JXB Advance Access originally published online on February 13, 2004
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Journal of Experimental Botany, Vol. 55, No. 397, pp. 613-622, March 1, 2004
© 2004 Oxford University Press
Cell and Molecular Biology, Biochemistry and Molecular Physiology |
Changes in gene expression during meristem activation processes in Solanum tuberosum with a focus on the regulation of an auxin response factor gene*
Received 6 August 2003; Accepted 25 November 2003
1 Quality, Health and Nutrition, Scottish Crop Research Institute, Invergowrie, Dundee DD2 5DA, UK
2 Computational Biology, Scottish Crop Research Institute, Invergowrie, Dundee DD2 5DA, UK
* The sequences reported in this article will appear in the EMBL database dbEST with the accession numbers CK565–CK565637. To whom correspondence should be addressed. Fax: +44 (0)1382 562426. E-mail: mtaylo{at}scri.sari.ac.uk
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Abstract |
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A suppression subtractive hybridization approach (SSH) was used to generate a cDNA library enriched in clones representing genes that are up-regulated in the potato tuber apical bud on dormancy release. The sequences of cDNAs representing 385 different genes were determined. This study focuses on the characterization of one of these cDNAs. On the basis of sequence similarity, the cDNA was identified as encoding a member of the auxin response factor family (ARF6). The expression pattern of potato ARF6 was determined by in situ hybridization. In apical tuber buds in the early stages of sprouting, relatively high levels of ARF6-specific transcripts were detected, especially in the peripheral zones of the tunica and corpus of the apical meristems. Expression was also detected in procambial and early vascular tissues, both subtending the meristem and in adjacent leaf primordia. By contrast, in dormant buds no expression of ARF6 could be detected. The expression pattern was also determined during the tuberization process; steady-state expression levels decreased c. 10-fold in the apical region as tuberization proceeded. In non-growing buds, exhibiting correlative inhibition, ARF6-specific transcript levels were relatively low, but rapidly increased when apical dominance was removed by excision of the apical bud. The effects of gibberellin and auxin on axillary bud growth and ARF6 expression are described.
Key words: Auxin response factor, bud dormancy, meristem activation, Solanum tuberosum.
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionSupplementary dataReferences |
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The manipulation of the potato tuber life cycle in order to improve the timing of tuberization, tuber-size distribution, and dormancy characteristics is a major economic target (reviewed in Fernie and Willmitzer, 2001). An understanding of the processes that lead to stop–start cycles in the growth of the potato tuber apical meristem is important to achieve this aim. It is generally accepted that tuberization of stolon tips is accompanied by the inactivation of the stolon apical meristem and, thus, tuber dormancy is actually initiated at tuberization (Claassens and Vreugdenhil, 2000). The length of the post-harvest dormancy period depends on both the genetic background of the cultivar and the prevailing environmental conditions during tuber development (Kotch et al., 1992). Commercial practices often require storage of potato tubers for periods beyond that of natural dormancy (generally 1–15 weeks). Sprouting of stored potatoes is detrimental for many utilization processes, as it leads to quality deterioration, loss of dry matter, and onset of disease (Wiltshire and Cobb, 1996). Practical control of premature sprouting in storage is achieved through the use of low temperatures or treatment with chemical sprout suppressants. However, low temperature storage has negative repercussions on tuber processing quality (e.g. low temperature sweetening) and the chemicals employed as sprout suppressants increasingly raise economic and environmental concerns. These concerns have stimulated interest in the identification of mechanisms underpinning dormancy bud break in order to develop a targeted approach to the control of sprouting in stored potatoes.
