AOBPreview originally published online on December 13, 2005
Annals of Botany 2006 97(5):857-866; doi:10.1093/aob/mcj603
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The Response of Spartium junceum Roots to Slope: Anchorage and Gene Factors
1 Dipartimento di Scienze e Tecnologie per l'Ambiente ed il Territorio, Università degli Studi del Molise, Via Mazzini 8, 86170 Isernia, Italy, 2 Dipartimento di Scienze Chimiche ed Ambientali, Università degli Studi dell'Insubria, Via Valleggio 11, 22100 Como, Italy and 3 Institute of Plant Genetics, CNR, Via Università 133, 80055 Portici, Italy
* For correspondence. E-mail scippa{at}unimol.it
Received: 18 August 2005 Returned for revision: 12 September 2005 Accepted: 7 October 2005 Published electronically: 13 December 2005
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSLITERATURE CITED |
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• Background and Aims Plant anchorage is governed by complex, finely regulated mechanisms that occur at a morphological, architectural and anatomical level. Spanish broom (Spartium junceum) is a woody plant frequently found on slopes—a condition that affects plant anchorage. This plant grows throughout the Mediterranean area where it plays an important role in preventing landslides. Spanish broom seedlings respond promptly to slope by altering stem and root morphology. The aim of this study was to investigate the mechanisms whereby the root system of Spanish broom seedlings adapts to ensure anchorage to the ground.
• Methods Seedlings were grown in tilted and untilted pots under controlled conditions. The root apparatus was removed at different times of growth and subjected to morphological, biomechanical and molecular analyses.
• Key Results In slope-grown seedlings, changes in root system morphology, pulling strength and chemical lignin content, all features related to plant anchorage in the soil, were related to seedling age. cDNA-AFLP analysis revealed changes in the expression of several genes in root systems of slope-grown plants. BLAST analysis showed that some differentially expressed genes are homologues of genes induced by environmental stresses in other plant species, and/or are involved in the production of strengthening materials.
• Conclusion Plants use various mechanisms/strategies to respond to slope depending on their developmental stage.
Key words: Anchorage, cDNA-AFLP, lignin, pulling, root morphology, root, slope, Spartium junceum
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSLITERATURE CITED |
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Terrestrial plants are anchored in the ground by a complex, highly organized root system. In nature, a wide variety of environmental factors (i.e. gravity, touch, wind, soil density, soil compaction and grazing) may affect the stability of plants, thereby triggering profound alterations in their development. Anchorage-related changes in response to wind, touch, rain, rubbing and soil impedance are well documented (Jaffe and Forbes, 1993
; Telewski, 1995). These changes, termed ‘thigmomorphogenesis’ (Jaffe and Biro, 1979), could serve to improve plant anchorage when mechanical stress affects plant stability (Mitchell et al., 1975; Jaffe and Biro, 1979). Studies of herbaceous (Neel and Harris, 1971; Beyl and Mitchell, 1977; Biddington, 1986; Jones and Mitchell, 1989) and woody species (Stokes et al., 1995, 1997b; Telewski and Pruyn, 1998) showed that the stems of mechanically stimulated plants tend to be less elongated and have greater radial growth than those of control plants (Jacobs, 1954; Larson, 1965; Goodman and Ennos, 1997).
The root systems of plants subjected to mechanical perturbations alter their morphology, anatomy and biomechanics to prevent uprooting (Gartner, 1994; Stokes et al., 1995, 1997a, b; Goodman and Ennos, 1996, 1997, 1998; Stokes and Guitard, 1997; Niklas, 1998). For instance, the roots of mechanically stressed plants are thicker and more numerous (Goodman and Ennos, 1996, 1997), and they change their mechanical properties by altering the shape (Nicoll, 2000), elongation rate (Goodman and Ennos, 1999) and diameters (Atwell, 1988) of the taproot and/or lateral roots (Stokes and Guitard, 1997).
Slope is a frequent, complex environmental condition that profoundly affects plant stability. The roots of many tree species growing on slopes are orientated uphill and stabilize the soil (Coutts and Nicoll, 1991). However, although it is well recognized that plants help prevent landslides (Watson et al., 1995), little is known about the effects exerted by slope on root system growth and development. Studies of four woody species (Quercus pubescens, Q. cerris, Fraxinus ornus, Spartium junceum) growing on slopes, under natural conditions, revealed morphological and architectural alterations in the orientation and density of lateral roots (Chiatante et al., 2001, 2003a, b). In particular, they had an asymmetrical root system, designated ‘bilateral-fan shape’, in which lateral roots developed both downslope and upslope (Chiatante et al., 2001). These root changes could serve to ensure plant stability (Chiatante et al., 2001, 2003a, b; Di Iorio et al., 2005).
