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Keywords:

  • MYB;
  • transcription;
  • det3;
  • lignin;
  • photomorphogenesis

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Results and discussion
  5. Conclusion
  6. Experimental procedures
  7. Acknowledgements
  8. References

Overexpression of a pine MYB, PtMYB4, in Arabidopsis caused ectopic lignin deposition and allowed the plants to undergo photomorphogenesis even when they were grown in the dark. The phenotype caused by PtMYB4 overexpression was reminiscent of the previously characterised dark-photomorphogenic mutant, de-etiolated 3 (det3); consequently, we tested the hypothesis that MYB misexpression may explain aspects of the det3 phenotype. We show here that AtMYB61, a member of the Arabidopsis R2R3-MYB family, is misexpressed in the det3 mutant. Semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) experiments suggested that AtMYB61 was misexpressed in a det3 background relative to wild-type plants. Examination of AtMYB61 promoter activity in a det3 background showed that the spatial control of AtMYB61 expression was lost. In order to determine if such misexpression could explain the mutant phenotype, AtMYB61 was overexpressed in wild-type Arabidopsis plants. Transgenic plants that overexpressed AtMYB61 had the same ectopic lignification and dark-photomorphogenic phenotype as that of the det3 mutant. In order to test if AtMYB61 was necessary for these aspects of the det3 phenotype, AtMYB61 expression was downregulated in det3 plants in both antisense and sense suppression experiments. Suppression of AtMYB61 in a det3 mutant background restored all mutant phenotypes of the det3 mutant associated with development in the dark. Taken together, these results suggest that AtMYB61 misexpression was both sufficient and necessary to explain the ectopic lignification and dark-photomorphogenic phenotypes of the det3 mutant.


Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Results and discussion
  5. Conclusion
  6. Experimental procedures
  7. Acknowledgements
  8. References

Plants constantly monitor endogenous and environmental cues and use this information to make adjustments in resource allocation, both to maximise resource acquisition and to minimise exposure to deleterious phenomena (Gilroy and Trewavas, 2001; Sultan, 2000). To accomplish this, plants possess mechanisms that integrate and interpret the information provided by internal and environmental signals (Gilroy and Trewavas, 2001; Trewavas and Malho, 1997). Mutations that have pleiotropic effects on plant growth and development have enabled the identification of the molecular machinery that interprets endogenous and exogenous signals to modulate plant resource allocation (McCarty and Chory, 2000).

The de-etiolated 3 (det3) mutation of Arabidopsis thaliana has pleiotropic effects on plant resource allocation. The det3 mutant was originally identified on the basis of the fact that it was photomorphogenic when grown in the dark (Cabrera y Poch et al., 1993). In contrast to wild-type Arabidopsis plants, which exhibit skotomorphogenic development in the dark, the det3 mutant has a light-grown morphology even when grown in the dark. Dark photomorphogenesis is only one component of the det3 mutant phenotype. When grown in the light, the det3 mutant is also significantly dwarfed relative to wild-type plants. Another striking aspect of the det3 phenotype is that lignin distribution is ectopic (Cano-Delgado et al., 2000). In wild-type stems, lignin deposition is normally limited to xylem cells and the interfascicular fibres (Altamura et al., 2001; Dharmawardhana et al., 1992), where it plays important roles in the support of the plant body and in the transport of water and solutes from the roots to the aerial tissues (Esau, 1965). In contrast, in the det3 mutant, in addition to the xylem cells and interfascicular fibres, lignin also accumulates in the cell walls of pith cells and phloem fibres (Cano-Delgado et al., 2000).

The DET3 locus encodes the C-subunit of the V-type ATPase (Schumacher et al., 1999), which is implicated in the maintenance of pH homeostasis in the plant body (Taiz, 1992). The det3 mutant has a 60% reduction in V-type ATPase activity because of a point mutation that destroys a branch-point consensus sequence within the gene that encodes the C-subunit (Schumacher et al., 1999). In the det3 mutant, a reduction in V-type ATPase activity is believed to affect solute uptake into the vacuole, which results in a decrease in turgor pressure. In both dark- and light-grown det3 plants, the decreased turgor pressure is believed to result in reduced cell expansion and elongation (Schumacher et al., 1999). Although this hypothesis argues for a defect in cell elongation and expansion, it does not account for all aspects of the dark-photomorphogenic phenotype of the det3 mutant. For example, it is not known how the mutation in the C-subunit of the V-type ATPase results in an ectopic lignification phenotype in the det3 mutant. The det3 mutant provides an opportunity to identify components of the plant regulatory machinery that function at the interface between signal integration and the modulation of resource allocation in plants.

The R2R3-MYB family of transcription factors are good candidates for pleiotropic regulators that may function to integrate signals and modulate disparate components of plant resource allocation. The R2R3-MYB family is one of the largest groups of transcriptional regulators in plants (Riechmann et al., 2000), with over 120 family members in Arabidopsis (Stracke et al., 2001). Members of the R2R3-MYB family have demonstrable roles in processes ranging from the determination of epidermal cell fate (Glover et al., 1998; Kirik et al., 2001; Lee and Schiefelbein, 1999, 2001) to the regulation of phenylpropanoid metabolism (Borevitz et al., 2000; Jin et al., 2000; Tamagnone et al., 1998), from seed development and germination (Diaz et al., 2002; Gubler et al., 1995, 1999; Penfield et al., 2001) to stress responses (Abe et al., 1997, 2003; Denekamp and Smeekens, 2003; Vailleau et al., 2002) and from anther development (Higginson et al., 2003; Murray et al., 2003; Steiner-Lange et al., 2003) to photomorphogenesis (Ballesteros et al., 2001; Seo et al., 2003). Recently, we found that a member of the R2R3-MYB transcription factor family from pine, Pinus taeda MYB4 (PtMYB4), was able to induce ectopic lignification when overexpressed in transgenic tobacco plants (Patzlaff et al., submitted). Here, we show that the overexpression of PtMYB4 in transgenic Arabidopsis plants gives rise to a phenotype that resembles the det3 mutant, including ectopic lignin deposition and a dark-photomorphogenic response. These results suggested that an Arabidopsis MYB might be involved in generating at least some aspects of the det3 mutant phenotype. Consistent with this hypothesis, we show that the Arabidopsis R2R3-MYB, AtMYB61, is both sufficient and necessary to explain aspects of the det3 phenotype.

