechnology, Faculty of Science, Mahidol University, Rama 6 Rd.,Rajthewee, Phayathai, Bangkok, 10400, Thailand.

Cassava (Manihot esculenta Crantz) is one of the most important crops of Thailand. Its storage roots are used as food, feed, starch production, and be the important source for biofuel and biodegradable plastic production. Despite the importance of cassava storage roots, little is known about the mechanisms involved in their formation. This present study has focused on comparison of the expression profiles of cassava root proteome at various developmental stages using two-dimensional gel electrophoresis and LC-MS/MS. Based on an anatomical study using Toluidine Blue, the secondary growth was confirmed to be essential during the development of cassava storage root. To investigate biochemical processes occurring during storage root maturation, soluble and membrane proteins were isolated from storage roots harvested from 3-, 6-, 9-, and 12-month-old cassava plants. The proteins with differential expression pattern were analysed and identified to be associated with 8 functional groups: protein folding and degradation, energy, metabolism, secondary metabolism, stress response, transport facilitation, cytoskeleton, and unclassified function. The expression profiling of membrane proteins revealed the proteins involved in protein folding and degradation, energy, and cell structure were highly expressed during early stages of development. Integration of these data along with the information available in genome and transcriptome databases is critical to expand knowledge obtained solely from the field of proteomics. Possible role of identified proteins were discussed in relation with the activities during storage root maturation in cassava.
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http://dx.doi.org/10.1007/s10265-015-0761-4DOI Listing
January 2016

Gene Co-Expression Analysis Inferring the Crosstalk of Ethylene and Gibberellin in Modulating the Transcriptional Acclimation of Cassava Root Growth in Different Seasons.

PLoS One 2015 14;10(9):e0137602. Epub 2015 Sep 14.

Systems Biology and Bioinformatics Research Group, Pilot Plant Development and Training Institute, King Mongkut's University of Technology Thonburi, Thakham, Bangkhunthian, Bangkok, Thailand; Department of Chemical Engineering, Faculty of Engineering, King Mongkut's University of Technology Thonburi, Bangkok, Thungkhru, Bangmod, Bangkok, Thailand.

Cassava is a crop of hope for the 21st century. Great advantages of cassava over other crops are not only the capacity of carbohydrates, but it is also an easily grown crop with fast development. As a plant which is highly tolerant to a poor environment, cassava has been believed to own an effective acclimation process, an intelligent mechanism behind its survival and sustainability in a wide range of climates. Herein, we aimed to investigate the transcriptional regulation underlying the adaptive development of a cassava root to different seasonal cultivation climates. Gene co-expression analysis suggests that AP2-EREBP transcription factor (ERF1) orthologue (D142) played a pivotal role in regulating the cellular response to exposing to wet and dry seasons. The ERF shows crosstalk with gibberellin, via ent-Kaurene synthase (D106), in the transcriptional regulatory network that was proposed to modulate the downstream regulatory system through a distinct signaling mechanism. While sulfur assimilation is likely to be a signaling regulation for dry crop growth response, calmodulin-binding protein is responsible for regulation in the wet crop. With our initiative study, we hope that our findings will pave the way towards sustainability of cassava production under various kinds of stress considering the future global climate change.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0137602PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4569563PMC
June 2016

Genome-wide analysis reveals phytohormone action during cassava storage root initiation.

Plant Mol Biol 2015 Aug 29;88(6):531-43. Epub 2015 Jun 29.

Department of Biotechnology, Center for Cassava Molecular Biotechnology, Faculty of Science, Mahidol University, Bangkok, 10400, Thailand,

Development of storage roots is a process associated with a phase change from cell division and elongation to radial growth and accumulation of massive amounts of reserve substances such as starch. Knowledge of the regulation of cassava storage root formation has accumulated over time; however, gene regulation during the initiation and early stage of storage root development is still poorly understood. In this study, transcription profiling of fibrous, intermediate and storage roots at eight weeks old were investigated using a 60-mer-oligo microarray. Transcription and gene expression were found to be the key regulating processes during the transition stage from fibrous to intermediate roots, while homeostasis and signal transduction influenced regulation from intermediate roots to storage roots. Clustering analysis of significant genes and transcription factors (TF) indicated that a number of phytohormone-related TF were differentially expressed; therefore, phytohormone-related genes were assembled into a network of correlative nodes. We propose a model showing the relationship between KNOX1 and phytohormones during storage root initiation. Exogeneous treatment of phytohormones N (6) -benzylaminopurine and 1-Naphthaleneacetic acid were used to induce the storage root initiation stage and to investigate expression patterns of the genes involved in storage root initiation. The results support the hypothesis that phytohormones are acting in concert to regulate the onset of cassava storage root development. Moreover, MeAGL20 is a factor that might play an important role at the onset of storage root initiation when the root tip becomes swollen.
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http://dx.doi.org/10.1007/s11103-015-0340-zDOI Listing
August 2015

Involvement of miR160/miR393 and their targets in cassava responses to anthracnose disease.