In an attempt to shed light on the mechanisms governing plant anchorage, the morphological, biomechanical and molecular changes that occur in response to slope in S. junceum, a species that grows on hilly ground, were studied. Seedlings were grown in tilted and untilted pots under controlled conditions in which seedlings must respond to their own weight (Fig. 1) and gravitropism. Other mechanical factors (i.e. wind, weight and strains of moving soil, soil impedance, grazing, etc.) that may affect plant stability under natural slope conditions were excluded.
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It was observed that young seedlings perceive and promptly respond to slope early during development by altering the morphology of their root systems, by changes in their biomechanical properties and by increases in their lignin content. Lastly, in the roots of slope-grown plants, changes were found in the expression of several genes, some of which may be homologues of genes involved in environmental stress responses and/or in the metabolic pathways regulating plant biomechanical properties.
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MATERIALS AND METHODS |
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Plant material
Seeds of S. junceum L. (Ansaloni Florasilva, Bologna) were sown in a mixture of peat and vermiculite (3 : 1) in untilted and in tilted (45°) pots. After 20 d of germination, uniform seedlings were transferred to pots (112 x 20 x 45 cm, length x depth x width), untilted or tilted, with the same soil composition as before germination. Seedlings were grown in a greenhouse under a 16 : 8-h light–dark regime, a quantum flux of 300 µmol m–2 s–1, temperature of 24/18 °C and 45/70 % humidity. They were watered with the same amount of tap water every week. The roots were cleansed of soil with a paintbrush dipped in iced-water in order to prevent damage to root tissue. Finally, roots were washed in 10 % SDS, immediately frozen in liquid nitrogen and stored at –80 °C until required for RNA isolation. Two-, 3- and 8-month-old plants were used for pulling experiments. Before the plants were pulled up, pots were watered from above with tap water until saturated and then allowed to drain for 1 h.
Analysis of root traits
The root systems of 1-, 2-, 4- and 8-month-old seedlings were excavated intact and their morphological and topological traits were analysed by mean of a computerized system consisting of a scanner for image acquisition and the WinRhizo Pro 2003b software (Regent Ltd, Canada) for image analysis. The morphometric parameters recorded were: length and diameter of the taproot, and number, length and diameter of all lateral roots of the first three orders. The root systems of ten plants for each condition and age were used for statistical analysis.
Pulling test
Twenty plants for each condition (plane and slope) and age (1, 2, 4 and 8 months) were pulled out with a portable dynamometer, by applying a force normal to the soil. To estimate the anchorage force of a plant to the soil under plane and slope conditions, displacement of the plant from the soil was recorded as the plant was pulled up and the force applied to the plant until uprooted (breaking load). Root and shoot dry weights were measured after drying in an oven at 70 °C for 1 week.
Lignin extraction and analysis
To measure the lignin content of S. junceum root systems of 1-, 2-, 4- and 8-month-old plants grown on a plane or on a slope, about 1 g of each sample was boiled in ethanol for 30 min, pulverized in liquid nitrogen and thawed in 10·0 mL of homogenization buffer (50 mM Tris–HCl, 10 g L–1 Triton X-100, 1 M NaCl pH 8·3). The suspension was vortexed and centrifuged at 2000g for 10 min. The cell-wall pellet was washed twice with 4 mL of the homogenization buffer, 80 % acetone and pure acetone, and dried in a concentrator. Each pellet was then treated with 0·4 mL thioglycolic acid and 2 mL 2 M HCl for 4 h at 95 °C, centrifuged at 15 000g for 10 min and washed three times with distilled water. The lignothioglycolic acid from each pellet was extracted with 2 mL 0·5 M NaOH by agitating for 16 h at 20 °C. The supernatant was acidified with 0·4 mL concentrated HCl. Lignothioglycolic acid was precipitated for 3·5 h at 4 °C, recovered by centrifugation at 15 000g for 20 min, and dissolved in 1 mL 0·5 mol L–1 NaOH. The amount of lignin was calculated from the absorbance at 280 nm using a specific absorbance coefficient of 6·0 L g–1 cm–1. Because this specific absorbance coefficient provides only an approx-imate conversion (the absorbance of lignothioglycolic acid from different sources can vary considerably; see Doster and Bostock, 1988), the specimen with the highest lignin content was used as an internal standard in measurements of the percentage lignin content of the other samples. The results of three assays were used for statistical analysis.
