Suppression subtractive hybridization to enrich low-abundance and submergence-enhanced transcripts of adventitious root primordia of Sesbania rostrata
Introduction
Sesbania rostrata is an annual legume from the Sahel region of West Africa that is adapted to grow under waterlogged conditions during the rain season and that carries dormant adventitious root primordia on vertical rows along the stem. Upon submergence, the quiescent apical meristem of the root primordia is activated and roots develop adventitiously. Alternatively, upon inoculation with the microsymbiont Azorhizobium caulinodans, the root primordia can develop into nitrogen-fixing nodules [1], in which case, nodule primordia originate from root cortical cells that re-enter the cell cycle. Thus, preformed adventitious root primordia are a good subject for studying adventitious root outgrowth. Here, molecular markers were isolated that are associated with dormant primordia and with the submergence response.
Growth that is stimulated by flooding has been studied mostly in deepwater rice. Internodes are induced to grow rapidly upon submergence to prevent the plants from drowning by keeping part of the foliage above the rising floods. The initial signal for internode elongation is a reduced partial oxygen pressure [2]; subsequently, the process is regulated by ethylene, abscisic acid and gibberellin [3], [4]. Submergence stimulates the expression of several genes, such as members of the 1-aminocyclopropane-1-carboxylate synthase gene family [5], [6] and expansin genes [7], [8].
When S. rostrata stems are immersed in water, the dormancy of adventitious rootlets is broken. The cells of the quiescent meristems are diploid and have a null mitotic index [9]. After 24 h of submergence, part of the nuclear population is tetraploid and mitotic activity is resumed [9]. By in situ hybridizations, no B1-type cyclin transcripts could be detected in the quiescent apical meristem cells [10], whereas histone H4 transcripts are abundant [10], indicating an arrest in the S phase of the cell cycle, possibly as a preparation for massive histone synthesis and fast division upon waterlogging.
Previously, differential display [11] has been used to identify genes involved in stem nodule initiation [12], [13]. The aim of the present work was to identify a subset of molecular markers for the primordia. Suppression subtractive hybridization (SSH) is a polymerase chain reaction (PCR)-based method that has been developed to enrich rare transcripts and low-abundance genes in animal systems [14]; recently, several applications have been reported in plants [15], [16], [17], [18], [19]. Here, the method was combined with cDNA arrays to collect low-abundant messengers of root primordia and to enrich for genes with differential expression upon submergence of adventitious rootlets of S. rostrata.
Section snippets
Plant material and RNA preparation
S. rostrata Brem seeds were surface-sterilized and germinated as described [20]. Five-week-old plants were immersed in water for 4 h. Adventitious root primordia were harvested and frozen in liquid nitrogen. RNA was prepared using a LiCl precipitation method [12]. Polyadenylated (A+) RNA was prepared using Dynabeads oligo(dT)25 (Dynal, Oslo, Norway).
Construction of a forward cDNA library
A forward cDNA library was constructed with the PCR-Select cDNA subtraction kit (Clontech Laboratories, Palo Alto, CA) according to the
Construction and differential screening of a subtracted cDNA library
The response of S. rostrata to waterlogging can be compared with that of deepwater rice. In deepwater rice reactivated cells enter the S phase between 2 and 6 h after submergence [21]. In both rice and Sesbania, root elongation is enhanced 8–10 h after submergence. To generate an S. rostrata cDNA library enriched for submergence response sequences, two RNA populations were prepared: a control from a pool of untreated root primordia and a target sample from primordia submerged in water for 4 h.
Acknowledgements
The authors thank Dr Pieter Rottiers (Department Molecular Biology, Ghent University), for help, discussions, and advice on SSH, Sylvia Herman for excellent technical assistance, Jan Gielen for sequencing, Sofie Goormachtig for critical reading of the manuscript, and Martine De Cock for help in preparing it. This work was supported by a grant from the Flemish Community (‘Bilaterale wetenschappelijke en technologische samenwerking Vlaanderen-Chili’ BIL99/68). MC is indebted to the European
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Present address: Department of Ecophysiology, Biochemistry and Toxicology, University of Antwerp, Groenenborgerlaan 171, B-2020 Antwerpen, Belgium.