Results and discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Results and discussion
  5. Conclusion
  6. Experimental procedures
  7. Acknowledgements
  8. References

Overexpression of PtMYB4 in Arabidopsis phenocopies aspects of the dark-photomorphogenic mutant, det3

The pine MYB, PtMYB4, was overexpressed in Arabidopsis plants under the control of the cauliflower mosaic virus (CaMV) 35S promoter. Arabidopsis plants overexpressing PtMYB4 had reduced stature, with an obvious dwarf phenotype and reduced apical dominance (Figure 1a). In order to determine if the PtMYB4 overexpressors (PtMYB4OEs) had an overall impairment in cell elongation, the hypocotyl lengths of dark-grown PtMYB4OE seedlings were analysed. Dark-grown Arabidopsis seedlings undergo skotomorphogenesis, which is characterised by etiolation, rapid hypocotyl elongation, and arrested cotyledon and leaf development (Figure 1b). In contrast to wild-type plants, dark-grown PtMYB4OE seedlings had short hypocotyls, open and expanded cotyledons, and, in some cases, well-developed leaves (Figure 1b). In this respect, PtMYB4OE lines exhibited the dark-photomorphogenic phenotype reminiscent of well-known mutants such as det1, det2, det3, and brassinosteroid insensitive 1 (bri1; Figure 1b).

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Figure 1. Transgenic Arabidopsis plants overexpressing PtMYB4 are dwarfed, dark-photomorphogenic and have altered spatial deposition of lignin.

(a) Whole-plant phenotype of wild-type plants (Col-0) in comparison to representative plants that overexpressed the pine MYB, PtMYB4, under the control of the CaMV 35S promoter (PtMYB4OE). The latter show the dwarf-phenotype characteristic of these transgenic plants. Bar equals 2 cm.

(b) Comparison of Col-0 and PtMYB4 overexpressing (PtMYB4OE) seedlings with known photomorphogenic mutants, det1, det2, det3 and bri1. All plants were grown in the dark for 14 days on horizontal MS sucrose-containing plates. Dark-grown wild-type (Col-0) seedlings display skotomorphogenic development with long hypocotyls, closed cotyledons and an apical hook. Dark-photomorphogenic seedlings have short hypocotyls, open and expanded cotyledons, and developed leaves. Bar equals 5 mm.

(c–e) Stem sections from 5-week-old plants stained with phloroglucinol-HCl. Phloroglucinol-HCl stains lignifed cells red. (c) Wild-type (Col-0) stem section showing lignification in the xylem and interfascicular fibres. Ectopic lignin deposition was observed in pith and phloem fibres in stems from (d) PtMYB4OE plants and (e) det3 mutants. P, pith; X, xylem; PF, phloem fibres; IF, interfascicular fibres. Bar equals 20 µm.

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As overexpression of PtMYB4 in transgenic tobacco had previously been shown to induce ectopic lignification (Patzlaff et al., submitted), the spatial distribution of lignin was assessed in the Arabidopsis PtMYB4OE lines. Stem sections were stained with phloroglucinol-HCl; a histochemical dye that stains hydroxycinnamaldehyde residues within the lignin polymer red. In wild-type plants, lignin is typically found in the xylem and interfascicular fibres (Figure 1c). Cross-sections of PtMYB4OE stems revealed that lignified cells were not restricted to the xylem and interfascicular fibres, but were also found in the pith and phloem fibres as well (Figure 1d).

Previously, the dark-photomorphogenic mutant det3 was shown to have alterations in the spatial control of lignin deposition (Cano-Delgado et al., 2000, 2003). These changes in lignin deposition are similar to those observed in the PtMYB4OE lines. Cross-sections of the det3 mutant showed that it also deposited lignin in the pith and phloem fibres, in addition to the xylem and sclerified parenchyma, just like the PtMYB4OE lines (Figure 1e). Furthermore, similar to PtMYB4OE lines, the ectopic lignification within the stems of the det3 mutant occurred in patches along the length of the primary inflorescence (data not shown). Thus, PtMYB4OEs share some phenotypic traits with the det3 mutant. Most notably, both PtMYB4OE lines and det3 mutants have a dwarfed stature, a dark-photomorphogenic response and altered spatial control of lignin deposition.

An Arabidopsis MYB that is related to PtMYB4, AtMYB61, is misexpressed in det3 mutants

Given that several aspects of the det3 phenotype were shared with PtMYB4OE lines, the det3 mutant was analysed to determine if any of the genes encoding R2R3-MYB family members related to PtMYB4 were misexpressed. On the basis of amino acid sequence similarity, PtMYB4 is most similar to the Arabidopsis R2R3-MYB family subgroup 13 (Kranz et al., 1998). This particular R2R3-MYB subgroup is defined by two conserved amino acid sequence motifs, GIDPxTHKPxSEV and DVFxKDLQRMA (Kranz et al., 1998). There are three R2R3-MYB family members in subgroup 13: AtMYB50, AtMYB61 and AtMYB86. There are four other R2R3-MYB family members that are very similar to PtMYB4 and subgroup 13 but not included within the subgroup, as they do not share the conserved subgroup-specific amino acid sequence motifs. These R2R3-MYB family members are AtMYB35, AtMYB40, AtMYB46 and AtMYB67 (Kranz et al., 1998). PtMYB4 is most similar to AtMYB46 (Patzlaff et al., submitted).

To determine if any of the Arabidopsis R2R3-MYB family members that are similar to PtMYB4 were misregulated in the det3 mutant, their expression patterns were analysed in both light- and dark-grown Columbia (Col-0) and det3 plants in a preliminary screen using reverse transcriptase-polymerase chain reaction (RT-PCR). Tubulin was also simultaneously amplified in the multiplexed reactions as an internal control. Results from the RT-PCR suggested that only AtMYB61 was misregulated in a det3 background, where its expression was greater in the dark (Figure 2). While AtMYB86 also appeared to be upregulated in the dark, this was independent of the genotypic background (Figure 2).

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Figure 2. Expression analysis of related R2R3-MYB family members in det3 and wild-type plants.