J Plant Physiol 2015 Feb 5;174:26-35. Epub 2014 Oct 5.

Department of Biotechnology, Faculty of Science, Mahidol University, Bangkok, Thailand. Electronic address:

Cassava is a starchy root crop for food and industrial applications in many countries around the world. Among the factors that affect cassava production, diseases remain the major cause of yield loss. Cassava anthracnose disease is caused by the fungus Colletotrichum gloeosporioides. Severe anthracnose attacks can cause tip die-backs and stem cankers, which can affect the availability of planting materials especially in large-scale production systems. Recent studies indicate that plants over- or under-express certain microRNAs (miRNAs) to cope with various stresses. Understanding how a disease-resistant plant protects itself from pathogens should help to uncover the role of miRNAs in the plant immune system. In this study, the disease severity assay revealed different response to C. gloeosporioides infection in two cassava cultivars. Quantitative RT-PCR analysis uncovered the differential expression of the two miRNAs and their target genes in the two cassava cultivars that were subjected to fungal infection. The more resistant cultivar revealed the up-regulation of miR160 and miR393, and consequently led to low transcript levels in their targets, ARF10 and TIR1, respectively. The more susceptible cultivar exhibited the opposite pattern. The cis-regulatory elements relevant to defense and stress responsiveness, fungal elicitor responsiveness and hormonal responses were the most prevalent present in the miRNAs gene promoter regions. The possible dual role of these specific miRNAs and their target genes associated with cassava responses to C. gloeosporioides is discussed. This is the first study to address the molecular events by which miRNAs which might play a role in fungal-infected cassava. A better understanding of the functions of miRNAs target genes should greatly increase our knowledge of the mechanism underlying susceptibility and lead to new strategies to enhance disease tolerance in this economically important crop.
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http://dx.doi.org/10.1016/j.jplph.2014.09.006DOI Listing
February 2015

Involvement of ethylene-responsive microRNAs and their targets in increased latex yield in the rubber tree in response to ethylene treatment.

Plant Physiol Biochem 2014 Nov 30;84:203-212. Epub 2014 Sep 30.

Department of Biotechnology, Faculty of Science, Mahidol University, Bangkok, Thailand; Rubber Technology Research Centre, Faculty of Science, Mahidol University, Thailand. Electronic address:

The rubber tree is an economically important plant that produces natural rubber for various industrial uses. The application of ethylene contributes to increased latex production in rubber trees; however, the molecular biology behind the effects of ethylene on latex yield remains to be elucidated. Recently, the intersection between microRNA (miRNA) regulation and phytohormone responses has been revealed. Insight into the regulation of miRNAs and their target genes should help to determine the functional importance of miRNAs as well as the role of miRNAs in signaling under ethylene stimulation in the rubber tree. In this study, hbr-miR159 and hbr-miR166 were down-regulated in bark under ethylene treatment. The ethylene also down-regulated ATHB15-like (Class III Homeodomain Leucine Zipper, HD-ZIP III) which have been extensively implicated in the regulation of primary and secondary vascular tissue pattern formation. The strong negative-regulation of ARF6/ARF8 caused by hbr-miR167 involved in an attenuation of vascular development and may gradually lead to bark dryness syndrome in the long term ethylene treatment. The negative correlation of hbr-miR172 and its target REF3 in the inner soft bark under ethylene treatment results in dramatic increases in latex yield in the ethylene-sensitive clone of the rubber tree. The overall results suggested that the differential expression of HD-ZIP III, miR167/ARF6, ARF8, and miR172/REF3 and related genes may play possible roles in the response to ethylene treatment, resulting in longer latex flow and increased latex yield.
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http://dx.doi.org/10.1016/j.plaphy.2014.09.016DOI Listing
November 2014

Differential expression of microRNAs and their targets reveals a possible dual role in physiological bark disorder in rubber tree.

J Plant Physiol 2014 Aug 10;171(13):1117-26. Epub 2014 May 10.

Department of Biotechnology, Faculty of Science, Mahidol University, Bangkok, Thailand; Rubber Technology Research Centre, Faculty of Science, Mahidol University, Thailand. Electronic address:

Trunk phloem necrosis (TPN), a physiological bark disorder of the rubber tree (Hevea brasiliensis), is a serious problem that affects the yield of natural rubber. The resultant bark dryness occurs in up to half of a plantation's trees in almost every rubber tree plantation region, causing a great annual loss of dry rubber for natural rubber production. Different types of injury and physical damage caused by mechanical activation as well as environmental stresses cause physiological bark disorder in tree. Due to the essential role of miR166, miR393 and miR167 in vascular development and abiotic stress response in diverse plant species, it was interesting to investigate the role of these miRNAs in rubber trees, particularly during development of a physiological bark disorder. In this study, the expression pattern of miR166, miR393 and miR167; and their target genes, HD-ZIP III; TIR1 and ARF8, respectively; was demonstrated in healthy tree and different TPN trees. Their existence and function in vivo was validated using RNA ligase-mediated 5' rapid amplification of cDNA ends. Taken together, the results suggest a possible dual role of these three miRNAs in maintaining normal bark regeneration in healthy trees, coping with overtapping by affecting the wound healing system leading to abnormal bark regeneration in overtapped-TPN trees, and act as additional forces that enhance the attenuation of vascular development resulting in bark necrosis and cell death in the natural-TPN tree. This is the first study to address the molecular events of miRNAs involved in the physiological bark disorder TPN in rubber tree. Further study will open the possibility to better understanding of physiological and molecular perspectives during TPN development, and lead to improvement of monitoring the exploitation of rubber tree plantations.
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http://dx.doi.org/10.1016/j.jplph.2014.05.001DOI Listing
August 2014

Characterization of rubber tree microRNA in phytohormone response using large genomic DNA libraries, promoter sequence and gene expression analysis.

