Semiviviparous embryo development and dehydrin ex

Authors: Flora AbdulRahman Ismail Lisette M C Nitsch Mieke M C WoltersArts Celestina Mariani Jan W M Derksen

Publish Date: 2010/01/19

Volume: 23, Issue: 2, Pages: 95-103

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Abstract

Rhizophora mucronata Lam is a tropical mangrove with semiviviparous cotyledon body protrusion before shedding nonquiescent and nondesiccating recalcitrant seeds As recalcitrance has been thought to relate to the absence of desiccationrelated proteins such as dehydrins we for the first time systematically described and classified embryogenesis in R mucronata and assessed the presence of dehydrinlike proteins Embryogenesis largely follows the classic pattern till stage eight the torpedo stage with the formation of a cotyledonary body Ovule and embryo express radical adaptations to semivivipary in the saline environment 1 A large highly vacuolated and persistent endosperm without noticeable food reserves that envelopes the developing embryo 2 Absence of vascular tissue connections between embryo and maternal tissue but instead transfer layers in between endosperm and integument and endosperm and embryo Dehydrinlike proteins 55–65 kDa were detected by the Western analysis in the ovules till stage 10 when the integuments are dehisced An additional 50 kDa band was detected at stages 6–8 Together these results suggest a continuous flow of water with nutrients from the integument via the endosperm to the embryo circumventing the vascular route and probably suppressing the initially induced dehydrin expressionMost angiosperms disperse their seeds in a dormant dry and resting condition so called orthodox seeds In contrast viviparous plants develop embryos that do not enter dormancy nor desiccate and germinate whilst attached to the maternal plant Members of the Rhizophoraceae show continuing embryo growth prior to dispersal albeit not vivipary in sensu stricto Instead they develop a cotyledon body that protrudes from the seed with the radicle germinating and protruding only after shedding We characterize this process as “semivivipary” in this work The semiviviparous seed which remains attached to the mother plant is termed a “propagule” Embryos of such species either omit or modify a number of postfertilization events In angiosperms this behaviour is restricted to some mangroves and members of the sea grass genera Amphibolis and Thalassodendron Elmqvist and Cox 1996 Mangroves are woody species found in brackish or saline tropical and subtropical tidal areas Representative genera of semi or trueviviparous mangroves include Rhizophora Bruguiera Ceriops and Kandelia of the tribe Rhizophoreae family Rhizophoraceae Tomlinson 2000In orthodox seeds quiescence is acquired during the later stages of maturation at the same time that a considerable quantity of water ~90 is gradually lost by desiccation Finkelstein et al 2002 Desiccation tolerance acquisition is marked by a variety of factors such as an increase in low molecular weight solutes and the appearance of dehydrins the D11 family Dure 1993a 1993b of late embryogenesis abundant LEA proteins Battaglia et al 2008 Close 1997 Finkelstein et al 2002 Ingram and Bartels 1996 Wise and Tunnacliffe 2004 Dehydrins are detected in several tissues during stress and development They are suggested to be involved in resisting environmental stresses by stabilizing proteins and membrane structures at low water conditions Allugulova et al 2003 Arora et al 1997 Campbell and Close 1997 Ingram and Bartels 1996 Rorat 2006 Wise and Tunnacliffe 2004 Dehydrins are in general thermostable hydrophilic and contain at their carboxyterminus a “Kmotif” which in angiosperms includes the amino acid residue consensus sequence EKKGIMDKIKEKLPG Close 1996Absence of dehydrins in mature mangroves seeds has been related to vivipary Farrant et al 1996 however there are no reports on their expression during embryogenesis Presently we systematically described and staged embryo development in Rhizophora mucronata and assessed the presence of dehydrins in ovules and embryos through development from fertilization to the point of emergence from the micropyleFlowers and ovaries from R mucronata Lam were collected during February–March from Mbweni fishing village 40 km from Dar es Salaam Tanzania In R mucronata several stages of development from flower bud to propagule can often be found on a single plant The trees grow almost exclusively in single stands and samples were collected from ~25 trees at a single site to ensure that developmental stages were representative of the local population Synchrony of each developmental stage is at best an estimate since the sample population is wild and the environment unsettledUnless stated otherwise all chemicals PA grade were from Merck Darmstadt Germany The polyclonal antidehydrin antiserum and peptide–salt containing the dehydrin Ksegment consensus peptide were kind gifts from Dr T Close Riverside CA USAAll materials were prepared according to standard techniques Ovules were immediately dissected with much of the testa removed before fixation using 2 glutaraldehyde and 1 paraformaldehyde in buffer 100 mM PIPES pH 70 2 sucrose 005 CaCl2·2H20 and 01 Tween 20 The samples were infiltrated with Paraplast via a water–ethanol–xylene series Sections 5–7 μm thick were cut using an AO 820 microtome American Optical Ramsey MN USA were deparaffinated rehydrated and stained with 01 toluidine blue O in borate buffer pH 8 In addition 1μm thick sections were made using Spurr’s resinembedded material see below and stained with 1 aqueous toluidine blue Micrographs were made using a Leitz Orthoplan microscope Leica Microsystems GmbH Wetzlar Germany equipped with a Coolsnap digital camera Roper Scientific Tucson AZ and Meta Vue Universal Imaging Corporation West Chester PA USA imaging software

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