RNA extraction and cDNA synthesis
Total RNA was extracted from the whole root systems of 4-month-old S. junceum by grinding 2 g of root tissue in liquid nitrogen. Buffer A (0·1 M LiCl, 0·01 M EDTA, 1 % SDS, 0·1 M Tris pH 9·0)/phenol mixture (80 °C) in a 1 : 2 powder-to-liquid ratio and 1 volume of chloroform were added to the root tissue. The sample was shaken for 30 min at room temperature, and RNA was precipitated with one-third volume of 8 M LiCl overnight at 4 °C. The RNA pellet was dissolved in 2 M LiCl and then washed twice with 70 % ethanol and once with 100 % ethanol. The air-dried pellet was finally dissolved in DEPC-treated water. Only high-quality RNA, i.e. with an A260/A280 ratio of 1·8 : 2·0 as shown by spectrophometry, was treated with DNase and used for cDNA synthesis, which was obtained using a SuperScript One-Step Reverse Transcription (RT)-PCR with Platinum Taq (Life Technologies, San Diego, CA), according to the manufacturer's recommendations. Finally cDNA was phenol/chloroform (3 : 1)-extracted, ethanol-precipitated and taken-up in a final volume of 20 µL DEPC-treated water. Double-strand cDNA was obtained from the single-strand cDNA with DNA polymerase I and RNase H.
cDNA-AFLP analysis
An AFLP Analysis System I Kit (Life Technologies) was used for cDNA-AFLP, according to the manufacturer's recommendations. In brief, the double-stranded cDNA was digested with EcoRI (as a ‘rare cutter’ enzyme) and MseI (as a ‘frequent cutter’ enzyme) and ligated to the respective oligo primers (adaptors). The digested-ligated products were diluted three-fold and used as a template for preselective amplifications (20 cycles at 94 °C for 30 s, at 56 °C for 1 min and at 72 °C for 1 min) using primers with one restrictive base at the 3' end of the primers. The pre-amplification product was diluted ten-fold and used as starting material for the second amplification reaction. We used primers with three restrictive bases at the 3' end for this selective amplification (a total of 64 primer combinations were used). One of the primers (EcoRI) was end-labelled with [
33P]dATP. The generic primers were EcoRI: 5'-GACTGCGTACCAATTCNN-3' and MseI: 5'-GATGATTCCTGAGTAANNN-3'. Pre-amplification was as follows: 20 cycles of denaturation (30 °C for 30 s), annealing (56 °C for 1 min) and denaturation (72 °C for 1 min). For selective PCR amplification we used 13 cycles at 94 °C for 30 s, at 65 °C for 30 s and at 72 °C for 1 min, with the annealing temperature decreasing by 0·7 °C per cycle, and 23 cycles at 94 °C for 30 s, at 56 °C for 30 s and at 72 °C for 1 min. After amplification, products were resolved on a 5 % polyacrylamide gel, run at 1600 V for 3 h. Gels were dried on 3MM Whatman paper (Whatman, Maidstone, UK) on a slab gel dryer and exposed to Kodak Biomax film (Sigma) overnight. cDNA fragments were visualized by autoradiography after positionally marking gel and film.
Isolation and sequencing of transcript derived fragments (TDFs)
The film and gel were aligned and the bands of interest were cut from the gel with a razor blade. The gel slices were hydrated in 100 µL of water for 15 min, incubated at 100 °C for 20 min and precipitated with glycogen at –20 °C overnight. The eluted cDNA was amplified with the same primers and under the same conditions as used for the cDNA-AFLP analysis. Two microlitres of the PCR reaction was analysed on an agarose gel to verify correct fragment size. The fragments were purified from PCR products with a QIAquick PCR purification kit (Ambion, Austin, TX) and sequenced. They were identified with the BLAST sequence alignment program of the National Centre for Biotechnology Information (http://www.ncbi.nlm.nih.gov/BLAST) (Altschul et al., 1997). Any similarity with a score of over 46 or an E-value of less than 10–3 was considered a hit.