As with many plant developmental processes, roles for the phytohormones in the control of dormancy have been investigated (reviewed in Claassens and Vreugdenhil, 2000; Fernie and Willmitzer, 2001). For example, in potato, recent data describes the progressive increase in both free and conjugated IAA levels in tuber apical meristems during the dormancy period, which rapidly falls on sprouting (Sorce et al., 2000). Two related families of proteins, the Aux/IAA proteins and the auxin response factors (ARFs) are key regulators of auxin-reponsive gene expression (reviewed in Liscum and Reed, 2002). In Arabidopsis there are up to 29 members of the Aux/IAA family, whereas the ARF gene family has up to 23 members. Recently, a model for the action of these proteins has been developed (reviewed in Hagen and Guilfoyle, 2002). ARF proteins contain an amino terminal DNA binding domain that binds to a specific sequence (TGTCTC) in the promoters of auxin-responsive genes (Ulmasov et al., 1999a). Whether the ARF protein acts as a repressor or activator of transcription is dependent on the middle region of the ARF protein; Q-rich middle regions activate transcription whereas P/S/T-rich middle regions confer repressor activity (Ulmasov et al., 1999b). In most ARFs there are two conserved carboxyl-terminal protein–protein interaction motifs (domains III and IV). These motifs are involved in ARF dimerization, which facilitates stable binding to DNA (Ulmasov et al., 1999a). Aux/IAA proteins do not bind to DNA, but exert their effects by interacting with ARFs, thus modulating ARF activity (Guilfoyle et al., 1998). Generally, Aux/IAA proteins have domains similar to the ARF domains III and IV and so can bind to them, disrupting the formation of ARF dimers (Ulmasov et al., 1999a). Consequently, Aux/IAA proteins can modulate gene activation or repression. In general, Aux/IAA proteins are short-lived, targeted to the nucleus and are induced by auxin (Abel and Theologis, 1996). The equivalent ARF and aux/IAA proteins remain to be characterized in other species such as potato, but may have key regulatory roles.
An additional complexity in the study of bud dormancy is presented by the influence of factors external to the bud itself, a phenomenon known as paradormancy (Lang et al., 1987). The mechanisms implicated in the control of axillary bud growth by apical meristems remains to be determined (Shimizu-Sato and Mori, 2001). In some plant species, the physiological, genetic, and hormonal factors that influence axillary meristem growth have been characterized. For example, some of the genes which influence hormonal balance and which control the growth of specific organs at different developmental stages have been identified (Sussex and Kerk, 2001). Examples include the Lateral suppressor gene of tomato (Schumacher et al., 1999) and the Petunia dad gene (Napoli, 1996). The mechanism of action of these genes must now be determined, and possibilities for modifying meristem activation to provide agronomic benefits explored.
A detailed study of changes in gene expression in the potato tuber meristem during release from dormancy has not previously been reported. In this study, suppression subtractive hybridization has been used to produce a library of genes enriched in those up-regulated on the release of dormancy. The detailed expression pattern of one of these candidate genes is described during the tuber life-cycle and during other meristem activation processes. Physiological experiments describe the effects of gibberellin and auxin on axillary bud growth and ARF6 expression. These data suggest that the gene can be used as a molecular marker for meristem activity in the tuber apical and axillary buds.
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Materials and methods |
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Plant material
Potato tubers (Solanum tuberosum, cultivar Desirée) were harvested from fully mature plants grown in the field. Tubers of a similar size (c.100 g FW) were stored at 4 °C in the dark. Endodormant apical buds were excised from tubers stored for 2–4 weeks, whereas visibly growing apical buds were excised from tubers stored for 8–10 weeks. Stolons and tubers were harvested from plants grown in a glasshouse. For some experiments, tissue was harvested from tissue-culture plants. These plants were micropropagated on MS medium (Murashige and Skoog, 1962) supplemented with 3% (w/v) sucrose and 0.8% (w/v) microagar (Duchefa). The plants were grown in plastic magenta jars (350 ml) covered by a vented plastic closure (Sigma) under a 16 h photoperiod at a light intensity of 70 µmol m–2 s–1 and constant temperature of 22 °C. Leaves, petioles, and stems were sampled from these plants. Axillary and apical buds were also harvested from the etiolated shoot produced by tubers following long-term (6–9 month) storage at 4 °C. Nodes were excised when sufficient internode was accessible to allow 10–15 mm of stem either side of a bud, but before the axillary bud had grown to more than 1 mm in length. They were placed in sterile 10 ml tubes containing 1 ml water, maintaining the orientation of the cutting. One hundred microlitres of 10 µM solutions of GA3 or NAA were applied to the apical side to compare the relative effectiveness of these hormones on bud growth and ARF6 expression.