Mol Genet Genomics 2014 Oct 26;289(5):921-33. Epub 2014 May 26.

Department of Biotechnology, Faculty of Science, Mahidol University, Rama 6 Rd, Rajthewee, Bangkok, 10400, Thailand.

The para rubber tree is the most widely cultivated tree species for producing natural rubber (NR) latex. Unfortunately, rubber tree characteristics such as a long life cycle, heterozygous genetic backgrounds, and poorly understood genetic profiles are the obstacles to breeding new rubber tree varieties, such as those with improved NR yields. Recent evidence has revealed the potential importance of controlling microRNA (miRNA) decay in some aspects of NR regulation. To gain a better understanding of miRNAs and their relationship with rubber tree gene regulation networks, large genomic DNA insert-containing libraries were generated to complement the incomplete draft genome sequence and applied as a new powerful tool to predict a function of interested genes. Bacterial artificial chromosome and fosmid libraries, containing a total of 120,576 clones with an average insert size of 43.35 kb, provided approximately 2.42 haploid genome equivalents of coverage based on the estimated 2.15 gb rubber tree genome. Based on these library sequences, the precursors of 1 member of rubber tree-specific miRNAs and 12 members of conserved miRNAs were successfully identified. A panel of miRNAs was characterized for phytohormone response by precisely identifying phytohormone-responsive motifs in their promoter sequences. Furthermore, the quantitative real-time PCR on ethylene stimulation of rubber trees was performed to demonstrate that the miR2118, miR159, miR164 and miR166 are responsive to ethylene, thus confirmed the prediction by genomic DNA analysis. The cis-regulatory elements identified in the promoter regions of these miRNA genes help augment our understanding of miRNA gene regulation and provide a foundation for further investigation of the regulation of rubber tree miRNAs.
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http://dx.doi.org/10.1007/s00438-014-0862-0DOI Listing
October 2014

Arabidopsis-derived shrimp viral-binding protein, PmRab7 can protect white spot syndrome virus infection in shrimp.

J Biotechnol 2012 Sep 29;161(1):60-7. Epub 2012 May 29.

Department of Biotechnology, Faculty of Science, Mahidol University, Bangkok 10400, Thailand.

White spot syndrome virus is currently the leading cause of production losses in the shrimp industry. Penaeus monodon Rab7 protein has been recognized as a viral-binding protein with an efficient protective effect against white spot syndrome infection. Plant-derived recombinant PmRab7 might serve as an alternative source for in-feed vaccination, considering the remarkable abilities of plant expression systems. PmRab7 was introduced into the Arabidopsis thaliana T87 genome. Arabidopsis-derived recombinant PmRab7 showed high binding activity against white spot syndrome virus and a viral envelope, VP28. The growth profile of Arabidopsis suspension culture expressing PmRab7 (ECR21# 35) resembled that of its counterpart. PmRab7 expression in ECR21# 35 reached its maximum level at 5 mg g(-1) dry weight in 12 days, which was higher than those previously reported in Escherichia coli and in Pichia. Co-injection of white spot syndrome virus and Arabidopsis crude extract containing PmRab7 in Litopenaeus vannamei showed an 87% increase in shrimp survival rate at 5 day after injection. In this study, we propose an alternative PmRab7 source with higher production yield, and cheaper culture media costs, that might serve the industry's need for an in-feed supplement against white spot syndrome infection.
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http://dx.doi.org/10.1016/j.jbiotec.2012.05.007DOI Listing
September 2012

Genome-wide analysis of microRNAs in rubber tree (Hevea brasiliensis L.) using high-throughput sequencing.

Planta 2012 Aug 10;236(2):437-45. Epub 2012 Mar 10.

Department of Biotechnology, Faculty of Science, Mahidol University, Rama 6 Rd., Rajthewee, Bangkok, 10400, Thailand.