Transcript accumulation corresponding to seven related R2R3 MYB family members were analysed using RT-PCR. First-strand cDNA was synthesised from 5 µg of total RNA from 2-week-old light- and dark-grown wild-type (Col-0) and det3 mutant seedlings. The R2R3 MYB family members and an Arabidopsis tubulin control were then PCR amplified using gene-specific primers. AtMYB61 was misregulated in dark-grown det3 plants.

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A further RT-PCR experiment was undertaken to confirm and extend the preliminary RT-PCR results. This second experiment took advantage of a conditional aspect of the det3 phenotype. As is the case with wild-type seedlings, the extent of lignification in dark-grown det3 mutants is contingent on the presence of exogenously supplied sugar. That is, in the absence of an exogenous sugar source, dark-grown seedlings, which are not undergoing photosynthesis, do not accumulate appreciable amount of lignin, as determined by staining with phloroglucinol-HCl (Figure 3). While dark-grown det3 mutants appear to have greater amounts of lignified cells even in the absence of exogenous sugar, there is dramatic increase in phloroglucinol staining when these seedlings are grown in the presence of a carbohydrate such as sucrose (Figure 3). As had been shown previously by Penfield et al. (2001), RT-PCR revealed that the expression of AtMYB61 was sugar-regulated in wild-type plants (Figure 3). AtMYB61 expression was also sugar-regulated in the det3 mutant background, but transcript levels were much higher in the dark-grown det3 mutants, even in the absence of sugar (Figure 3). As was the case for the dramatic increase in phloroglucinol-positive cell wall-bound material in the presence of sugar in the dark-grown det3 mutants, there was also a dramatic increase in the abundance of AtMYB61 transcripts, as detected by RT-PCR. The qualitative differences in AtMYB61 expression in det3 suggest that AtMYB61 is misexpressed in this mutant.

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Figure 3. The extent of ectopic lignification and AtMYB61 misexpression in the det3 mutant is sucrose responsive.

Dark-grown wild-type (Col-0) and det3 mutant seedlings were grown either in the absence (–sucrose) or in the presence (+sucrose) of sucrose for 2 weeks and then stained with phloroglucinol-HCl to detect lignins, which stain red (upper panels). RT-PCR was used to detect the abundance of AtMYB61 mRNA in dark-grown Col-0 and det3 plants, grown in the presence or absence of sucrose. RT-PCR was performed with 5 µg of total RNA template. Tubulin was used as an internal control and co-amplified with the AtMYB61 cDNA in a multiplexed reaction. RT-PCR results show that AtMYB61 transcript levels are more abundant in plants grown with exogenously supplied sucrose, and that this level is greater in the det3 mutant.

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The pattern of AtMYB61 expression was analysed in Col-0 and det3 backgrounds using a promoter::β-glucuronidase (GUS) fusion. The localisation of AtMYB61 expression was determined by driving the expression of a reporter gene from AtMYB61 sequences that comprised 2.5 kbp of putative AtMYB61 promoter together with the first 500 bp of the N-terminal coding sequence, which contains both of the introns for his gene. The AtMYB61 sequences were used in a translational fusion with the reporter gene, uidA, which encodes GUS. The AtMYB61 intron sequences were included because similar non-coding sequences have been shown to be important in the appropriate regulation of gene expression (Deyholos and Sieburth, 2000; Larkin et al., 1993; Sieburth and Meyerowitz, 1997). The AtMYB61 promoter::AtMYB61 N-terminus::GUS construct was transformed into both wild-type (Col-0) and det3 mutant plants.

As has been reported by Penfield et al. (2001), AtMYB61 gene expression was localised to the vascular tissue in 7-day-old Col-0 plants, as determined by GUS histochemical staining (Figure 4). In striking contrast, histochemical staining of GUS in det3 plants transformed with the AtMYB61 promoter::AtMYB61 N-terminus::GUS construct revealed that expression was no longer localised to the vasculature of 7-day-old seedlings, which were grown under long-day conditions (16-h light:8-h dark). Instead, GUS staining appeared in patches throughout the det3 mutant plant in a manner similar to the patches of ectopic lignification found in this mutant (Figure 4). These findings substantiate the results obtained by RT-PCR, and are consistent with the hypothesis that AtMYB61 is misregulated in the det3 mutant. Moreover, the extent and localisation of AtMYB61 misregulation coincides with both the extent and ectopic deposition of lignin in this mutant. Thus, AtMYB61 expression correlated with both the intensity and the localisation of at least one component of the det3 phenotype. It may be that AtMYB61 misexpression is related to this component of the det3 mutant phenotype.

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Figure 4. AtMYB61 misregulation in the det3 mutant corresponds to regions of lignification.

Localisation of lignin was observed by phloroglucinol-HCl staining of wild-type (Col-0) (a) and det3 mutant (d) plants grown under long-day conditions. The activity of the AtMYB61 promoter was detected in wild-type (Col-0) (b,c) and det3 mutant (e) plants using histochemical localisation of GUS activity expressed under the control of AtMYB61 regulatory sequences. (b) GUS activity was localised to the vasculature of developing Col-0 seedlings. (c) Closer inspection of vascular localised expression in Col-0 seedling. (e) Vascular-localised expression was supplanted by a ‘patchy’ expression pattern in det3 mutant seedlings, as was observed for lignification (d).

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AtMYB61 overexpression suggests that AtMYB61 misexpression may be sufficient to explain aspects of the det3 phenotype

The hypothesis that AtMYB61 misexpression is sufficient to account for facets of the det3 phenotype was tested by overexpressing the AtMYB61 cDNA in Arabidopsis under the control of the CaMV 35S promoter. Similar to plants overexpressing PtMYB4, plants overexpressing AtMYB61 had obvious changes in growth and development. As was the case for PtMYB4OE lines, AtMYB61 overexpressor (AtMYB4OE) lines had a dwarfed habit and reduced apical dominance when grown in the dark. The hypocotyls of dark-grown AtMYB61OEs were compared with the hypocotyl lengths of dark-grown Col-0 and det3 plants. Similar to the det3 mutant, the AtMYB61OE plants were dark-photomorphogenic, with the short hypocotyls, open and expanded cotyledons, and well-developed leaves characteristic of dark-photomorphogenic mutants (Figure 5a).