Suppression subtractive hybridization (SSH)
SSH, using the Clontech PCR-Select cDNA subtraction kit was used to generate a cDNA library enriched in sequences derived from genes that are up-regulated in the potato tuber apical meristem in the early stages of dormancy release. Total RNA was extracted from the meristems of dormant tubers (stored for 2–4 weeks at 4 °C) which served as ‘driver’ and from tubers in the early stages of dormancy release (8–10 weeks of storage at 4 °C) as assessed by visible growth of the meristem (‘tester’). Meristems were observed under a low power microscope, excised from the tubers with a scalpel and forceps, and were frozen immediately in liquid nitrogen. In order to obtain sufficient tissue for the 2 µg of poly A+ RNA required for the Clontech protocol, it was necessary to harvest approximately 300 tubers for each stage (approximately 60 mg FW tissue). Following SSH, amplification products were purified using a Qiaquick PCR purification kit (Qiagen) and cloned using the pGEM-T Easy cloning kit from Promega. E. coli strain DH5
was host for all recombinant plasmids.
DNA sequencing and sequence analysis
Sequencing reactions were performed using an Applied Biosytems Perkin Elmer Big Dye Terminator cycle sequencing reaction kit (PE Biosystems) and an ABI377 automated sequencer (PE Biosystems).
DNA base calling was carried out using phred (Ewing et al., 1998), vector masking using cross-match (Ewing and Green, 1998), and vector and quality trimming was carried out using a series of custom Perl scripts. EST assembly was based on the use of CAP3 (Huang and Madan, 1999) with the following set of parameters –p 95 –d 6 to 0 –f 100 –h 30.
Database searching was carried out using the NCBI Netblast client (http://www.ncbi.nlm.nih.gov/Web/Search/client.html) (Altschul et al., 1990) with each SSH sequence searched against the NCBI nr nucleotide and protein databases as well as dbEST at NCBI. Protein sequences were aligned using CLUSTAL W (Higgins et al., 1994) and the alignments manually refined using the BioEdit suite of programmes (www.mbio.ncsu.edu/BioEdit).
In situ hybridization
Hand-sections containing potato meristems were excised from tubers using a razor blade and fixed in neutral-buffered formaldehyde (formaldehyde 3.8–4.0% w/v). Following dehydration through an ethanol series, tissue was automatically embedded in paraffin wax in a Tissue-Tek VIP tissue processor. Sections (5 µm thick) were cut on a microtome and were mounted on positively charged slides (Fisher). In situ hybridization was carried out using the mRNAlocatorTM-Hyb kit (Ambion), using a 20 min proteinase K digestion and omitting the DNase treatment as recommended. Sense and anti-sense single-stranded RNA probes were generated by in vitro transcription using the MEGA scriptTM kit (Ambion) with either SP6 or T7 polymerase. A 449 bp fragment of the ARF6 gene, containing sequence encoding the 3' 140 amino acids and 29 base pairs of 3'UTR sequence (see Fig. 2 in the Results), cloned in pGEM-T Easy (Invitrogen), and linearized prior to use, served as template for in vitro transcription. RNA was labelled post-synthesis, using Ambion’s BrightStarTM Psoralen-Biotin Kit.
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Following hybridization and probe detection, according to Ambion’s recommendations, slides were dehydrated, cleared, and mounted, prior to analysis. Samples were viewed under an Optiphot II light microscope (Nikon) equipped with Nikon achromat lenses, and digital images were captured using a Coolview digital CCD camera (Photonic Science).
