Journal of Experimental Botany, Vol. 51, No. 346, pp. 847-852,
May 2000
© 2000 Oxford University Press
Differential expression of two barley XET-related genes during coleoptile growth
URGAP, INRA, BV1540, 21034 Dijon Cedex, France
Received 30 November 1999; Accepted 17 January 2000
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Abstract |
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Plant cell elongation depends on the physical properties of the primary cell wall. Because xyloglucan endotransglycosylases (XETs) are enzymes that mediate cleavage and rejoining of the ß(1-4)-XG backbone of primary cell wall, they are potentially involved in cell elongation. In this paper, the growth of the barley coleoptile was related to the expression patterns of two genes from this family (hvEXT, hvXEB) in experiments where coleoptile elongation varied according to light/dark treatments in order to assess the potential role of these genes in cell elongation. In dark-grown and light-grown coleoptiles, growth rate variations were associated with altered levels of expression of hvEXT and hvXEB: they were higher in dark-grown than in light-grown seedlings, and decreased after 5 d in darkness, and after 4 d in continuous light. In 4-d-old seedlings, coleoptile elongation decreased significantly 4 h after the onset of a continuous white- light irradiation, and hvXEB and hvEXT mRNA levels decreased, respectively, 2 h and 4 h after the onset of white-light irradiation. Moreover, the distribution of hvXEB and hvEXT along the coleoptiles of 4-d-old dark-grown seedlings were different. Altogether, these results suggest a complex pattern of temporal and positional expression for the different genes of the XET-related family.
Key words: Xyloglucan endotransglycosylase, coleoptile elongation, Hordeum vulgare.
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Plant cell elongation depends on the physical properties of the primary cell wall. A novel class of transglycosylase has been identified in plants (Fry, 1992) which is proposed to play a role in cell wall metabolism during cell expansion. This class of enzymes, called alternatively endo-xyloglucan transglycosylase (EXT) or xyloglucan endotransglycosylase (XET), modifies xyloglucan (XG) by cleavage and rejoining of the ß(1–4)-XG backbone. Such activity can potentially alter cell size and form through cell wall loosening or tightening. Enzymes with XET activity have been identified in rapidly growing tissues from various plant species (Fry et al., 1992
; Pritchard et al., 1993; Potter and Fry, 1994; Wu et al., 1994; Smith et al., 1996) and in parallel studies, multigene families related to XET have been identified (de Silva et al., 1994; Nishitani, 1995; Xu et al., 1996; Schünmann et al., 1997). These genes are expressed in various organs, during different phases of development, and are regulated by different hormonal and environmental stimuli (Saab and Sachs, 1996; Xu et al., 1996; Schünmann et al., 1997). In barley, five XET-related genes have been isolated from growing leaves (Smith et al., 1996; Schünmann et al., 1997). Three of them showed a close association between expression and leaf growth rate patterns: they were preferentially expressed in the elongation zone of the leaf blade, and the enhanced elongation rate of the blade following GA3 treatment of GA-deficient dwarf mutants was associated with an increase in mRNA levels.
In this study, the expression of two of these barley XET-related genes –hvEXT and hvXEB– was investigated in a different system: the growing coleoptile. The coleoptile, a sheath-like organ present in grass, is a good model system for studying growth and cell elongation: (i) its growth is rapid, occurring in the week following sowing, (ii) it is essentially due to cell elongation (Liptay and Davidson, 1972), (iii) its epidermal cells are uniform in contrast to leaves where different epidermal cell types occur (Wenzel et al., 1997), and (iv) its growth is rapidly regulated by light. The growth of the barley coleoptile was related to the expression of hvEXT and hvXEB in light/dark experiments.
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Materials and methods |
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Experimental conditions
All experiments were conducted on ‘Himalaya’ barley (Hordeum vulgare L.) plants, germinated at 20 °C in a perlite-vermiculite mixture and watered every second day. Watering was done under a green safelight for plants germinated in the dark. For studying the association between growth and expression of the XET-related genes in darkness and in light, a minimum of 15 seedlings were grown either in continuous light (600 µmol m-2 s-1) or in continuous dark. Coleoptiles were measured, separated from the enclosed first leaf and harvested in bulk for RNA preparation daily from 3–6 d after sowing. They were only measured on days 7 and 10. This experiment was carried out three times for the coleoptile. In the second experiment, seedlings were grown in the dark for 4 d after sowing, and at time T0 on day 4, 50% of the plants were transferred to continuous light. At T0, and after 2, 4, 6, 8, and 24 h of exposure to white light, samples of 20 seedlings each were measured and harvested for Northern analysis. This experiment was carried out twice. All measurements and harvesting on dark-grown plants were performed under green safelight. In the third experiment, 20 4-d-old dark grown seedlings were sectioned into six equally long segments for northern analysis of gene expression along the coleoptile.