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Figure 5. Arabidopsis plants overexpressing AtMYB61 plants resemble the dark-photomorphogenic mutant, det3.

(a) A comparison of 14-day-old dark-grown Col-0, det3 and AtMYB61OE. AtMYB61OE seedlings are representative of five independently transformed lines.

(b–d) Hypocotyls of 14-day-old dark-grown (b) Col-0, (c) det3 and (d) Col-0 overexpressing AtMYB61. Seedlings were stained with phloroglucinol-HCl in order to analyse the pattern of lignification. The hypocotyls of the det3 mutant (c) and AtMYB61OE transgenic seedlings (d) lignified ectopically. Bar equals 50 µm for (b) Col-0 and (c) det3 hypocotyls, and 100 µm for (d) AtMYB61OE hypocotyls.

(e) Phloroglucinol-HCl stained cross-section through a stem of an AtMYB61OE, showing the ectopic lignification in the pith. P, pith; X, xylem; PF, phloem fibres; IF, interfascicular fibres.

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AtMYB61OE plants were also analysed for alterations in lignin deposition. Hypocotyls of 2-week-old, dark-grown Col-0, det3, and AtMYB61OE seedlings were stained with phloroglucinol-HCl (Figure 5b–d). Comparison of lignification patterns revealed that, similar to the det3 mutant, AtMYB61OEs also ectopically lignified (Figure 5c,d). Analysis of the pattern of lignification within AtMYB61OE stem sections revealed that lignified cells extended beyond the xylem and interfascicular fibres, to include the pith (Figure 4e). Furthermore, similar to det3 mutants, the ectopic lignin deposition appeared to be very sporadic throughout the stem of the AtMYB61OE plants (data not shown).

The results of the overexpression experiments support the hypothesis that AtMYB61 misexpression explains at least two components of the pleiotropic det3 phenotype. As AtMYB61 overexpression in Arabidopsis allows dark-photomorphogenesis and induces ectopic lignification, it may be that these two facets of the det3 phenotype are attributable to AtMYB61 misregulation in this mutant.

Suppression of AtMYB61 expression in det3 mutants indicates that AtMYB61 activity is necessary for manifestation of facets of the det3 phenotype

AtMYB61 expression was suppressed in the det3 mutant to test the hypothesis that AtMYB61 function is necessary to give rise to at least a portion of the det3 phenotype. Two different approaches were taken to suppress AtMYB61 expression; co-suppression and antisense suppression. The det3 mutants were stably transformed with either a sense or an antisense construct to downregulate the endogenous AtMYB61 gene. Arabidopsis plants transformed with an AtMYB61 sense construct contained a 6.2-kbp fragment that consisted of 4.2 kbp of AtMYB61 promoter and 5′ untranslated region (UTR), 1.7-kbp genomic AtMYB61 coding sequence (cds), and 300 bp AtMYB61 3′ UTR. The sense construct was designated gAtMYB61. Arabidopsis plants were also transformed with a construct containing an antisense AtMYB61 cDNA, and designated AS-AtMYB61. det3 plants were also transformed with a binary vector that served as a transformation control.

To ensure that the transgenes reduced mRNA transcript levels in det3 plants transformed with either the gAtMYB61 or AS-AtMYB61 constructs, RT-PCR was performed. Expression levels of AtMYB61 and an internal control, tubulin, were analysed. RT-PCR results revealed that AtMYB61 transcript levels were reduced in 6-week-old det3 plants transformed with either the gAtMYB61 or the AS-AtMYB61 transgene (Figure 6). Furthermore, results from the RT-PCR seemed to suggest that only AtMYB61 mRNA-specific transcripts were downregulated as the closely related AtMYB50 and AtMYB86 expression levels remained unaltered within the det3 AS-AtMYB61 plants (Figure 6).

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Figure 6. Expression of AtMYB61 and related family members in putatively co-suppressed and antisense det3 plants.

Accumulation of transcripts corresponding to AtMYB50, AtMYB61 and AtMYB86 was analysed using RT-PCR of RNA isolated from independently transformed lines of putatively co-suppressed (gATMYB61 O-2.6, gATMYB61 O-2.9) and antisense (AS-AtMYB61T B-13, AS-AtMYB61T E-19) AtMYB61 in a det3 mutant background as well as corresponding control plants (Col-0, det3 and a transformed control, pBIN+ D-1). Total RNA was isolated from 6-week-old plants of which 5 µg were used for first-strand cDNA synthesis. The use of equal amounts of RNA was confirmed by ethidium bromide staining of ribosomal RNAs (bottom panel). An oligo d(T)12−18 primer was used to amplify first-strand cDNAs from all samples excluding the antisense plants. Synthesis of first-strand cDNA from RNA isolated from the antisense plants was obtained by using a lower primer specific to AtMYB61 (AtMYB61-LEX2). AtMYB61, AtMYB61-related family members and an Arabidopsis tubulin control were PCR amplified using gene-specific primers. RT-PCR results show that AtMYB61 transcript levels are reduced in co-suppressed and antisense transgenic plants.

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The spatial deposition of lignin in dark-grown wild-type plants (Figure 7a) and det3 mutants (Figure 7b) was compared with the pattern of lignification in det3 gAtMYB61 and det3 AS-AtMYB61 plants, as determined by staining with phloroglucinol-HCl. The ectopic deposition of lignin in det3 seedlings was rectified if AtMYB61 expression was suppressed by either sense or antisense constructs (Figure 7c,d). Any lignin that was detected in the det3 gAtMYB61 or det3 AS-AtMYB61 hypocotyls was largely restricted to the vasculature; however, some staining could still be detected in a few cells that were adjacent to the vasculature (Figure 7c,d), which is consistent with the fact that suppression of gene expression by either sense or antisense methods was unlikely to be absolute.

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Figure 7. Suppression of AtMYB61 expression rectifies altered lignin deposition and dark-photomorphogenic phenotype in the det3 mutant.