Semi-quantitative RT-PCR
Total RNA was extracted from apical and axillary buds using the RNeasy kit from Qiagen. Following DNase treatment, RT-PCR was carried out essentially as described by Simpson et al. (1996) using 4.0 µg total RNA following the Life Technologies Superscript reverse transcriptase protocol. The 5' primer was designed to a non-conserved region of the potato ARF6 sequence (TCCCCTT AACTCAGACATGAC) and the 3' sequence was TCCAACAA GCCTTCTAGCC (primer binding sites indicated in Fig. 2 in the Results). Samples were removed from cycle 17 onwards, the products separated on 1.5% agarose gels and blotted onto a Hybond N+ membrane (Amersham Pharmacia Biotech). After probing with a 32P-labelled ARF6-specific probe, the filters were stringently washed. The ARF6-specific signal was quantified with the Molecular Image Storage Phosphor Imaging System (Model GS-525, Bio-Rad), using the Molecular Analyst software (version 1.4, Bio-Rad).
DNA extraction and Southern analysis
Plant genomic DNA was extracted from leaves as described previously (Draper et al., 1988). Ten micrograms of DNA was digested with EcoRI or HindIII and resolved by electrophoresis on 0.8% agarose gels. DNA was transferred to nylon membranes (Hybond-N+, Amersham). Filters were hybridized with the 449 bp ARF6 cDNA labelled to high specific activity (c. 1x109 cpm µg–1) with [-32P] dCTP using random primers (HiPrime, Boehringer). Following hybridization, filters were washed at low stringency (0.5xSSC, 0.1% SDS at 45 °C) and exposed to X-ray film for 48 h at –70 °C with intensifying screens.
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Results |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionSupplementary dataReferences |
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Gene expression changes during dormancy release in the potato tuber apical meristem
Several hundred cDNAs were sequenced from an SSH generated library enriched for sequences that are up-regulated on release from dormancy in the potato tuber apical bud. The CAP3 assembly of SSH EST sequences resulted in 109 contigs and a further 276 ESTs were unique singletons, giving a total of 385 different sequences. These sequences have been deposited on the EMBL database (accession numbers CK565–CK565637). BLASTX and/or BLASTN searches against the NCBI nr databases were used to identify and categorize the sequences (Fig. 1) (supplementary material can be found at Journal of Experimental Botany online). A relatively high proportion of the sequences (36%) did not show significant similarity (BLAST score less than 80) to other sequences or matched unknown proteins (13%). Approximately 12% of the sequences gave good hits to ribosomal protein genes, which may reflect a dramatic increase in biosynthetic activity on release from dormancy. Heat shock proteins or those with similarity to chaperones made up some 9% of the sequences. A relatively high percentage of carrier proteins (c. 4%) and transcription factors (c. 3%) were also identified in the set of sequences.
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Cloning and sequence analysis of potato ARF6
In this study the focus was on one clone that showed strong sequence similarity to a class of auxin response factors. The partial cDNA encodes the C-terminal portion of a protein with strong sequence similarity to Arabidopsis auxin-responsive transcription factors (Fig. 2). The best sequence match at the amino acid level is with the Arabidopsis ARF6 sequence (78% identity in an 111 amino acid overlap). Based on the high degree of similarity the potato cDNA was tentatively identified as encoding potato ARF6. The C-terminal regions of ARFs contain some well-conserved domains (Ulmasov et al., 1999a) and so other Arabidopsis ARFs (ARF7 and ARF8) also show significant sequence identity (Fig. 2). In addition, an Aux/IAA protein shares some sequence similarity with these proteins (Fig. 2).
Southern analysis
In order to obtain an indication of ARF6 copy number in the potato genome, Southern analysis of genomic DNA isolated from tetraploid S. tuberosum cv. Desirée was carried out using the putative ARF6 449 bp cDNA. For the EcoRI and HindIII digests only one strongly hybridizing DNA fragment could be detected (Fig. 3). For the EcoRI digest, a second fragment that hybridized much more weakly could be detected under conditions of low stringency washing. This indicates that the ARF6 gene is present at low copy number (possibly single copy) in the tetraploid potato genome and that the 449 bp probe was unlikely to detect multiple genes when used in hybridization-based expression analysis.