Reverse transcription-PCR analysis
Equal amounts (1 µg) of total RNA were reverse-transcribed with RT-PCR Kit QuantumRNA oligo (dT) primers (Ambion, Inc., Austin, TX), used according to the manufacturer's instructions. Amplifications were performed with preliminary denaturation at 94 °C for 5 min, 35 cycles at 94 °C for 30 s, at 55 °C for 30 s and at 72 °C for 1 min, and a final extension at 72 °C for 10 min, with primers specific for the gene of interest. The position and length of the primers were chosen based on their thermodynamic parameters by means of version 0·2x of the Primer3 Input Software (http://www-genome.wi.mit.edu/cgi-bin/primer/primer3.cgi). PCR products were separated by electrophoresis in a 1 % agarose gel and analysed.
Statistical analysis
We used the Student's t-test and the SPSS software package (SPSS Inc., version 8·0) to evaluate morphological data; P < 0·05 and P < 0·001 were used as levels of significance.
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RESULTS |
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Shoot and root morphology
As shown in Fig. 2A, after 2 months of growth, slope-grown seedlings had shorter shoots than plane-grown seedlings. The difference in shoot length was significant in 8-month-old plants. Shoot branching and leaf number had a similar growth pattern (Fig. 2B and C), and both were lower in slope-grown seedlings. Again, the difference was significant after 8 months of growth (Fig. 2B and C). Shoot and root biomass were significantly higher in slope-grown plants than in plane-grown plants after 8 months of growth (Fig. 3A and B).
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In the analysis of root system morphology, only the taproot and lateral roots of the first three orders were measured, because larger lateral roots were too few and not well developed at the growth stages considered. Taproot length was similar in slope- and plane-grown plants except after 4 months of growth, when it was longer in slope-grown seedlings (Fig. 4A). Taproot diameter was similar in both conditions throughout the study (Fig. 4B).
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The numbers of first-, second- and third-order lateral roots of slope- and plane-grown seedlings were similar at 1 and 2 months, and significantly greater in slope-grown seedlings at 4 months (Fig. 5A–C). Lateral root length was significantly greater in 4-month-old plants grown on a slope. After 8 months of growth, the numbers of first- and second-order laterals were higher in plane-grown seedlings. Lateral length had the same trend as lateral number (Fig. 5D–F). The diameter of first-order lateral roots was larger in slope-grown seedlings up to 4 months, and larger in plane-grown seedlings at 8 months (Fig. 5G). The diameters of second-order lateral roots were similar under both conditions at 1, 2 and 8 months, and larger in slope-grown seedlings at 4 months (Fig. 5H). The diameters of third-order lateral roots were similar in the two groups of plants throughout the study (Fig. 5I).
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Pulling test
Pulling tests were used to evaluate whether slope influences plant anchorage. Pulling strength is the angular coefficient of the line obtained by plotting the load necessary to uproot seedlings versus their displacement. Pulling strength increased with age under both conditions (Fig. 6). Moreover, after 1 month of growth, pulling strengths were higher in plants growing on a slope, whereas the opposite was observed in 2-, 4- and 8-month-old plants (Fig. 6). Breaking loads in slope-grown seedlings were lower at 1 and 2 months and higher at 4 and 8 months versus plane-grown plants (Fig. 6). Maximum plant displacement in response to pulling showed the same trend as the breaking load (Fig. 6).
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Lignin analysis
Lignin content in seedlings was measured using a chemical procedure (see Materials and methods), and differences between the two conditions at each growth point analysed were found. Differences became significantly higher in 8-month-old slope-grown plants (Fig. 7).
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Differential gene expression in slope-grown plants measured by cDNA-AFLP
The cDNA-AFLP technique was used to evaluate whether there were changes in gene expression in roots growing on a slope. Figure 8 shows a section of AFLP fingerprinting where cDNA fragments were amplified with 64 primer enzyme combinations for selective amplification. Because of the stringent cDNA-AFLP conditions, band resolution was high and the background was acceptable (Fig. 8). The radical changes in the intensity of individual bands between the plane and slope conditions did not affect the other bands in the lane. This indicates that accumulation of the product was not affected by the concentration of substrates in the reaction mix. The size arrangement of the expression pattern in the window of a 5 % AFLP polyacrylamide gel ranged from around 800 bp at the top to about 80 bp at the bottom (Fig. 8). From 50 to 90 TDFs were obtained for each primer enzyme combination, which resulted in analysis of about 5000 cDNA fragments. Three expression patterns were identified in plants growing on a slope: induced expression (Fig. 8, arrow a), decreased expression (arrow b) and constitutive expression (arrow c). About 6·0 % (300) of the TDFs were differentially expressed in the unstressed versus the slope-induced stress condition. Some fragments displayed a qualitative variation (presence/absence) and some a quantitative variation. Of the differentially expressed TDFs, only those greater than 300 bp were analysed, i.e. about 2 %, which accounted for 95 TDFs.