MicroRNAs (miRNAs) are short RNAs with essential roles in gene regulation in various organisms including higher plants. In contrast to the vast information on miRNAs from many economically important plants, almost nothing has been reported on the identification or analysis of miRNAs from rubber tree (Hevea brasiliensis L.), the most important natural rubber-producing crop. To identify miRNAs and their target genes in rubber tree, high-throughput sequencing combined with a computational approach was performed. Four small RNA libraries were constructed for deep sequencing from mature and young leaves of two rubber tree clones, PB 260 and PB 217, which provide high and low latex yield, respectively. 115 miRNAs belonging to 56 known miRNA families were identified, and northern hybridization validated miRNA expression and revealed developmental stage-dependent and clone-specific expression for some miRNAs. We took advantage of the newly released rubber tree genome assembly and predicted 20 novel miRNAs. Further, computational analysis uncovered potential targets of the known and novel miRNAs. Predicted target genes included not only transcription factors but also genes involved in various biological processes including stress responses, primary and secondary metabolism, and signal transduction. In particular, genes with roles in rubber biosynthesis are predicted targets of miRNAs. This study provides a basic catalog of miRNAs and their targets in rubber tree to facilitate future improvement and exploitation of rubber tree.
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http://dx.doi.org/10.1007/s00425-012-1622-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3405184PMC
August 2012

Computational identification of microRNAs and their targets in cassava (Manihot esculenta Crantz.).

Mol Biotechnol 2013 Mar;53(3):257-69

Department of Biotechnology, Faculty of Science, Mahidol University, Bangkok, Thailand.

MicroRNAs (miRNAs) are a newly discovered class of noncoding endogenous small RNAs involved in plant growth and development as well as response to environmental stresses. miRNAs have been extensively studied in various plant species, however, only few information are available in cassava, which serves as one of the staple food crops, a biofuel crop, animal feed and industrial raw materials. In this study, the 169 potential cassava miRNAs belonging to 34 miRNA families were identified by computational approach. Interestingly, mes-miR319b was represented as the first putative mirtron demonstrated in cassava. A total of 15 miRNA clusters involving 7 miRNA families, and 12 pairs of sense and antisense strand cassava miRNAs belonging to six different miRNA families were discovered. Prediction of potential miRNA target genes revealed their functions involved in various important plant biological processes. The cis-regulatory elements relevant to drought stress and plant hormone response were identified in the promoter regions of those miRNA genes. The results provided a foundation for further investigation of the functional role of known transcription factors in the regulation of cassava miRNAs. The better understandings of the complexity of miRNA-mediated genes network in cassava would unravel cassava complex biology in storage root development and in coping with environmental stresses, thus providing more insights for future exploitation in cassava improvement.
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http://dx.doi.org/10.1007/s12033-012-9521-zDOI Listing
March 2013

Proteomic analysis of salinity-stressed Chlamydomonas reinhardtii revealed differential suppression and induction of a large number of important housekeeping proteins.

Planta 2012 Mar 26;235(3):649-59. Epub 2012 Jan 26.

Department of Biochemistry, Kasetsart University, Bangkok 10900, Thailand.

Salinity stress is one of the most common abiotic stresses that hamper plant productivity worldwide. Successful plant adaptations to salt stress require substantial changes in cellular protein expression. In this work, we present a 2-DE-based proteomic analysis of a model unicellular green alga, Chlamydomonas reinhardtii, subjected to 300 mM NaCl for 2 h. Results showed that, in addition to the protein spots that showed partial up- or down-regulation patterns, a number of proteins were exclusively present in the proteome of the control cells, but were absent from the salinity-stressed samples. Conversely, a large number of proteins exclusively appeared in the proteome of the salinity-stressed samples. Of those exclusive proteins, we could successfully identify, via LC-MS/MS, 18 spots uniquely present in the control cells and 99 spots specific to NaCl-treated cells. Interestingly, among the salt-exclusive protein spots, we identified several important housekeeping proteins like molecular chaperones and proteins of the translation machinery, suggesting that they may originate from post-translational modifications rather than from de novo biosynthesis. The possible role and the salt-specific modification of these proteins by salinity stress are discussed.
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http://dx.doi.org/10.1007/s00425-012-1594-1DOI Listing
March 2012

Ectopic gene expression and organogenesis in Arabidopsis mutants missing BRU1 required for genome maintenance.

Genetics 2011 Sep 29;189(1):83-95. Epub 2011 Jul 29.

Bioscience and Biotechnology Center, Nagoya University, Chikusa, Nagoya 464-8601, Japan.

Chromatin reconstitution after DNA replication and repair is essential for the inheritance of epigenetic information, but mechanisms underlying such a process are still poorly understood. Previously, we proposed that Arabidopsis BRU1 functions to ensure the chromatin reconstitution. Loss-of-function mutants of BRU1 are hypersensitive to genotoxic stresses and cause release of transcriptional gene silencing of heterochromatic genes. In this study, we show that BRU1 also plays roles in gene regulation in euchromatic regions. bru1 mutations caused sporadic ectopic expression of genes, including those that encode master regulators of developmental programs such as stem cell maintenance and embryogenesis. bru1 mutants exhibited adventitious organogenesis, probably due to the misexpression of such developmental regulators. The key regulatory genes misregulated in bru1 alleles were often targets of PcG SET-domain proteins, although the overlap between the bru1-misregulated and PcG SET-domain-regulated genes was limited at a genome-wide level. Surprisingly, a considerable fraction of the genes activated in bru1 were located in several subchromosomal regions ranging from 174 to 944 kb in size. Our results suggest that BRU1 has a function related to the stability of subchromosomal gene regulation in the euchromatic regions, in addition to the maintenance of chromatin states coupled with heritable epigenetic marks.
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http://dx.doi.org/10.1534/genetics.111.130062DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3176131PMC
September 2011

Hormonal treatment of the bark of rubber trees (Hevea brasiliensis) increases latex yield through latex dilution in relation with the differential expression of two aquaporin genes.