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digested with HindIII and their positions are indicated by arrows (sizes in kb).The expression pattern of ARF6 during meristem activation
The expression pattern of ARF6 was determined in potato tuber apical buds and adjacent tissues during the dormancy-breaking period by in situ hybridization. An antisense probe for the ARF6 gene was synthesized using the 449 bp cDNA as template. In sprouting buds from tubers that were stored for 8–10 weeks at 4 °C, visible growth of the bud had occurred (Fig. 4A). Relatively high levels of transcript were detected in sections from these tubers. ARF6 was highly expressed in cells within the apical meristem (Fig. 4A, B), and was found in both the tunica and corpus and especially in the peripheral zones. Transcript was also detected in procambial and early vascular tissue, both subtending the meristem and in adjacent leaf primordia. Transcript was not detected in either ground or dermal tissues. Control samples from sprouting tubers (Fig. 4C) were hybridized to sense control probes and showed no blue colouration in any region of the meristem, or in leaf primordia, scale leaves, or subtending tuber tissue, indicating a lack of probe hybridization and, therefore, absence of artefactual signal. In dormant buds that were from tubers stored for 2–4 weeks at 4 °C, no visible growth of the bud had occurred (Fig. 4D). In these buds, probed with either antisense (Fig. 4D, E) or sense probes (Fig. 4F) no expression of ARF6 could be detected in any tissue. Very low levels of background staining were detected (Fig. 4E, arrows), however, examination of these slides at higher magnification indicated that this signal was only present in nuclei and there was no cytoplasmic signal (data not shown)..
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Expression pattern of the ARF6 gene in different potato tissues during the potato tuber life cycle
The steady-state level of the ARF6-specific transcript was determined in a range of potato tissues. The ARF6 transcript was barely detectable by northern blot analysis and so a semi-quantitative RT-PCR approach was used. The 5' primer was designed to a region of ARF6 sequence that was not conserved in other ARF or Aux/IAA genes and so the use of this primer only detected ARF6-specific transcript. Cloning and sequencing the RT-PCR products from sprouting tuber buds (six independent clones) demonstrated that only the expected ARF6-derived product was obtained. Using cDNA derived from leaf tips, a plot of log product concentration against cycle number demonstrated a linear relationship up to 24 cycles when the signal saturated (Fig. 5A). Thus 22 amplification cycles were used for all RT-PCR experiments. As a control, RT-PCR analysis was performed using primers designed to amplify the potato 18S ribosomal RNA gene from an aliquot of the same cDNA sample used for ARF6-specific amplification. For all experiments, a similar signal was obtained from the 18S RNA control, indicating that similar quantities of total RNA were used in the analysis of the different samples, thus enabling a comparison of expression levels in the different tissues.Although ARF6-specific transcript could be detected in all tissues tested, there was a wide variation in the transcript level (Fig. 5B, C). Relatively high levels were present in leaf tips compared with the levels in apical stems, petioles and basal stems (Fig. 5B). The ARF6 transcript level was also measured in tissues from the different stages of the tuber life cycle. Expression levels were highest in stolon tips prior to visible signs of tuberization. At the onset of tuberization, in swelling stolon tips, the level decreased by 2.5-fold (Fig. 5C) and as tuberization progressed the level continued to fall to a 10.3-fold and 8.3-fold lower level in small tubers (approximately 2 g FW) and large tubers (approximately 20 g FW), respectively. Following dormancy break in apical buds, the expression level increased from barely detectable levels in fully dormant buds to levels 3.4-fold higher in buds from the early stages of sprouting (Fig. 5D), confirming the expression pattern shown by in situ hybridization analysis (Fig. 4).
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In order to investigate other situations in which meristem growth is repressed, a potato sprout system was used. Potato tubers that are stored for 6–8 months at 4 °C produce a single, long, etiolated sprout, which develops from the tuber apical meristem. Due to apical dominance, other buds on the sprout are repressed. The mean length of the buds was measured and the smallest buds were consistently observed to be the first, counting from the apex (data not shown). Figure 5E shows the levels of ARF6 transcript in buds from etiolated sprouts. The level of ARF6 transcript was found to be higher in the apical bud compared with buds from the first or fifth nodes. Excised nodal sections containing the first bud and 10–15 mm of stem either side were placed in a sterile 10 ml tube, maintaining the orientation of the cutting. The lower end of the stem was immersed in sterile water taking care to ensure that the bud and upper part of the stem were clear of the water. Under these conditions, the bud is released from the effects of apical dominance, and growth of the bud can be observed within 12 h (similar data for growth of buds from the first and third nodes are presented in Fig. 6). ARF6 expression was studied during the time-course of bud growth in this system. The levels of ARF6 transcript increased considerably (by 4.9-fold) over a 24 h period as the buds are released from the effects of apical dominance.