Northern analysis
Total RNA was purified as described previously (Chandler et al., 1983), electrophoresed in formaldehyde gels and transferred to nylon membrane (Hybond-N, Amersham) as described previously (Smith et al., 1996). Probes were prepared by random priming (MegaPrime kit, Amersham) 3' specific regions of about 200 bp for the genes hvXEB and hvEXT as described previously (Schünmann et al., 1997). Hybridization and washing of the filters were carried out as described in Smith et al. (Smith et al., 1996). The filters were exposed to a phosphor- screen after hybridization with the probe, and again after re-hybridization with an 18S ribosomal probe. The signal was quantified using a PhosphorImager (Molecular Dynamics). The mRNA signal was corrected for loading using the 18S ribosomal signal. Relative mRNA levels were computed as transcript levels divided by the mRNA signal of 4-d-old dark-grown seedlings for the first experiment, and by the mRNA signal of dark-grown seedlings 2 h after T0 for the second experiment. These relative transcript levels were then averaged over replicate experiments.
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Results |
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HvEXT and hvXEB transcript levels along the coleoptile
HvXEB and hvEXT were expressed all over the coleoptile but had slightly different patterns of expression: hvEXT mRNA level was maximal in the three upper segments, declined towards the basal segments, and was low in the most basal segment, while hvXEB was preferentially expressed at the tip of the coleoptile (Fig. 1
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Light regulation of growth rate and transcript levels
In constant light, seeds germinated earlier than in the dark, and the elongation rate of the coleoptile was initially higher. However, by 3 d after sowing, the coleoptiles of dark-grown seedlings had a higher elongation rate, which persisted until growth of the coleoptile ceased (Fig. 2). The coleoptile elongation rate was maximal between 4 and 5 d after sowing (1 mm h-1) and was higher than the maximum elongation rate in the light (0.36 mm h-1), that occurred between 3 and 4 d after sowing. At 7 d after sowing, epidermal cells were correspondingly longer in the dark than in light (data not shown). In parallel, RNAs were extracted from the coleoptiles of light- and dark-grown seedlings, and the content of mRNAs assessed. The relative abundance of hvEXT and hvXEB mRNAs was greater in dark-grown coleoptiles (Fig. 3), and declined at about the same time as coleoptile growth rate (i.e. between 4 and 5 d in the light, and between 5 and 6 d in the dark).
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Because of the significant effect of light on coleoptile growth and mRNA levels, the relationship between coleoptile elongation rate and mRNA levels was investigated in greater detail, by using dark-grown seedlings submitted to continuous white-light irradiation. Seedlings were grown in darkness for 4 d, and then transferred to continuous light. The elongation of the coleoptile was monitored and the coleoptile elongation rate declined 4 h after the transfer to white-light (Fig. 4
). Four hours after transfer, it was about 5 times higher in the dark than in the light. mRNA levels from coleoptiles of 4-d-old dark-grown seedlings transferred to continuous white light were compared to those from dark-grown seedlings, over a period of 24 h. The light treatment affected the relative levels of hvXEB and hvEXT transcripts. Within 2 h the hvXEB transcript light treatment level had fallen to 40% of the dark treatment level (Fig. 5B), and this decline continued for 2 h. HvEXT transcript light treatment level fell to 50% of the dark treatment level after 4 h of light irradiation (Fig. 5A).
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Discussion |
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This study extends previous work to a new experimental system. The coleoptile is a good model system to study the relationship between growth and XET-related gene expression because the effect of white light on its growth is extremely rapid. This rapid regulation of coleoptile growth is adaptive: the coleoptile is a specialized embryonic structure present in grasses, that guides and provides physical protection to the first leaf during seed germination (O'Brien and Thimann, 1965
). On reaching the soil surface and being exposed to light, the coleoptile growth is inhibited, while the first leaf continues to grow and emerges through the pore located at the apex of the coleoptile. As expected, light/dark regulation of coleoptilar elongation was observed in the present study. It was found to be related to the expression of the two barley XET-related genes studied: there was a close correspondence between the duration of elongation and the duration of expression of XET-related genes, and transcript levels were higher in dark-grown coleoptiles, and associated with greater elongation rates. In light/dark treatments on 4-d-old dark-grown seedlings, the reduction in coleoptile elongation rate became significant 4 h after the onset of white light irradiation, and was associated with an even more rapid reduction in the level of the hvXEB transcript (60% decrease after 2 h of exposure). Thus, changes in the level of hvXEB transcripts may potentially relate to changes in elongation rate. There was also a reduction in the level of the hvEXT transcripts in response to light, but this was observed at later times.