Hypocotyls of 14-day-old dark-grown (a) Col-0, (b) det3 untransformed seedlings, (c) det3 seedlings transformed with the gAtMYB61 transgene and (d) det3 seedlings transformed with the AS-AtMYB61 construct. Seedlings were derived from the independently transformed lines characterised in Figure 6. Seedlings were stained with phloroglucinol-HCl in order to analyse their pattern of lignification. (b) Cells within the hypocotyls of dark-grown det3 mutant seedlings ectopically lignified. (c,d) Ectopic lignification was partially suppressed in hypocotyl cells of det3 plants transformed with either the gAtMYB61 or AS-AtMYB61. (a–d) Bar represents 50 mm. (e,f) Comparison of hypocotyl elongation phenotype of 14-day-old dark-grown (e) Col-0, det3 and det3/gAtMYB61 plants and (f) Col-0, det3 and det3/AS-AtMYB61 plants.

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Suppression of lignification was also observed in the xylem in the hypocotyls of the det3 gAtMYB61 or det3 AS-AtMYB61 plants. This finding is consistent with the fact that suppression of AtMYB61 expression by these constructs should occur throughout the plant body, thereby suppressing AtMYB61 expression in cells where it would normally be found, such as the xylem. The fact that the lignification of xylem cells was also reduced in the det3 gAtMYB61 or det3 AS-AtMYB61 plants suggests that AtMYB61 may play a role in normal lignification in xylem cells.

The suppression of AtMYB61 expression in det3 mutants was able to rescue another component of the det3 phenotype. The hypocotyls of dark-grown det3 plants transformed with either the gAtMYB61 or the AS-AtMYB61 transgene were more similar to wild-type plants, with the elongated hypocotyls characteristic of skotomorphogenesis, than they were to the dark-photomorphogenic det3 mutant (Figure 7e,f). Although the hypocotyls of the det3 gAtMYB61 and det3 AS-AtMYB61 seedlings did not elongate as much as wild-type hypocotyls, they elongated to a greater extent than the hypocotyls of the det3 control (Figure 8). Moreover, in addition to partially elongated hypocotyls, the dark-grown det3 gAtMYB61 and det3 AS-AtMYB61 seedlings also had closed cotyledons, similar to the skotomorphogenic, dark-grown, wild-type seedlings (Figure 7e,f). The observation that the cotyledons remained closed and that there was no evidence of leaf primordia initiation suggest that skotomorphogenic development was restored in det3 mutants when AtMYB61 expression had been suppressed by either the gAtMYB61 or AS-AtMYB61 constructs.

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Figure 8. Suppression of AtMYB61 activity in the det3 mutant rectifies the dark-grown hypocotyl length phenotype.

Hypocotyl lengths were measured for 7-day-old dark-grown det3 mutants and wild-type (Col-0) plants, and compared with two independently transformed transgenic det3 lines where AtMYB61 expression was co-suppressed by a sense construct (gAtMYB61) and seven independently transformed lines where the expression of AtMYB61 was suppressed by an antisense construct (AS-AtMYB61). Each bar represents the mean of 10 hypocotyls for each genotype, with SE indicated by the error bars.

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Taken together, these findings are consistent with the hypothesis that AtMYB61 activity is not only sufficient but also necessary for the manifestation of components of the det3 mutant phenotype. Suppression of AtMYB61 expression in the det3 mutant rectifies two key features of the det3 phenotype: dark photomorphogenesis and ectopic lignification. This suggests that misregulation of AtMYB61 activity is necessary for these two facets of the det3 phenotype.

AtMYB61 activity only impacts some components of the det3 mutant phenotype

To determine if AtMYB61 activity was both sufficient and necessary to explain other aspects of the det3 mutant phenotype, the transgenic plants described above were characterised at the gross morphological level. Overexpression of AtMYB61 was sufficient to cause dwarfing and reduced apical dominance in transgenic Arabidopsis plants (Figure 9a); therefore, the dwarf stature of the det3 mutant may also be attributable to AtMYB61 misexpression. If this is the case, then suppression of AtMYB61 activity in the det3 mutant should rectify the dwarf phenotype. All of the transgenic AS-AtMYB61 det3 lines had a det3 phenotype (Figure 9b), and only two of the gAtMYB61 det3 lines had a partially rectified phenotype, with greater apical dominance and longer stems than the det3 mutant (Figure 9b). Even with the two gAtMYB61 det3 lines where the ectopic lignification and dark photomorphogenesis phenotypes were rectified, stem elongation and apical dominance were not restored to wild-type levels. Consequently, AtMYB61 misexpression alone does not appear to be sufficient to account for the dwarf stature of det3 mutants, while it is sufficient for dark photomorphogenesis and ectopic lignification in this mutant.

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Figure 9. AtMYB61 misexpression cannot account for all of the phenotypic chances in the det3 mutant.

The gross morphological features are shown for representative plants, grown in the light, that are AtMYB61OE(a), as well as det3 mutants where AtMYB61 expression was suppressed with the sense construct (gAtMYB61) or by an antisense construct (AS-AtMYB61) (b). Seedlings were derived from the independently transformed lines characterised in Figure 6. Wild-type (Col-0) and det3 mutant plants are shown for comparison. The bars equal 2 cm.

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Conclusion

  1. Top of page
  2. Summary
  3. Introduction
  4. Results and discussion
  5. Conclusion
  6. Experimental procedures
  7. Acknowledgements
  8. References

The results presented herein suggest that AtMYB61 misexpression is both sufficient and necessary to account for two components of the det3 mutant phenotype, dark photomorphogenesis and ectopic lignification. AtMYB61 is the second Arabidopsis MYB gene to be implicated in processes related to photomorphogenesis. AtMYB21 was recently implicated in photomorphogenesis on the basis of the fact that it is misregulated in the dark photomorphogenic mutant, cop1 (Shin et al., 2002). AtMYB21 expression is normally restricted to developing flowers, but in the cop1 mutant, it is ectopically expressed throughout the plant (Shin et al., 2002). Overexpression of AtMYB21 in transgenic plants demonstrated that ectopic expression of this MYB gene was sufficient to account for at least a subset of the cop1-associated phenotypes, including dark photomorphogenesis (Shin et al., 2002). Shin et al. (2002) did not test the hypothesis that AtMYB21 misexpression was necessary to give rise to the cop1 phenotype, but their findings do provide evidence that MYB misexpression may be a common theme in the derivation of mutant phenotypes, particularly when that phenotype is pleiotropic, as is the case with cop1 and det3.