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Effects of GA3 and NAA on axillary bud growth and expression of ARF6 transcript level
The growth rates of the first and third buds counting from the apex, in excised nodal sections, were measured following apical treatment with either water, 10 µM GA3, or 10 µM NAA. Bud growth rate was stimulated on treatment with GA3 compared with the water-treated controls, whereas NAA caused a decrease in bud growth rate (Fig. 6A, B). After 4 d of growth the bud length was 0.95±0.11 and 0.68±0.09 cm for the first and third nodes, respectively, in samples treated with GA3. The corresponding buds from water-treated samples were 0.54±0.07 and 0.44±0.04, respectively. Conversely, after 4 d of NAA treatment, the first and third bud lengths were 0.33±0.04 cm and 0.32±0.04, respectively.
In parallel with these experiments, ARF6 expression level was determined in buds from the third node (Fig. 7). Eight hours after excising the nodal sections, levels of ARF6 transcript increased compared with the beginning of the experiment whatever the treatment. ARF6 levels were higher with GA3 treatment than those with NAA or H2O throughout the 4 d time-course, whereas NAA treatment resulted in lower ARF6 transcript levels compared with the controls.
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Discussion |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionSupplementary dataReferences |
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SSH was used to generate a cDNA library enriched for sequences up-regulated in the potato tuber apical meristem during the early stages of dormancy release. Some indication of the efficiency of the SSH process in achieving targeted gene discovery can be seen from the contrast between the relative sensitivity of detection of the AFR6 homologue in the SSH process as opposed to its distribution in other non-selected libraries. By sequence comparison, identification of many of the cDNAs was possible enabling the focus to be on those encoding proteins with possible regulatory roles. Amongst several transcription factors identified was one with strong sequence similarity to a family of auxin response factors.
The functions of several ARF genes have been demonstrated in Arabidopsis by the study of loss-of-function mutants. Whereas mutations in some ARF genes give quite distinct phenotypes (e.g. ARF3, ARF5, and ARF7), mutations in other ARF genes give rise to less dramatic phenotypes. This indicates that some ARFs have specific and unique roles, whereas there may be redundancy in the roles of other ARFs. Mutations in the MONOPTEROUS (MP)/ARF5 gene disrupt embryo axis formation and vascular development (Hardtke and Berleth, 1998). Mutations in the ETTIN (ETT)/ARF3 gene lead to impaired flower development (Sessions et al., 1997) and lesions in the NPH4 gene encoding ARF7 result in plants with abnormal differential growth (Harper et al., 2000).