J Plant Physiol 2011 Feb 15;168(3):253-62. Epub 2010 Jul 15.

Department of Biotechnology, Faculty of Science, Mahidol University, Rama VI Road, Bangkok 10400, Thailand.

Natural rubber is synthesized in laticifers in the inner liber of the rubber tree (Hevea brasiliensis). Upon bark tapping, the latex is expelled due to liber turgor pressure. The mature laticifers are devoid of plasmodesmata; therefore a corresponding decrease in the total latex solid content is likely to occur due to water influx inside the laticifers. Auxins and ethylene used as efficient yield stimulants in mature untapped rubber trees, but, bark treatments with abscisic acid (ABA) and salicylic acid (SA) could also induce a transient increase latex yield. We recently reported that there are three aquaporin genes, HbPIP2;1, HbTIP1;1 and HbPIP1;1, that are regulated differentially after ethylene bark treatment. HbPIP2;1 was up-regulated in both the laticifers and the inner liber tissues, whereas HbTIP1;1 was up-regulated in the latex cells, but very markedly down-regulated in the inner liber tissues. Conversely, HbPIP1;1 was down-regulated in both tissues. In the present study, HbPIP2;1 and HbTIP1;1 showed a similar expression in response to auxin, ABA and SA, as seen in ethylene stimulation, while HbPIP1;1 was slightly regulated by auxin, but neither by ABA nor SA. The analysis of the HbPIP1;1 promoter region indicated the presence of only ethylene and auxin responsive elements. In addition, the poor efficiency of this HbPIP1;1 in increasing plasmalemma water conductance was confirmed in Xenopus oocytes. Thus, an increase in latex yield in response to all of these hormones was proposed to be the major function of aquaporins, HbPIP2;1 and HbTIP1;1. This study emphasized that the circulation of water between the laticifers and their surrounding tissues that result in latex dilution, as well as the probable maintenance of the liber tissues turgor pressure, favor the prolongation of latex flow.
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http://dx.doi.org/10.1016/j.jplph.2010.06.009DOI Listing
February 2011

AFLP-based transcript profiling for cassava genome-wide expression analysis in the onset of storage root formation.

Physiol Plant 2010 Oct;140(2):189-98

Center for Cassava Molecular Biotechnology, Faculty of Science, Mahidol University, Bangkok, Thailand.

Cassava (Manihot esculenta Crantz) is a root crop that accumulates large quantities of starch, and it is an important source of carbohydrate. Study on gene expressions during storage root development provides important information on storage root formation and starch accumulation as well as unlock new traits for improving of starch yield. cDNA-Amplified Fragment Length Polymorphism (AFLP) was used to compare gene expression profiles in fibrous and storage roots of cassava cultivar Kasetsart 50. Total of 155 differentially expressed transcript-derived fragments with undetectable or low expression in leaves were characterized and classified into 11 groups regarding to their functions. The four major groups were no similarity (20%), hypothetical or unknown proteins (17%), cellular metabolism and biosynthesis (17%) and cellular communication and signaling (14%). Interestingly, sulfite reductase (MeKD82), calcium-dependent protein kinase (CDPK) (MeKD83), ent-kaurene synthase (KS) (MeKD106) and hexose transporter (HT) (MeKD154) showed root-specific expression patterns. This finding is consistent with previously reported genes involved in the initiation of potato tuber. Semi-quantitative reverse transcription polymerase chain reaction of early-developed root samples confirmed that those four genes exhibited significant expression with similar pattern in the storage root initiation and early developmental stages. We proposed that KS and HT may involve in transient induction of CDPK expression, which may play an important role in the signaling pathway of storage root initiation. Sulfite reductase, on the other hand, may involve in storage root development by facilitating sulfur-containing protein biosynthesis or detoxifying the cyanogenic glucoside content through aspartate biosynthesis.
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http://dx.doi.org/10.1111/j.1399-3054.2010.01389.xDOI Listing
October 2010

Involvement of HbPIP2;1 and HbTIP1;1 aquaporins in ethylene stimulation of latex yield through regulation of water exchanges between inner liber and latex cells in Hevea brasiliensis.

Plant Physiol 2009 Oct 5;151(2):843-56. Epub 2009 Aug 5.

Department of Biotechnology, Faculty of Science, Mahidol University, Bangkok 10400, Thailand.