AtMYB61 was previously implicated in the extrusion of seed coat mucilage (Penfield et al., 2001). Recessive loss-of-function mutations of AtMYB61 resulted in a loss of the ability of the mutants to deposit and extrude seed coat-derived rhamnogalacturonan (Penfield et al., 2001). Despite the fact that AtMYB61 gene expression was observed in developing seeds and differentiating xylem, Penfield et al. (2001) were only able to observe a seed coat phenotype in mutants harbouring loss-of-function AtMYB61 alleles. Consequently, it was hypothesised that AtMYB61 function was limited to some aspect of seed coat formation, either the synthesis of the seed coat rhamnogalacturonan itself or some process that affects the appropriate deposition of this extracellular polymer (Penfield et al., 2001). The findings described herein suggest that AtMYB61 has the capacity to play a role in the regulation of a much wider range of developmental and biochemical processes. When AtMYB61 expression was suppressed throughout the plant body in det3 mutants, by either co-suppression or antisense constructs, both ectopic lignification and vascular lignification were suppressed (Figure 7). These findings suggest that AtMYB61 may play a role in cell wall deposition in normal vascular development. In keeping with this hypothesis, analysis of vascular development in AtMYB61 loss-of-function mutants has revealed that AtMYB61 is important in establishing the ratio of xylem to phloem during secondary growth (Dubos and Campbell, unpublished). Future work should aim to examine the roles played by AtMYB61 that extend beyond the deposition of seed coat mucilage.

The signalling pathway that causes misregulation of AtMYB61 expression in the det3 mutant remains to be determined. Recently, both ethylene and jasmonic acid have been implicated in the derivation of ectopic lignification phenotypes (Cano-Delgado et al., 2003; Ellis et al., 2002; Zhong et al., 2002), but it remains to be determined if they are involved in the misregulation of AtMYB61 in the det3 ectopic lignification mutant. Many other signalling molecules may not be localised properly in the det3 mutant. As the det3 mutant is impaired in V-ATPase function, cellular pH homeostasis is likely to be disrupted, with knock-on effects on localisation of many molecules. For example, the proton motive force established by the V-ATPase is thought to be important in loading the vacuole with calcium (Cheng et al., 2003), proteins (Matsuoka et al., 1997) and sugars (Taiz, 1992). Perturbations in the proper targeting of some of these molecules to the vacuole can have profound effects on development. For example, disruption of vacuolar calcium loading by insertional mutagenesis of the vacuolar proton-calcium antiport resulted in plants with pleiotropic phenotypes including the reduced apical dominance reminiscent of the det3 mutant (Cheng et al., 2003). The det3 mutant is known to have altered calcium-signalling responses (Allen et al., 2000), and this may affect a plethora of processes regulated by this second messenger, which may include the expression of AtMYB61. It is tempting to speculate that sugar may be the key to the misregulation of AtMYB61 in the det3 mutant. Sugar has been linked to lignin deposition (Anterola et al., 2002; Nose et al., 1995), dark photomorphogenesis (Roldan et al., 1999) and AtMYB61 expression (Penfield et al., 2001), and may provide the link between these disparate processes in the det3 mutant. Regardless of which molecules are implicated, elucidation of the molecular machinery involved in the misregulation of AtMYB61 in the det3 mutant should provide useful insights into the signalling processes controlling resource allocation in plants.

Experimental procedures

  1. Top of page
  2. Summary
  3. Introduction
  4. Results and discussion
  5. Conclusion
  6. Experimental procedures
  7. Acknowledgements
  8. References

Plant material, plant propagation, and growth conditions

Arabidopsis thaliana ecotype Col-0 seed and det3-1 seed in a Col-0 background were obtained from the Nottingham Arabidopsis Stock Centre (NASC). All seeds were initially propagated under sterile on MS agar plates (Murashige and Skoog, 1962) containing appropriate antibiotics. Seeds that were to be germinated and grown in the dark were wrapped in three layers of aluminum foil and placed in a box that was enclosed within a dark plastic bag. The seed was then stratified for 2 days at 4°C to ensure uniform germination. All light- and dark-grown in vitro plant material was grown at 19.5°C under long-day conditions. Long-day growth conditions consisted of an 8-h dark/16-h light photoperiod. All light-grown in vitro plant material was exposed to an average light intensity of 60.58 µmol m−2 sec−1. All in vitro plant material was either harvested after 14 days or transferred to soil after 21–28 days. Seedlings were either transferred into 4 cm × 4 cm or 8.5 cm × 8.5 cm pots that were filled with Levington's Universal soil and Vermiperl vermiculite (2 : 1). Soil-propagated plants were grown in a temperature-controlled Sanyo Gallenkamp growth room at 22°C under long-day conditions. All light-grown soil-propagated plant material was exposed to an average light intensity of 130 µmol m−2 sec−1.

