JXB Advance Access originally published online on September 21, 2006
Journal of Experimental Botany 2006 57(14):3717-3726; doi:10.1093/jxb/erl124
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RESEARCH PAPER |
Tissue-specific expression of tomato Ribonuclease LX during phosphate starvation-induced root growth
Martin Luther University Halle-Wittenberg, Biocenter, D-06120 Halle, Germany
* To whom correspondence should be addressed. E-mail: margret.koeck{at}biozentrum.uni-halle.de
Received 19 April 2006; Accepted 17 July 2006
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Abstract |
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Ribonuclease LX (RNaseLX) from tomato (Solanum lycopersicum L.) belongs to the RNase T2/S-RNase superfamily of plant endoribonucleases and this is a report on the characterization of the RNaseLX gene and its encoded protein as a member of the phosphate starvation response in tomato. RNaseLX gene sequences were cloned by a PCR-assisted approach. RNaseLX promoter sequences contained the conserved binding motif of the transcription factor PHR1 known to mediate phosphate starvation-dependent gene expression. The increase of RNaseLX transcript levels in roots during phosphate starvation correlated with high promoter activity in transgenic plants carrying a PromLX::uidA gene construct and pointed to transcriptional control of RNaseLX expression. Histochemical staining for ß-glucuronidase activity and immunodetection of RNaseLX protein revealed striking RNaseLX expression in main and lateral root tips of phosphate-starved transgenic plants, specifically in epidermal cells, as well as in lateral and adventitious root primordia. Induced RNaseLX expression in roots correlated with stimulated growth and elongation of primary and lateral roots during phosphate deprivation. Phosphate-starvation-induced RNaseLX transcript levels in roots were not modulated by auxin or ethylene. These data indicate that the role of intracellular RNaseLX in the phosphate starvation response is connected with specific RNA turnover processes at the root tip.
Key words: Abscission, phosphate starvation response, PHR motif, promoter, root elongation, tissue specificity, T2-type/S-like RNase, tomato
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Introduction |
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Environmental availability of phosphorus is considered one of the important factors that limit growth in natural ecosystems and in agriculture. Inorganic phosphate (Pi) plays a pivotal role in primary metabolism by modulating enzyme activity, gene transcription, or substrate properties. Maintenance of phosphorus homeostasis inside plants includes uptake, intracellular partitioning, storage, translocation, and phosphate mobilization from diverse substrates. Plants respond to phosphate limitation with different but co-ordinated strategies to conserve phosphate, to reduce metabolic phosphate consumption, and to maximize its acquisition from the rhizosphere (Mimura, 1999; Plaxton and Carswell, 1999; Raghothama, 1999; Vance et al., 2003; Karandashov and Bucher, 2005). To sustain phosphate-requiring processes, plants respond to Pi limitation by scavenging Pi from macromolecules. After activating the respective enzymes plants actively change the membrane lipid composition and replace phospholipids by glycolipids (Essigmann et al., 1998; Frentzen, 2004). Nucleic acids, especially RNAs, are also an important source of Pi in plants and co-ordinated RNA degradation could be part of the plant response to Pi deficiency. Biochemical and developmental adaptations to enhance Pi acquisition from the rhizosphere include (i) induction of transporters and extracellular enzymes, (ii) increased release of organic acids and protons, (iii) association with mycorrhizal fungi, and (iv) remodelling of root system architecture (RSA) and formation of proteoid roots (Harrison, 1999; Raghothama 1999; Williamson et al., 2001; Vance et al., 2003). Changes in the internal and external phosphate availability were found to influence main root growth, lateral root and root-hair formation (Borch et al., 1999; Linkohr et al., 2002; López-Bucio et al., 2002; Ma et al., 2003). This raises questions about the cross-talk between nutritional and hormonal control of root development and their transduction pathways. Involvement of auxin, ethylene, abscisic acid, and nutrients such as nitrate in the regulation of root architecture is well established for Arabidopsis (Casimiro et al., 2003; López-Bucio et al., 2003). In tomato, lateral and adventitious root development is dependent on auxin and ethylene signalling (Clark et al., 1999; Coenen et al., 2003). Although tomato seedlings respond to Pi limitation with a pronounced increase of the root-to-shoot ratio (Bosse and Köck, 1998) the impact of Pi starvation on root architecture and the role of hormones has not yet been investigated. An interesting morphological adaptation in response to Pi and Fe starvation is the formation of transfer cells in the epidermis of tomato roots possessing extensive ingrowths along their outer walls and being enriched in mitochondria and rough ER. Most likely they are engaged in the export or import of ions (Schikora and Schmidt, 2002).