Natural rubber is synthesized in specialized articulated cells (laticifers) located in the inner liber of Hevea brasiliensis. Upon bark tapping, the laticifer cytoplasm (latex) is expelled due to liber tissue turgor pressure. In mature virgin (untapped) trees, short-term kinetic studies confirmed that ethylene, the rubber yield stimulant used worldwide, increased latex yield, with a concomitant decrease in latex total solid content, probably through water influx in the laticifers. As the mature laticifers are devoid of plasmodesmata, the rapid water exchanges with surrounding liber cells probably occur via the aquaporin pathway. Two full-length aquaporin cDNAs (HbPIP2;1 and HbTIP1;1, for plasma membrane intrinsic protein and tonoplast intrinsic protein, respectively) were cloned and characterized. The higher efficiency of HbPIP2;1 than HbTIP1;1 in increasing plasmalemma water conductance was verified in Xenopus laevis oocytes. HbPIP2;1 was insensitive to HgCl(2). In situ hybridization demonstrated that HbPIP2;1 was expressed in all liber tissues in the young stem, including the laticifers. HbPIP2;1 was up-regulated in both liber tissues and laticifers, whereas HbTIP1;1 was down-regulated in liber tissues but up-regulated in laticifers in response to bark Ethrel treatment. Ethylene-induced HbPIP2;1 up-regulation was confirmed by western-blot analysis. The promoter sequences of both genes were cloned and found to harbor, among many others, ethylene-responsive and other chemical-responsive (auxin, copper, and sulfur) elements known to increase latex yield. Increase in latex yield in response to ethylene was emphasized to be linked with water circulation between the laticifers and their surrounding tissues as well as with the probable maintenance of liber tissue turgor, which together favor prolongation of latex flow.
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http://dx.doi.org/10.1104/pp.109.140228DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2754619PMC
October 2009

Purification and characterization of rice DNA methyltransferase.

Plant Physiol Biochem 2009 Aug 9;47(8):671-80. Epub 2009 Apr 9.

Department of Biotechnology, Faculty of Science, Mahidol University, Rama VI Road, Bangkok 10400, Thailand.

Epigenetic modification is essential for normal development and plays important roles in gene regulation in higher plants. Multiple factors interact to regulate the establishment and maintenance of DNA methylation in plant genome. We had previously cloned and characterized DNA methyltransferase (DNA MTase) gene homologues (OsMET1) from rice. In this present study, determination of DNA MTase activity in different cellular compartments showed that DNA MTase was enriched in nuclei and the activity was remarkably increased during imbibing dry seeds. We had optimized the purification technique for DNA MTase enzyme from shoots of 10-day-old rice seedlings using the three successive chromatographic columns. The Econo-Pac Q, the Hitrap-Heparin and the Superdex-200 columns yielded a protein fraction of a specific activity of 29, 298 and 800 purification folds, compared to the original nuclear extract, respectively. The purified protein preferred hemi-methylated DNA substrate, suggesting the maintenance activity of methylation. The native rice DNA MTase was approximately 160-170 kDa and exhibited a broad pH optimum in the range of 7.6 and 8.0. The enzyme kinetics and inhibitory effects by methyl donor analogs, base analogs, cations, and cationic amines on rice DNA MTase were examined. Global cytosine methylation status of rice genome during development and in various tissue culture systems were monitored and the results suggested that the cytosine methylation level is not directly correlated with the DNA MTase activity. The purification and characterization of rice DNA MTase enzyme are expected to enhance our understanding of this enzyme function and their possible contributions in Gramineae plant development.
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http://dx.doi.org/10.1016/j.plaphy.2009.03.014DOI Listing
August 2009

Evaluation of a morphological marker selection and excision system to generate marker-free transgenic cassava plants.

Plant Cell Rep 2009 Mar 18;28(3):445-55. Epub 2008 Dec 18.

Department of Biotechnology, Faculty of Science, Mahidol University, Bangkok, Thailand.

The efficacy of the ipt-type Multi-Auto-Transformation (MAT) vector system to transform the extensively grown cassava cultivar "KU50" was evaluated. This system utilizes the isopentenyltransferase (ipt) gene as morphological marker for visual selection of transgenic lines. The extreme shooty phenotype (ESP) of transgenic lines is lost due to the removal of ipt gene mediated by the yeast Rint/RS system. As a result, phenotypically normal shoots, considered marker-free transgenic plants, could be obtained. When transforming KU50 cassava cultivar with two different ipt-type MAT vectors, transformation frequency at 19-21% was observed. Among the total number of ESP explants, 32-38% regained normal extended shoot phenotype and 88-96% of which were confirmed to represent the marker-free transgenic plants. This is the first demonstration of the efficacy of Rint/RS system in promoting excision of ipt marker gene in cassava specie, with the consequent rapid production of marker-free transgenic plants. The high efficiency of this system should facilitate pyramiding a number of transgenes by repeated transformation without having to undergo through laborious, expensive and time-consuming processes of sexual crossing and seed production. The generation of marker-free, thus environmentally safe, genetically modified cassava clones should also ease the public concerns regarding the use of transgenic cassava in both food and nonfood industries.
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http://dx.doi.org/10.1007/s00299-008-0658-yDOI Listing
March 2009

Development of genotype-independent regeneration system for transformation of rice (Oryza sativa ssp. indica).