RT-PCR

A minimum of two independently prepared RNA extractions for each genotype and for each growth condition were used for first-strand cDNA synthesis (Goldsbrough and Cullis, 1981; Kirby, 1956). Any contaminating genomic DNA was eliminated from the RNA samples by a DNase treatment. Five micrograms of total plant RNA was reverse transcribed using Superscript II Reverse Transcriptase according to the manufacturer's instructions (Gibco BRL Life Technologies; Paisley, UK). First-strand cDNA was generated using either an oligo (dT)12−18 primer or a gene-specific primer (AtMYB61-LEX2, 5′-GTCGGATCCTCATTGTTTCAGTTTCTTCTT-3′). One to two microlitres of the first-strand cDNA was used as a template in a subsequent PCR reaction. Gene-specific primers were used to amplify AtMYB35 (AtMYB35-U, 5′-TTGGCAGCAGGTGGTCTTC-3′; AtMYB35-L, 5′-CGGCTTCGATGTTGAAATGA-3′), AtMYB40 (AtMYB40-U, 5′-GGCAACCGGTGGTCTAAAAT-3′; AtMYB40-L, 5′-CAAGATTGGCGAAGAGGTCC-3′), AtMYB46 (AtMYB46-U, 5′-TCATTCGCTTTCATTCCATC-3′; AtMYB46-L, 5′-CCGGATCCATTAGTGTTCAA-3′), AtMYB50 (AtMYB50-U, 5′-GGAAACAGATGGTCGCAAAT-3′; AtMYB50-L, 5′-AGGCAATGTTGGAGTTGAAA-3′), AtMYB61 (AtMYB61-U2, 5′-AGACTGCAGTTCTTCTTCTCTTTTACTGTT-3′; AtMYB61-LEX2), AtMYB67 (AtMYB67-U, 5′-TTGGCAGCTGGACCTTTTAG-3′; AtMYB67-L, 5′-TGCTTTGCCTTCGCTCTTC-3′), AtMYB86 (AtMYB86-U, 5′-AGGCAACAGATGGTCTCAAA-3′; AtMYB86-L, 5′-GGCAATGTCTGGCTTCACT-3′), and tubulin (AtTUB-U, 5′-GATCACACCGGTCAATACGTC-3′; AtTUB-L, 5′-GTGAACTCCATCTCGTCCAT-3′). PCR components consisted of 4 µl of 100 mg ml−1 BSA, 3 µl of 10× long amplification (LA) PCR buffer (160 µl ml−1 of 1 m NH4SO4, 200 µl ml−1 of Tris–HCl (pH 9.0)), 4 µl of 25 mm MgCl2, 1 µl of 10 mm dNTPs, 1 µl of each primer, and template DNA in a 25-µl reaction volume. The reaction was overlaid with 50 µl of mineral oil. The DNA and primers were denatured prior to the addition of the recombinant Taq polymerase (rTaq) with a hot start programme that consisted of a 10-min denaturation step at 95°C. Following denaturation, the reaction was held at 85°C while 5 µl of an LA PCR mix that contained the recombinant Taq polymerase (rTAQ) (0.5 µl of 10× LA PCR buffer, 0.25 µl of rTAQ, and 4.25 µl of dH2O) was added under the mineral oil overlay. PCR conditions consisted of three steps that were performed as follows: an initial denaturation (1 min, 95°C) followed by 30 cycles of denaturation (1 min, 94°C), annealing (1 min, 55°C) and primer extension (2 min, 72°C). The final cycle was completed with a 5-min extension at 72°C.

Construction of binary vectors

The construction of binary vectors that drove the expression of the PtMYB4 cDNA under the control of the CaMV 35S promoter is described elsewhere by Patzlaff et al. (submitted). As a first step in the creation of vectors to alter AtMYB61 expression, an RT-PCR product corresponding to a full-length AtMYB61 cDNA was generated using gene-specific primers (AtMYB61-U2, 5′-AGACTGCAGTTCTTCTTCTCTTTTACTGTT-3′; AtMYB61-LEX1, 5′-GAAGGATCCAAAGAAAAAAGCTAAAGGGAC-3′). The AtMYB61 cDNA was directionally cloned into the pBSKS plasmid, generating a 4.1-kbp pBSKS::AtMYB61 construct. The AtMYB61 cDNA that had been cloned into the pBSKS plasmid was directionally cloned into the pUC2X35S vector (L.J. Newman, D.Phil., University of Oxford, 2001). Insertion of the AtMYB61 cDNA into the PstI and BamHI sites of the pUC2X35S vector generated a 5.5-kbp pUC2X35S::AtMYB61 construct, which contained a fusion of the 2X CaMV 35S promoter, the AtMYB61 cDNA, and the CaMV poly A terminator. The 2X CaMV 35S promoter, the AtMYB61 cDNA, and the CaMV poly A terminator fusion were then excised from the pUC2XS35S::AtMYB61 construct by digestion with the PacI and AscI restriction endonucleases. The liberated fragment was directionally cloned into the pBIN+ binary vector (van Engelen et al., 1995), generating a 13.5-kbp pBIN+::AtMYB61 construct.

Gene-specific primers were designed in order to amplify a full-length antisense AtMYB61 cDNA (AS-AtMYB61-ASU2, 5′-AGAGGATCCTTCTTCTTCTCTTTTACTGTT-3′; AtMYB61-ASLEX1, 5′-GAACTGCAGAAAGAAAAAAGCTAAAGGGAC-3′). An RT-PCR product corresponding to the full-length antisense cDNA (AS-AtMYB61) was generated and directionally cloned into the pBSKS plasmid, generating a 4.1-kbp pBSKS::AS-AtMYB61 construct. The construct was 3′ end sequenced using the T7 primer in order to confirm cloning of the antisense AtMYB61 cDNA. The full-length AtMYB61 cDNA that had been cloned into the pBSKS plasmid was then directionally cloned into the pUC2X35S vector (L.J. Newman, D.Phil., University of Oxford, 2001). Insertion of the antisense AtMYB61 cDNA into the PstI and BamHI sites of the pUC2X35S vector generated a 5.5-kbp pUC2X35S::AS-AtMYB61. The 2X CaMV 35S promoter, the antisense AtMYB61 cDNA and the CaMV poly A terminator were then excised from the pUC2XS35S::AtMYB61 construct by digestion with the PacI and AscI restriction endonucleases. The liberated fragments were directionally cloned into the pBIN+ binary vector (van Engelen et al., 1995), generating a 13.5-kbp pBIN+::AtMYB61.

Bacterial artificial chromosome (BAC) preparation and gAtMYB61 isolation

A BAC that contained the entire AtMYB61 gene was obtained from the Arabidopsis Biological Resource Center (ABRC; Ohio State University, Columbus, OH, USA). The BAC, F14J9, was isolated and subsequently digested with the restriction endonuclease, EcoRI. The resulting F14J9 BAC fragments were separated on a 0.8% agarose gel, and a band corresponding to an 11.0-kbp fragment was gel purified. Contained within the purified 11.0-kbp fragment were two genes: the AtMYB61 gene and a gene encoding a pectin acetylesterase. The 11.0-kbp fragment was subcloned into the EcoRI restriction site of the pGEM7Zf vector (Promega, Southampton, UK) creating a 14.0-kbp pGEM7Zf::F14J9 construct. The pGEM7Zf::F14J9 construct was digested with the restriction endonucleases EcoRI and NaeI to liberate a 6.2-kbp fragment that contained the entire AtMYB61 gene. The 6.2-kbp fragment contained 4.2 kbp of AtMYB61 promoter and 5′ UTR, 1.7 kbp of genomic AtMYB61 cds, and 300 bp of AtMYB61 3′UTR. The 6.2-kbp fragment was subcloned into the EcoRI and SmaI restriction sites of the pBSKS vector generating a 9.2-kbp pBSKS::gAtMYB61 construct. The construct was 3′ end sequenced using the T7 primer in order to confirm cloning of AtMYB61. The 6.2-kbp fragment was subcloned into the EcoRI and SmaI restriction sites of the pBIN+ binary vector, generating an 18.6-kbp pBIN+::gAtMYB61 construct that was used to transform det3 mutant plants.