Most of the Arabidopsis ARF genes that have been studied are expressed in most major organs, although the tissue-specificity (if any) of expression within the organs has not been reported. However, a recently identified tomato gene (DR12) encoding an auxin response factor, is differentially expressed during fruit ripening. Down-regulation of DR12 using an antisense approach resulted in tomato plants modulated in several morphological and physiological processes (Jones et al., 2002). This phenotype was characterized by dark-green immature fruit, unusual cell division in the fruit pericarp, blotchy ripening, enhanced fruit firmness, upward curling leaves, and increased hypocotyls and cotyledon growth. The current study provides evidence that the potato ARF6 gene is also expressed in a developmentally regulated manner. Expression is strongly up-regulated on release from dormancy and is particularly high in the files of cells associated with the developing vascular tissue leading to the meristem. The evidence provided here indicates that changes in ARF6 expression in this tissue occur early in the meristem activation process and may be of primary importance. In particular, ARF6 expression levels showed a marked decrease upon tuber initiation, when stolon growth changes from a longitudinal expansion due to cell division to a lateral expansion (Xu et al., 1998), and there is a marked decline in the mitotic index in the shoot apical meristem. On the other hand, ARF6 expression was strongly induced in buds upon endo-dormancy release when meristematic activity recommences. Interestingly, the expression of ARF6 also increases strongly in axillary buds on release from the effects of apical dominance (paradormancy or correlative inhibition). Thus it may be that there are common mechanisms in the release of endodormancy and paradormancy. Under several other conditions ARF6 transcript level appeared to correlate with the rate of meristem growth, ARF6 transcript levels were lowest in buds at the first node of etiolated stems, the smallest paradormant buds having the lowest level of ARF6 transcript. Moreover, on release from correlative inhibition ARF6 levels increased in parallel with the bud growth rates as shown by treatments with GA3 or NAA. Consistent with these data is the recent observation that the rice ARF1 gene (OsARF1) is auxin-regulated and acts as a primary auxin responsive gene (Waller et al., 2002). However in the case of rice, ARF1 is up-regulated by exogenous auxin whereas in potato, auxin appears to repress the expression of ARF6. In Arabidopsis, ARF6 functions as an activator of gene expression (Tiwari et al., 2003) and, if the same is the case in potato, ARF6-induced changes in gene expression may be important in the control of dormancy release. In apical and axillary buds the spatial and temporal expression patterns are consistent with this function. Further studies will determine the extent of ARF6 involvement in these processes. Nevertheless, the data presented here clearly demonstrate that ARF6 expression level is a marker for meristem activation status in potato. It will be interesting to see if similar results are found on bud activation in other species.
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Supplementary data |
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Supplementary data showing the results of the BLASTX and BLASTN searches against the NCBI nr databases can be found at Journal of Experimental Botany online.
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Acknowledgements |
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The authors are grateful to Fiona Hunter for embedding and sectioning potato buds and to Drs Alison Roberts and Brian Williamson for help with image analysis. This work was funded in part by the Scottish Executive Environment and Rural Affairs Department. OFR is grateful to the EU for her post-doctoral Marie Curie fellowship.
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References |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionSupplementary dataReferences |
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Abel S, Theologis A. 1996. Early genes and auxin action. Plant Physiology 111, 9–17.[CrossRef][Web of Science][Medline]
Altschul SF, Gish W, Miller W, Myers EW, Lipman D. 1990. Basic and local alignment search tool. Journal of Molecular Biology 215, 403–410.[CrossRef][Web of Science][Medline]
Claassens MMJ, Vreugdenhil D. 2000. Is dormancy breaking of potato tubers the reverse of tuber initiation? Potato Research 43, 347–369.[CrossRef]
Draper J, Scott RJ, Armitage P, Walden R. 1988. Plant genetic transformation and gene expression: a laboratory manual. London, UK: Blackwell Scientific Publications.
Ewing B, Green P. 1998. Base-calling of automated sequencer traces using phred. II. Error probabilities. Genome Research 8, 186–194.
Ewing B, Hillier L, Wendl MC, Green P. 1998. Base-calling of automated sequencer traces using phred. I. Accuracy assessment. Genome Research 8, 175–185.
Fernie AR, Willmitzer L. 2001. Molecular and biochemical triggers of potato tuber development. Plant Physiology 127, 1459–1465.
Guilfoyle TJ, Hagen G, Ulmasov T, Murfett J. 1998. How does auxin turn on genes? Plant Physiology 118, 341–347.
Hagen G, Guilfoyle TJ. 2002. Auxin-responsive gene expression: genes, promoters and regulatory factors. Plant Molecular Biology 49, 373–385.[CrossRef][Web of Science][Medline]
Hardtke CS, Berleth T. 1998. The Arabidopsis gene MONOPTEROS encodes a transcription factor mediating embryo axis formation and vascular development. EMBO Journal 17, 1405–1411.[CrossRef][Web of Science][Medline]
Harper RM, Stowe-Evans EL, Luesse DR, Muto H, Tatematsu K, Watahiki MK, Yamamoto K, Liscum E. 2000. The NPH4 locus encodes the auxin response factor ARF7, a conditional regulator of differential growth in aerial Arabidopsis tissue. The Plant Cell 12, 757–770.