J Plant Res 2007 Mar 1;120(2):237-45. Epub 2006 Dec 1.

Department of Biotechnology, Faculty of Science, Mahidol University, Rama VI Road, Bangkok, 10400, Thailand.

Rice (Oryza sativa ssp. indica) is an important economic crop in many countries. Although a variety of conventional methods have been developed to improve this plant, manipulation by genetic engineering is still complicated. We have established a system of multiple shoot regeneration from rice shoot apical meristem. By use of MS medium containing 4 mg L(-1) thidiazuron (TDZ) multiple shoots were successfully developed directly from the meristem without an intervening callus stage. All rice cultivars tested responded well on the medium and regenerated to plantlets that were readily transferred to soil within 5-8 weeks. The tissue culture system was suitable for Agrobacterium-mediated transformation and different factors affecting transformation efficiency were investigated. Agrobacterium strain EHA105 containing the plasmid pCAMBIA1301 was used. The lowest concentration of hygromycin B in combined with either 250 mg L(-1) carbenicillin or 250 mg L(-1) cefotaxime to kill the rice shoot apical meristem was 50 mg L(-1) and carbenicillin was more effective than cefotaxime. Two-hundred micromolar acetosyringone had no effect on the efficiency of transient expression. Sonication of rice shoot apical meristem for 10 s during bacterial immersion increased transient GUS expression in three-day co-cultivated seedlings. The gus gene was found to be integrated into the genome of the T(0) transformant plantlets.
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http://dx.doi.org/10.1007/s10265-006-0046-zDOI Listing
March 2007

Gene activation in plastids by the CRE site-specific recombinase.

Plant Mol Biol 2006 Jul;61(4-5):711-8

Waksman Institute, Rutgers, the State University of New Jersey, Piscataway, NJ 08854-8020, USA.

We developed a novel system for gene activation in plastids that uses the CRE/loxP site-specific recombination system to create a translatable reading frame by excision of a blocking sequence. To test the system, we introduced an inactive gfp* gene into the tobacco plastid genome downstream of the selectable spectinomcyin resistance (aadA) marker gene. The aadA gene is the blocking sequence, and is flanked by directly oriented loxP sites for excision by the CRE. In the non-activated state, gfp* is transcribed from the aadA promoter, but the mRNA is not translated due to the lack of an AUG translation initiation codon. Green Fluorescent Protein (GFP) expression is activated by excision of the aadA coding segment to link up the gfp* coding region with the translation initiation codon of aadA. Tobacco plants that carry the inactive gfp* gene do not contain detectable levels of GFP. However, activation of gfp* resulted in GFP accumulation, proving the utility of CRE-induced protein expression in tobacco chloroplasts. The gene activation system described here will be useful to probe plastid gene function and for the production of recombinant proteins in chloroplasts.
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http://dx.doi.org/10.1007/s11103-006-0044-5DOI Listing
July 2006

Isolation and characterization of an alpha-amylase gene in cassava (Manihot esculenta).

Plant Physiol Biochem 2005 Sep 4;43(9):821-7. Epub 2005 Oct 4.

National Center for Genetic Engineering and Biotechnology, 113 Phaholyothin Road, Klong 1, Klong Luang, Pathumthani 12120, Thailand.

The roots of cassava plants (Manihot esculenta Crantz) accumulate starch as their major form of carbohydrate reserve. Starch accumulation and properties are determined by a balance between starch biosynthesis and degradation processes. Alpha-amylases (EC 3.2.1.1) are alpha-1,4 endoglycolytic enzymes, responsible for the mobilization of stored carbohydrate reserves by initiating the degradation process. Alpha-amylase genes have been shown to be differentially expressed at various developmental stages and environmental conditions through the action of plant hormones such as abscisic acid (ABA) and gibberellic acid (GA). In this study, we isolated an alpha-amylase gene from cassava tuberous roots (designated as MEamy2, GenBank accession number DQ011041). The deduced product of MEamy2 is 407 amino acid residues in length, with a calculated molecular mass of 46.7 kDa and an isoelectric point of 8.66. Southern blot analysis showed that the MEamy2 is present as a single copy in cassava genome. It shares the highest homology with AMY8 from apple fruit. The predicted structural model of MEamy2 contains three domains, active sites and starch-binding domain that are common with other plant alpha-amylases. RT-PCR analysis showed that the MEamy2 gene expression was induced in cassava roots within 2 hours after treatment with GA, but not ABA.
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http://dx.doi.org/10.1016/j.plaphy.2005.07.014DOI Listing
September 2005

Molecular characterization of new members of the Hevea brasiliensis hevein multigene family and analysis of their promoter region in rice.

Biochim Biophys Acta 2005 Mar 20;1727(3):151-61. Epub 2005 Jan 20.

CIRAD, TA 80/03, Avenue Agropolis, 34398 Montpellier cedex, France.