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Compilation of sequences from slope-induced cDNA fragments
The 95 differentially expressed TDFs were excised from the gel, purified and re-amplified using the same PCR conditions as the cDNA-AFLP selective PCR, and the sequenced. DNA sequences were obtained for 38 fragments. The sequences were compared with those in the GenBank database using the latest version of the BLAST program (Altschul et al., 1997
). Twelve of the 38 TDFs corresponded to known functional genes; the sequences of the remaining fragments did not match any sequences in the public databases. Sequence similarity to known genes, the length and the GenBank accession numbers of the TDFs are listed in Table 1 (E-value threshold of 0·01). The proteins encoded by these fragments are homologues of the 26S ribosomal subunit, rDNA intergenic spacer DNA, the putative WD-40 repeat protein, an initiation factor, Arabidopsis chromosome 1 and 3, a transcription factor, cytosolic malate dehydrogenase, and the Myb transcription factor. Seven fragments with the best sequence quality as determined by RT-PCR were examined, and their differential expression was confirmed (Fig. 9).
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DISCUSSION |
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It is well recognized that vegetation prevents landslides, and that root morphology is important in increasing the shear strength of soil (Coppin and Richards, 1990
; Morgan and Rickson, 1995; Gray and Sotir, 1996). However, the mechanisms underlying the stability of plants growing on a slope have not previously been thoroughly investigated.
The mechanisms governing the anchorage of Spartium junceum plants growing on a slope were investigated. Seedlings were grown under controlled conditions of light, temperature, humidity and soil composition (see Materials and methods) in untilted and tilted (45°) pots. In this experimental set-up, which represents a simplification of slope, the forces acting on the plant–soil system are self-weight and gravity. Thus, plants are affected by the weight of the soil (
) and by the weight of the aerial part of the plant (
). Considering that the borders of the small pots do not exert any effect on the plant and the low density of the peat–agriperlite mixture, it is reasonable to assume that, in this experimental system, soil weight does not affect plant growth. Thus, in this system, the only physical effect exerted by the slope is the weight of the aerial part of the plant (‘self-loading’).
Here it was observed that seedlings perceive slope at an early stage of growth, and respond with changes in stem and root morphology. Some alterations occur before others. At 1 month, the shoot axis had grown a few centimetres along the normal to the soil, after which it curved up towards the stem, probably because of the negative gravitropism. In slope-growing plants, the inclination of the growing aerial part of the plant caused a bending moment at the stem base. The bending moment varied proportionally both to the intensity and the moment arm of the weight of the aerial part (Fig. 1), and hence changed as the plant grew.
After 2 months of growth, shoot height, shoot branching and leaf number were reduced in slope-grown seedlings (Fig. 2), which could help to reduce the bending moment. By contrast, shoot dry weight was greater in 8-month-old slope-grown seedlings (Fig. 3). Although not investigated further here, the increase in shoot dry weight together with the reduction of shoot height in slope-grown plants could be due to an increase in radial growth and/or density. The morphological alterations and increased shoot biomass may be strategies used by the plant to reduce and/or counteract the bending caused by the aerial part on the root system, and hence to limit uprooting forces, which are mainly represented by self-loading (Hunt and Jaffe, 1980; Jaffe, 1980; Cremer et al., 1982; Heuchert et al., 1983; Richter, 1984; Telewski, 1995).
To achieve anchorage on a slope, plants must transfer the loading forces exerted on the shoot into the ground via roots. Plants have developed various strategies to withstand mechanical loading. For instance, on steep slopes tree roots are generally orientated uphill (Coutts and Nicoll, 1991; Marler and Discekici, 1997; Di Iorio et al., 2005) and the morphology of root systems of old plants growing in natural slope conditions differs from that of plants growing on plane ground (Chiatante et al., 2001). Our study confirms that slope induces morphological alterations in the root system and suggests that different anchorage mechanisms may be required at different stages of plant growth.
