Expansins are a group of extracellular proteins that directly modify the mechanical properties of plant cell walls, leading to turgor-driven cell extension. Within the completely sequenced Arabidopsis genome, we identified 38 expansin sequences that fall into three discrete subfamilies. Based on phylogenetic analysis and shared intron patterns, we propose a new, systematic nomenclature of Arabidopsis expansins. Further phylogenetic analysis, including expansin sequences found here in monocots, pine (Pinus radiata, Pinus taeda), fern (Regnellidium diphyllum, Marsilea quadrifolia), and moss (Physcomitrella patens) indicate that the three plant expansin subfamilies arose and began diversifying very early in, if not before, colonization of land by plants. Closely related "expansin-like" sequences were also identified in the social amoeba, Dictyostelium discoidium, suggesting that these wall-modifying proteins have a very deep evolutionary origin.The availability of information from genome sequencing programs now offer a new route to understanding multigene families within and across different species. Several recent studies have demonstrated the usefulness of phylogenetic analysis to complement parallel investigations of gene function in vivo (Sanderfoot et al., 2000; Kellogg, 2001; Li et al., 2001; Ross et al., 2001). The present analysis makes use of the completely sequenced Arabidopsis genome (The Arabidopsis Genome Initiative, 2000), together with comprehensive searches of GenBank and expressed sequence tag (EST) databases (maintained at the National Center for Biotechnology Information, NCBI), to determine the phylogeny of the plant cell wall protein, expansin.Expansins play a variety of roles in vivo by modifying the cell wall matrix during growth and development (for review, see Cosgrove, 2000a; Darley et al., 2001). Initially identified by their unique ability to induce the pH-dependent extension of plant cell walls in vitro (McQueen-Mason et al., 1992), expansins appear to increase polymer mobility in the cell wall, allowing the structure to slide apart during extension (McQueen-Mason et al., 1993; McQueenMason and Cosgrove, 1994, 1995; Whitney et al., 2000). To date, expansin remains the only protein to demonstrate cell wall extension in vitro and in vivo. In addition to roles in plant cell growth, expansins are also now believed to play key roles in the early development of leaf primordia (Fleming et al., 1997), fruit softening (Civello et al., 1999; Rose et al., 2000), plant reproduction (Cosgrove et al., 1997), and wall disassembly (Cho and Cosgrove, 2000).Growing tissues from a wide range of plants, including dicotyledons (Rayle and Cleland, 1977), grasses (Kutschera, 1994), gymnosperms (Kim et al., 2000), and green algae (Metraux and Taiz, 1977), have been shown to undergo acid-induced extension. As it is now generally accepted that expansins are the chief agents responsible for acid-induced extension, these data suggest that expansins may be found in all land plants and probably algae. In suppor...

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SummaryThe semi-aquatic dicot Rumex palustris responds to complete submergence by enhanced elongation of young petioles. This elongation of petiole cells brings leaf blades above the water surface, thus reinstating gas exchange with the atmosphere and increasing survival in flood-prone environments. We already know that an enhanced internal level of the gaseous hormone ethylene is the primary signal for underwater escape in R. palustris. Further downstream, concentration changes in abscisic acid (ABA), gibberellin (GA) and auxin are required to gain fast cell elongation under water. A prerequisite for cell elongation in general is cell wall loosening mediated by proteins such as expansins. Expansin genes might, therefore, be important target genes in submergence-induced and plant hormone-mediated petiole elongation. To test this hypothesis we have studied the identity, kinetics and regulation of expansin A mRNA abundance and protein activity, as well as examined pH changes in cell walls associated with this adaptive growth. We found a novel role of ethylene in triggering two processes affecting cell wall loosening during submergence-induced petiole elongation. First, ethylene was shown to promote fast net H þ extrusion, leading to apoplastic acidification. Secondly, ethylene upregulates one expansin A gene (RpEXPA1), as measured with real-time RT-PCR, out of a group of 13 R. palustris expansin A genes tested. Furthermore, a significant accumulation of expansin proteins belonging to the same size class as RpEXPA1, as well as a strong increase in expansin activity, were apparent within 4-6 h of submergence. Regulation of RpEXPA1 transcript levels depends on ethylene action and not on GA and ABA, demonstrating that ethylene evokes at least three, parallel operating pathways that, when integrated at the whole petiole level, lead to coordinated underwater elongation. The first pathway involves ethylenemodulated changes in ABA and GA, these acting on as yet unknown downstream components, whereas the second and third routes encompass ethylene-induced apoplastic acidification and ethylene-induced RpEXPA1 upregulation.

Sodium at high millimolar levels in the cytoplasm is toxic to plant and yeast cells. Sequestration of Na(+) ions into the vacuole is one mechanism to confer Na(+)-tolerance on these organisms. In the present study we provide direct evidence that the Arabidopsis thaliana At-NHX1 gene and the yeast NHX1 gene encode low-affinity electroneutral Na(+)/H(+) exchangers. We took advantage of the ability of heterologously expressed At-NHX1 to functionally complement the yeast nhx1-null mutant. Experiments on vacuolar vesicles isolated from yeast expressing At-NHX1 or NHX1 provided direct evidence for pH-gradient-energized Na(+) accumulation into the vacuole. A major difference between NHX1 and At-NHX1 is the presence of a cleavable N-terminal signal peptide (SP) in the former gene. Fusion of the SP to At-NHX1 resulted in an increase in the magnitude of Na(+)/H(+) exchange, indicating a role for the SP in protein targeting or regulation. Another distinguishing feature between the plant and yeast antiporters is their sensitivity to the diuretic compound amiloride. Whereas At-NHX1 was completely inhibited by amiloride, NHX1 activity was reduced by only 20-40%. These results show that yeast as a heterologous expression system provides a convenient model to analyse structural and regulatory features of plant Na(+)/H(+) antiporters.

In all terrestrial and aquatic plant species the primary cell wall is a dynamic structure, adjusted to fulfil a diversity of functions. However a universal property is its considerable mechanical and tensile strength, whilst being flexible enough to accommodate turgor and allow for cell elongation. The wall is a composite material consisting of a framework of cellulose microfibrils embedded in a matrix of non-cellulosic polysaccharides, interlaced with structural proteins and pectic polymers. The assembly and modification of these polymers within the growing cell wall has, until recently, been poorly understood. Advances in cytological and genetic techniques have thrown light on these processes and have led to the discovery of a number of wall-modifying enzymes which, either directly or indirectly, play a role in the molecular basis of cell wall expansion.