Generation and selection of transgenic Arabidopsis plants

Transgenic plants were generated using vacuum infiltration-aided Agrobacterium tumefaciens transformation method of Bechtold et al. (1993), as modified by Bell-Lelong et al. (1997). Seed from transformed plants was surface sterilised in a calcium hypochlorite-saturated solution containing 0.1% Triton X-100, rinsed in sterile distilled water, and sown on MS agar (Murashige and Skoog, 1962) containing 1% sucrose and 50 mg l−1 kanamycin, 200 mg l−1 timentin and 10 µg ml−1 benomyl. Seedlings (T1) that were resistant to kanamycin were transferred to soil and allowed to self-pollinate and set seed. Similar selection regimes were carried out on later generations while examining lines for homozygosity. A minimum of 15 independently transformed lines were generated per construct. Detailed analysis was conducted on three lines of the PtMYB4OEs, three lines of the AtMYB61OEs, the two lines of the sense-suppressed AtMYB61/det3 (gAtMYB61/det3) plants that deviated from the det3 phenotype, and seven lines of antisense-suppressed det3 (AS-AtMYB61/det3) plants.

Verification of transformation into a det3 background

In order to verify that the pBIN+::AS-AtMYB61 construct had truly been transformed into a det3 background, a strategy was devised that took advantage of a polymorphism in the det3 mutant. The det3 allele contains a point mutation at a branch point splice site, which results in a unique ApoI restriction site. Primers were designed that amplified across the region where the point mutation was located within the det3 allele (det3-U, 5′-ATGACTTCGAGATATTGGGTG-3′; det3-L, 5′-GCAGATCATCGCCGAGAG-3′). Digestion of the amplified product yielded a restriction pattern that was unique to the det3 mutant. Thus, transformed plants in either a Col-0 or det3 background were distinguished from one another based on their restriction patterns. Comparison of the banding patterns from the digested DNA that had been isolated from Col-0 and from putatively transformed det3 plants revealed that all constructs had been transformed into a det3 background (data not shown).

Preparation and phloroglucinol-HCl histochemical staining of Arabidopsis stem sections and seedlings

Stems from 5-week-old Arabidopsis plants were fixed overnight in formaldehyde: acetic acid (FAA) at 4°C. Following the overnight incubation, the plants were transferred to fresh FAA and vacuum infiltrated for 2 × 20 min. The vacuum was released slowly, and a third 20-min infiltration was performed if the tissue did not sediment to the bottom of the tube. The tissue was vacuum infiltrated for 15 min in 70% EtOH and then incubated at room temperature for an additional hour. The fixed stems were cleared of all chlorophyll through a graded EtOH series. Semi-thin stem sections (50–100 µm) were then prepared from the cleared stems using a Series 1000 Vibratome (General Scientific, Surrey, UK). The sections were then rehydrated through a graded EtOH series before being stained with 1% phloroglucinol-HCl for 10 min, with gentle shaking prior to being mounted in 50% glycerol, 6N HCl (Gurr, 1965). Plant material was then immediately observed under dark-field illumination using a Leica DMRB microscope (Leica, Wetzlar, Germany). Dark-grown Col-0 and det3 seedlings were also stained with 1% phloroglucinol-HCl for 10 min prior to being mounted in 50% glycerol, 6N HCl and viewed under bright-field illumination.

Construction of an AtMYB61 promoter::GUS fusion and histochemical localisation of GUS activity

A GUS cassette that consisted of the CaMV 35S promoter, the uidA gene and the NOS terminator was excised from the pBI121 binary vector (Clonetech, Basingstoke, UK) and subcloned into the HindIII–EcoRI sites of the pUCAP vector (van Engelen et al., 1995). A GUS reporter fusion was then constructed by replacing the CaMV 35S promoter within the GUS cassette with the AtMYB61 promoter. The AtMYB61 promoter was cloned by PCR using gene-specific primers that amplified a region of the promoter that ranged from −2289 to +781, with +1 being equivalent to the transcriptional start site (AtMYB61-UPN, 5′-TTATTGAGCATGCTTTCTTTTTGGGTTCCT-3′ and AtMYB61-L3, 5′-GTCGGATCCTCCTCTTTGTTTGCAGTTTCTTCTT-3′). Following excision of the CaMV 35S promoter, the AtMYB61 promoter fragment was then directionally cloned into the SphI and BamHI restriction sites within the pUCAP GUS cassette containing vector. The AtMYB61 promoter::GUS cassette was then cloned into the binary vector pBINPLUS (van Engelen et al., 1995) using the unique restriction sites, AscI and PacI. The binary construct was then mobilised into the A. tumefaciens strain GV3101 (Holsters et al., 1980) by electroporation. Five independently transformed lines were generated for each genotype. The histochemical localisation of GUS activity was conducted as described by Jefferson et al. (1987).

Acknowledgements

  1. Top of page
  2. Summary
  3. Introduction
  4. Results and discussion
  5. Conclusion
  6. Experimental procedures
  7. Acknowledgements
  8. References

This work was supported by generous grants from the BBSRC and the University of Oxford to M.M.C. L.J.N. was supported by the Sam Wilson Bequest of the Oxford Forestry Institute, and by a studentship from Somerville College, University of Oxford. The authors are grateful to Mrs Christine Surman and Mr Adam Newman for their technical assistance, and to Mr John Baker for his assistance with photography.

References

  1. Top of page
  2. Summary
  3. Introduction
  4. Results and discussion
  5. Conclusion
  6. Experimental procedures
  7. Acknowledgements
  8. References