It has been suggested that the above-ground portion of a plant growing on a slope constantly alters self-loading and consequently the bending moment; this in turn triggers morphological and biomechanical changes in the root system to cope with the increased self-loading and so ensure plant stability (Chiatante et al., 2003a, b).
The morphology of the taproot and lateral roots differed in slope-growing versus plane-growing S. junceum seedlings; the difference was particularly evident at 4 months (Fig. 4). These changes could be related to the changes in the biomechanical properties involved in anchorage mechanisms. In fact, the pulling test revealed that slope-growing seedlings have significantly higher breaking loads than seedlings plane-growing at 4 and 8 months of growth (Fig. 6). At 4 months, the slope-induced increase in breaking loads could be related to the increase in taproot and lateral root morphological parameters. At 8 months, slope-growing plants had lower taproot, first- and second-order lateral root values (Fig. 5), and higher breaking loads (Fig. 6). At 8 months, the root system's biomechanical properties may be strengthened by changes in mechanical tissue organization and/or composition. Examination of the mechanical tissues via optical microscopy did not reveal any differences between the slope-grown and plane-grown plants (data not shown). However, the concentration of lignin was higher in the root systems of slope-grown plants. Moreover, the differences between slope- and plane-grown plants became significant after 8 months of growth (Fig. 7), when breaking loads were also higher. These findings are in line with reports that in order to increase stability and ensure plant anchorage, root systems respond to mechanical stresses by increasing the production of lignin, a strengthening material (Patel, 1971; Stokes et al., 1997a), and/or by altering the cell wall (Timell, 1986; Showalter et al., 1992; Telewski, 1995; Shirsat et al., 1996; Zipse et al., 1998; Jamet et al., 2000).
Little is known about the molecular mechanisms underlying anchorage in plants subjected to mechanical perturbations. cDNA-AFLP was used here to identify genes involved in root response to slope. This technique allows the simultaneous screening of a large number of genes, and it requires very little initial template nucleic acid and no prior knowledge of target nucleic sequence. This was crucial in the case of S. junceum, the genome sequence of which is unknown. Four-month-old seedlings were first examined, in which morphological alterations were greatest and in which the mechanisms involved in plant anchorage were more evident. The cDNA-AFLP fingerprints of the roots of plane- and slope-grown plants revealed three cDNA expression patterns: up- and down-regulated expression and constitutive expression (Fig. 8). Some of the TDFs up-regulated in slope-grown plants had high sequence similarities with genes involved in plant responses to other environmental stresses that may be involved in wood formation and organization. In particular, one of the slope up-regulated TDFs matched a malate dehydrogenase gene (Table 1). Malate dehydrogenase genes are involved in responses to several environmental stresses (Delgado et al., 1993; Soussi et al., 1998; Kalifa et al., 2004) and are overexpressed in reaction wood biosynthesis (Kärkönen et al., 2002). Other TDFs overexpressed in the slope condition had a high sequence similarity with an initiation factor, an Myb transcription factor and a 26S rRNA ribosomal gene, which seem to be involved in the formation of wood tissue (Le Provost et al., 2003) and in lignification processes (Plomion et al., 1999, 2000; Newman and Campbell, 2000). Some of the other differentially expressed TDFs aligned with a chromosome genomic DNA, an mRNA sequence and a cosmid DNA lacking a known function, although the majority had no matches in the database. This lack of alignment may be because similar genes have not been discovered in S. junceum or in other organisms, or because these sequences belong to non-codifying regions. In most cases, RT-PCR confirmed up-regulation of TDFs in slope-growing plants. Technical reasons (i.e. the presence of secondary metabolites) probably account for the cases in which RT-PCR did not confirm TDF overexpression.
In conclusion, the experimental system used in this work represents a simplified slope in which the only effects are gravity and self-weight. It was demonstrated that plant roots promptly perceive the slope condition and respond with changes that seem to be related to growth stage. In fact, at 4 months, the root apparatus begins to acquire the ability to resist up-rooting forces, which is reflected in morphological changes, in increases in the production of the strengthening material lignin and in the alteration of the expression of several genes.
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ACKNOWLEDGEMENTS |
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We thank Jean Gilder for editing the text. This work was partly funded by the Consiglio Nazionale delle Ricerche (CNR).
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LITERATURE CITED |
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