The cloning of hevein genes from Hevea brasiliensis was undertaken with the objective to isolate useful promoters to drive transgene expression in genetically engineered rubber tree. Four different full length genes were cloned by library screening and a fifth, a partial gene, by adaptor-anchored PCR. Sequence alignment revealed that hevein genes, although highly conserved in their transcribed region, diverged in two groups, with major differences in their promoter region, suggesting a more rapid evolution of the upstream regulatory functions of the genes than the downstream functions of their protein products. The promoter regions from two hevein genes representative of each group were isolated and analyzed in rice. Although both were functional, only the longest promoter sequence (PHev2.1) conferred a high level of expression to the transgene in various tissues of this heterologous host. It was in addition up-regulated by mechanical wounding and fungal infection in leaves. A number of potential cis-regulatory elements were identified in silico and are discussed in view of the expression profiles observed in rice.
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http://dx.doi.org/10.1016/j.bbaexp.2004.12.013DOI Listing
March 2005

Characterization of two rice DNA methyltransferase genes and RNAi-mediated reactivation of a silenced transgene in rice callus.

Planta 2004 Jan 25;218(3):337-49. Epub 2003 Sep 25.

Department of Biology, Texas A&M University, College Station, TX 77843-3155, USA.

Two genomic clones ( OsMET1-1, AF 462029 and OsMET1-2, TPA BK001405), each encoding a cytosine-5 DNA methyltransferase (MTase), were isolated from rice ( Oryza sativa L.) BAC libraries. OsMET1-1 has an open reading frame of 4,566 nucleotides with 12 exons and 11 introns while OsMET1-2 has an open reading frame of 4,491 nucleotides with 11 exons and 10 introns. Although OsMET1-1 and OsMET1-2 have high sequence similarity overall, they share only 24% identity in exon 1, and intron 3 of OsMET1-1 is absent from OsMET1-2. As for other eukaryotic DNA MTases of the Dnmt1/MET l class, the derived amino acid sequences of OsMET1-1 and OsMET1-2 suggest that they are comprised of two-thirds regulatory domain and one-third catalytic domain. Most functional domains identified for other MTases were present in the rice MET1 sequences. Amino acid sequence comparison indicated high similarity (56-75% identity) of rice MET1 proteins to other plant MET1 sequences but limited similarity (approx. 24% identity) to animal Dnmt1 proteins. Genomic blot and database analysis indicated the presence of a single copy of OsMET1-1 (on chromosome 3) and single copy of OsMET1-2 (on chromosome 7). Ribonuclease protection assays revealed expression of both OsMET1-1 and OsMET1-2 in highly dividing cells, but the steady-state level of OsMET1-2 was 7- to 12-fold higher than that for OsMET1-1 in callus, root and inflorescence. The functional involvement of the rice DNA MTases in gene silencing was investigated using an RNAi strategy. Inverted repeat constructs of either the N- or C-terminal regions of OsMET1-1 were supertransformed into calli derived from a rice line bearing a silenced 35S-uidA-nos transgene. Restoration of uidA expression in the bombarded calli was consistent with the inactivation of maintenance methylation and with previous evidence for the involvement of methylation in silencing of this line.
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http://dx.doi.org/10.1007/s00425-003-1112-6DOI Listing
January 2004

Studies on the effects of a flanking repetitive sequence on the expression of single-copy transgenes in Nicotiana sylvestris and in N. sylvestris-N. tomentosiformis hybrids.

Plant Mol Biol 2003 May;52(1):203-15

Institute of Molecular Biology, Austrian Academy of Sciences, Billrothstrasse 11, 5020 Salzburg, Austria.

To test the influence of a Nicotiana tomentosiformis repetitive sequence (R8.3) on transgene expression in N. sylvestris and in N. sylvestris-N. tomentosiformis hybrids, the R8.3 sequence was placed upstream of a nopaline synthase promoter (NOSpro)-NPTII reporter gene in a T-DNA construct. A number of transgenic N. sylvestris lines were produced and in most, the NPTII gene was expressed. In one line, however, the NPTII gene became silenced and methylated in the NOSpro region. The silenced locus was able to trans-inactivate and induce methylation of two stably expressed transgene loci comprising a similar construct. Nucleotide sequence analyses of the three transgene loci revealed that they each contained a single incomplete copy of the T-DNA, which had sustained deletions of varying sizes in the R8.3 region. Paradoxically, the R8.3 DNA upstream of the two active, unmethylated NOSpro-NPTII genes was highly methylated, whereas the R8.3 DNA upstream of the silenced, methylated NOSpro-NPTII gene was less methylated. The methylated portions of the R8.3 sequence corresponded to retroelement remnants. An active NOSpro-NPTII gene downstream of a nearly intact R8.3 sequence did not become methylated in N. sylvestris-N. tomentosiformis hybrids. Thus, methylation in the R8.3 sequence did not spread into adjoining transgene promoters and the effect of the R8.3 dispersed repeat family on transgene expression was negligible. The silencing phenomena observed with the three single-copy transgene loci are discussed in the context of other possible triggers of silencing.
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http://dx.doi.org/10.1023/a:1023937006311DOI Listing
May 2003