Higgins DG, Thompson JD, Gibson TJ. 1994. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Research 11, 4673–4680.
Huang X, Madan A. 1999. CAP3: A DNA sequence assembly program. Genome Research 9, 868–877.
Jones B, Frasse P, Olmos E, Zegzouti H, Li ZG, Latche A, Pech JC, Bouzayen M. 2002. Down-regulation of DR12, an auxin-response-factor homolog, in the tomato results in a pleiotropic phenotype including dark green and blotchy ripening fruit. The Plant Journal 32, 603–613.[CrossRef][Web of Science][Medline]
Kotch GP, Ortiz R, Peloquin SJ. 1992. Genetic analysis by use of potato haploid populations. Genome 35, 103–108.
Lang GA, Early JD, Martin GC, Darnell RL. 1987. Terminolgy and classification for dormancy research. Horticultural Science 22, 371–377.
Liscum E, Reed. 2002. Genetics of Aux/IAA and ARF action in plant growth and development. Plant Molecular Biology 49, 387–400.[CrossRef][Web of Science][Medline]
Murashige T, Skoog F. 1962. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiologia Plantarum 15, 473–497.[CrossRef]
Napoli C. 1996. Highly branched phenotype of the Petunia dad1-1 mutant is reversed by grafting. Plant Physiology 111, 27–37.[Abstract]
Schumacher K, Schmitt T, Rossberg M, Schmitz G, Theres K. 1999. The Lateral suppressor (Ls) gene of tomato encodes a new member of the VHIID protein family. Proceedings of the National Academy of Sciences, USA 96, 290–295.
Sessions A, Nemhauser JL, McColl A, Roe JL, Feldmann KA, Zambryski PC. 1997. ETTIN patterns the Arabidopsis floral meristem and reproductive organs. Development 124, 4481–4491.[Abstract]
Shimizu-Sato S, Mori H. 2001. Control of outgrowth and dormancy in axillary buds. Plant Physiology 127, 1405–1413.
Simpson CG, Clark G, Davidson D, Smith P, Brown JWS. 1996. Mutation of putative branchpoint consensus sequences in plant introns reduces splicing efficiency. The Plant Journal 9, 369–380.[CrossRef][Medline]
Sorce C, Lorenzi R, Ceccarelli N, Ranalli P. 2000. Changes in free and conjugated IAA during dormancy and sprouting of potato tubers. Australian Journal of Plant Physiology 27, 371–377.
Sussex IM, Kerk NM. 2001. The evolution of plant architecture. Current Opinion in Plant Biology 4, 33–37.[CrossRef][Web of Science][Medline]
Tiwari SB, Hagen G, Guilfoyle T. 2003. The roles of auxin response factor domains in auxin-responsive transcription. The Plant Cell 15, 533–543.
Ulmasov T, Hagen G, Guilfoyle TJ. 1999a. Dimerization and DNA binding of auxin response factors. The Plant Journal 19, 309–319.[CrossRef][Web of Science][Medline]
Ulmasov T, Hagen G, Guilfoyle TJ. 1999b. Activation and repression of transcription by auxin-response factors. Proceedings of the National Academy of Sciences, USA 96, 5844–5849.
Waller F, Furuya M, Nick P. 2002. OsARF1, an auxin response factor from rice, is auxin-regulated and classifies as a primary auxin responsive gene. Plant Molecular Biology 50, 415–424.[CrossRef][Web of Science][Medline]
Wiltshire JJJ, Cobb AH. 1996. A review of the physiology of potato tuber dormancy. Annals of Applied Biology 129, 553–569.
Xu X, van Lammeren AA, Vermeer E, Vreugdenhil D. 1998. The role of gibberellin, abscisic acid and sucrose in the regulation of potato tuber formation in vitro. Plant Physiology 117, 575–584.
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