Together with previous studies, a complex pattern of temporal and positional expression is emerging, suggesting specific regulatory patterns and functional roles for the gene products. The evolutionary significance of gene duplication is well known: this mechanism allows the emergence of new genes and hence of new functions, since a redundant duplicate gene may accumulate divergent mutations regardless of their selective value (Li, 1983). The differential, but overlapping, expression of the genes from the barley XET-related gene family, which were isolated from growing vegetative tissues, suggests that they may be involved in processes associated with early plant growth and cell elongation. In these experiments, hvXEB expression decreased, in dark-grown seedlings, 2 h after the onset of a white-light exposure, and hvEXT expression decreased after 4 h. HvXEB zone of expression was preferentially in the apex of the coleoptile, where cell elongation is assumed to start (O'Brien and Thimann, 1965), while hvEXT was maximally expressed over the upper 50% of the coleoptile. Could hvXEB be more involved in bound cleavage and hvEXT in bound rejoining and wall deposition? The HvEXT gene product was proved to have in vitro XET activity (Schünmann et al., 1997). Further functional analysis of the enzymes encoded by the different barley XET-related genes would be extremely useful to determine if these enzymes are preferentially involved in different processes required for cell wall expansion such as wall loosening or wall deposition. It has been suggested that different ‘xyloglucan-related proteins’ with slightly different catalytic activities could play specific roles in cell wall construction (Nishitani, 1995).
Functional analysis would also reveal differences in the substrate specificity or efficiency of the different enzymes. It has been shown that four XET-related enzymes from Arabidopsis thaliana perform the same function (transglycosylation) (Campbell and Braam, 1999), but have different functional properties as regards to substrate specificity and temperature optima. It has also been shown (Rose et al., 1996) that two EXT genes from nasturtium had mutually exclusive spatial patterns of expression, related to substrate specificity and substrate distribution. The significance of XET in grass cell wall metabolism has been questioned, since only small amounts of xyloglucan are present in these walls (Carpita, 1996). However, xyloglucan was found in the cell walls of oat coleoptiles (Labavitch and Ray, 1978), 4-d-old rice seedlings (Kato et al., 1982), and barley seedlings (Kato et al., 1981), and high levels of XET activity and/or XET-related transcripts were found in several monocots' growing organs (Fry et al., 1992; Pritchard et al., 1993; Smith et al., 1996; Palmer and Davies, 1996; Schünmann et al., 1997). Moreover, Carpita found that xyloglucan is present in maize coleoptiles and that the wall composition changes according to coleoptile age and to cell position along the coleoptile (Carpita, 1984). The distribution of mRNAs of hvXEB and hvEXT along the coleoptile could be related to this finding.
A comprehensive model should include the other enzymatic systems involved in the control of cell extension. XET activity is not sufficient for in vitro loosening of cell walls (McQueen-Mason et al., 1993). A summary model of wall extension has been presented (Cosgrove, 1999), involving primary and secondary wall-loosening agents. Primary wall-loosening agents would be able to induce wall loosening in vitro, and secondary agents would facilitate the action of primary agents by modifying the wall structure. Expansins are primary agents, since they can induce extension of isolated cell walls in acidic pH (McQueen-Mason et al., 1992; Li et al., 1993). They could work in concert with XETs as secondary agents. The spatial and temporal growth of oat coleoptiles has been related to wall properties associated with expansin action in experiments similar to the present experiments (Cosgrove and Li, 1993), in which cell extension varied according to the growth rate distribution along the coleoptile and to light/dark treatments. They showed that walls started to lose their ability to undergo acid-induced extension 4 h after the onset of the white-light treatment. In this experiment, hvXEB expression decreased as early as 2 h after the onset of the white-light treatment. If hvXEB encodes an XET with preferential hydrolytic activity, it could be involved in the change in cell wall susceptibility to expansin action that is of key importance for the control of growth in coleoptiles.
The question of what is the connection between changes in elongation rate and changes in XET-related gene expression is difficult to pursue experimentally until the XET gene families have been fully defined and the functionality of the gene products assessed. However, studying the differential regulation of related genes may provide helpful insights in the processes involved in cell wall loosening during cell elongation, a mechanism that is far from simple and involves the concerted action of several enzyme families.
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Acknowledgments |
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I wish to thank PM Chandler for helpful advice during the experiments and anonymous referees for helpful comments on the manuscript. I am grateful to PHD Schünmann for providing the hvEXT and hvXEB clones. This work was funded by the French Ministry of Research with a grant to J Burstin and was carried out in the laboratory of PM Chandler at CSIRO Plant Industry, Canberra, Australia.
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Notes |
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1 Fax: +33 3 80 69 31 59. E-mail: burstin{at}epoisses.inra.fr
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Abbreviations |
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XET, xyloglucan endotransglycosylase; XG, xyloglucan..
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