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      Cloning, characterization, and expression of xyloglucan endotransglucosylase/hydrolase and expansin genes associated with petal growth and development during carnation flower opening

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          Abstract

          Growth of petal cells is a basis for expansion and morphogenesis (outward bending) of petals during opening of carnation flowers ( Dianthus caryophyllus L.). Petal growth progressed through elongation in the early stage, expansion with outward bending in the middle stage, and expansion of the whole area in the late stage of flower opening. In the present study, four cDNAs encoding xyloglucan endotransglucosylase/hydrolase (XTH) ( DcXTH1–DcXTH4) and three cDNAs encoding expansin ( DcEXPA1–DcEXPA3) were cloned from petals of opening carnation flowers and characterized. Real-time reverse transcription-PCR analyses showed that transcript levels of XTH and expansin genes accumulated differently in floral and vegetative tissues of carnation plants with opening flowers, indicating regulated expression of these genes. DcXTH2 and DcXTH3 transcripts were detected in large quantities in petals as compared with other tissues. DcEXPA1 and DcEXPA2 transcripts were markedly accumulated in petals of opening flowers. The action of XTH in growing petal tissues was confirmed by in situ staining of xyloglucan endotransglucosylase (XET) activity using a rhodamine-labelled xyloglucan nonasaccharide as a substrate. Based on the present findings, it is suggested that two XTH genes ( DcXTH2 and DcXTH3) and two expansin genes ( DcEXPA1 and DcEXPA2) are associated with petal growth and development during carnation flower opening.

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          Rapid isolation of high molecular weight plant DNA.

          A method is presented for the rapid isolation of high molecular weight plant DNA (50,000 base pairs or more in length) which is free of contaminants which interfere with complete digestion by restriction endonucleases. The procedure yields total cellular DNA (i.e. nuclear, chloroplast, and mitochondrial DNA). The technique is ideal for the rapid isolation of small amounts of DNA from many different species and is also useful for large scale isolations.
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            The XTH family of enzymes involved in xyloglucan endotransglucosylation and endohydrolysis: current perspectives and a new unifying nomenclature.

            The polysaccharide xyloglucan is thought to play an important structural role in the primary cell wall of dicotyledons. Accordingly, there is considerable interest in understanding the biochemical basis and regulation of xyloglucan metabolism, and research over the last 16 years has identified a large family of cell wall proteins that specifically catalyze xyloglucan endohydrolysis and/or endotransglucosylation. However, a confusing and contradictory series of nomenclatures has emerged in the literature, of which xyloglucan endotransglycosylases (XETs) and endoxyloglucan transferases (EXGTs) are just two examples, to describe members of essentially the same class of genes/proteins. The completion of the first plant genome sequencing projects has revealed the full extent of this gene family and so this is an opportune time to resolve the many discrepancies in the database that include different names being assigned to the same gene. Following consultation with members of the scientific community involved in plant cell wall research, we propose a new unifying nomenclature that conveys an accurate description of the spectrum of biochemical activities that cumulative research has shown are catalyzed by these enzymes. Thus, a member of this class of genes/proteins will be referred to as a xyloglucan endotransglucosylase/hydrolase (XTH). The two known activities of XTH proteins are referred to enzymologically as xyloglucan endotransglucosylase (XET, which is hereby re-defined) activity and xyloglucan endohydrolase (XEH) activity. This review provides a summary of the biochemical and functional diversity of XTHs, including an overview of the structure and organization of the Arabidopsis XTH gene family, and highlights the potentially important roles that XTHs appear to play in numerous examples of plant growth and development.
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              Use of a new adhesive film for the preparation of multi-purpose fresh-frozen sections from hard tissues, whole-animals, insects and plants.

              A method for preparing thin fresh-frozen sections from large samples and hard tissues is described and the applications are shown. A new adhesive film is introduced to produce the frozen sections. The sample is frozen in a cooled hexane or liquid nitrogen, and then freeze-embedded with 4-5% carboxymethyl cellulose (CMC) in the coolant. A specially prepared adhesive film is fastened to the cut surface of the sample in order to support the section and cut slowly with a disposable tungsten carbide blade. The adhesive film is made of a thin plastic film and an adhesive before use. This method produces 2-microm thick fresh-frozen sections from a large sample, bone or tooth. The "film-section" i.e. the section attached to the adhesive film, can be used for many types of studies such as histology, general histochemistry, enzyme histochemistry, immunohistochemistry, in situ hybridization, elemental analysis, and autoradiography for water-soluble materials. Immunohistochemistry and in situ hybridization can be carried out with nonfixed and undecalcified sections. The section on the adhesive film can be transferred to a glass slide and mounted under a cover slip, and stained sections can be examined with an optical microscope at high magnification. This method is also useful for preparing frozen sections from samples of fish, insects, and plants. Furthermore, samples of particular areas can be collected from the film-section by means of a laser microdissection technique. The multiple possible applications of the adhesive film render it highly useful for studies in biological and medico-dental fields.
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                Author and article information

                Journal
                J Exp Bot
                jexbot
                exbotj
                Journal of Experimental Botany
                Oxford University Press
                0022-0957
                1460-2431
                January 2011
                19 October 2010
                19 October 2010
                : 62
                : 2
                : 815-823
                Affiliations
                [1 ]Laboratory of Genetic Engineering, Graduate School of Life and Environmental Sciences, Kyoto Prefectural University, Kyoto 606-8522, Japan
                [2 ]Kyoto Prefectural Institute of Agricultural Biotechnology, Seika-cho 619-0224, Kyoto Prefecture, Japan
                [3 ]Yamagata Integrated Agricultural Research Center, Sagae 991-0043, Yamagata Prefecture, Japan
                [4 ]Laboratory of Plant Cell Wall Biology, Graduate School of Life Sciences, Tohoku University, Sendai 980-8578, Japan
                Author notes
                []To whom correspondence should be addressed. E-mail: ssatoh@ 123456kpu.ac.jp
                [*]

                These authors contributed equally to this work.

                Article
                10.1093/jxb/erq319
                3003822
                20959626
                c339d81c-3c29-47a4-a601-4b0e4ad037f4
                © 2010 The Author(s).

                This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License ( http://creativecommons.org/licenses/by-nc/2.5), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

                This paper is available online free of all access charges (see http://jxb.oxfordjournals.org/open_access.html for further details)

                History
                : 27 March 2010
                : 17 August 2010
                : 20 September 2010
                Categories
                Research Papers

                Plant science & Botany
                flower opening,expansin,petal cell growth,carnation,gene expression,in situ xet staining,xyloglucan endotransglycosylase/hydrolase (xth)

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