The integration of nutrient-dependent and developmental signals requires a sophisticated regulatory system whose components including potential sensors are just beginning to be elucidated (Abel et al., 2002; Franco-Zorrilla et al., 2004; Ticconi and Abel, 2004). It is thought that many genes of the Pi starvation response such as Pi transporters, phosphatases, and putative riboregulators are controlled transcriptionally. Transcription factors have been characterized such as PSR1 (Wykoff et al., 1999), PHR1 (Phosphate starvation response1, Rubio et al., 2001), and homeodomain-leucine zipper proteins which may control distinct sets of Pi starvation induced (PSI) genes (Franco-Zorrilla et al., 2004). In Arabidopsis, expression of the PSI genes AtIPS1, At4, AtACP5, and AtPt1 was modulated by cytokinin and was dependent on functional cytokinin receptors (Martin et al., 2000; Franco-Zorrilla et al., 2002, 2005).
Being a member of the RNaseT2/S-RNase superfamily (Bariola and Green, 1997; Irie, 1999), the S-like RNaseLX is an RNA-dependent and single strand-specific endonuclease releasing mononucleotides as final reaction products (Löffler et al., 1993; Köck et al., 1995; Abel and Köck, 2001). RNaseLX expression is developmentally regulated and occurs in immature tracheary elements, in senescing leaves and flowers, as well as in seed endosperm during germination, supporting a role for RNaseLX in cell death processes (Lehmann et al., 2001). The increase of RNaseLX transcript levels upon ethylene treatment also points to involvement in senescence processes (Lers et al., 1998). Its intracellular accumulation in the ER is consistent with the presence of an N-terminal signal sequence and a C-terminal ER retention motif (Kaletta et al., 1998; Lehmann et al., 2001). RNaseLX was reported to be induced in cultivated tomato cells by Pi starvation as well as with another RNase (RNaseLE), a phosphodiesterase, and intra- and extracellular phosphatases (Köck et al., 1995; Abel et al., 2000; Stenzel et al., 2003). Its role, however, in tomato plants growing under Pi limitation has not yet been investigated. To get more information about the spatial and temporal expression profile of RNaseLX during Pi starvation, RNaseLX promoter sequences were cloned and a promoter-based histochemical expression analysis was carried out in transgenic tomato plants. Pi-starvation-dependent expression is restricted to the epidermal cell layer of the root tip, to adventitious and lateral root primordia, and is auxin-independent. The role of RNaseLX during Pi starvation is discussed in connection with its tissue- and organ-specific expression, intracellular location of RNaseLX and Pi-starvation-mediated root growth responses.
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Materials and methods |
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Plant material, anthocyanin content, and root growth
Tomato seeds (Solanum lycopersicum L. cv. Lukullus, nomenclature according to Peralta et al., 2005) were sterilized, germinated, and further cultivated in Hoagland solution for 14 d as described by Bosse and Köck (1998). On day 7, nutrient solutions were replaced and, if necessary, respective growth substances were added. Primary root length and distance from the first lateral root to the root tip were determined for each root with a ruler. All lateral roots emerging from the primary root and inspected under the stereomicroscope were taken into account for lateral root number data. Anthocyanin content was determined according to Bariola et al. (1999). Floral buds, flowers, fruits, and roots were collected from soil-grown plants cultivated in a greenhouse.
Cloning of genomic sequences by PCR amplification
Genomic DNA was isolated as described by Stenzel et al. (2003). To clone RNaseLX promoter sequences beneath the 5'-RACE abridged anchor primer 5'-GGCCACGCGTCGACTAGTAC[GGGII]3G-3' (Gibco BRL), the reverse primers LXrev I 5'-biotin-GTGAATATACCTCAGATTCATCCAAAG-3' (transition of exon II–intron II) and LXrev II GCTGGCCACTACAATAATATCTTAA-3' (transition intron I–exon II) were used. Both primers were derived from a cloned genomic DNA fragment of the coding region of RNaseLX containing three introns (pPCRLX, accession number Y17446). A PCR reaction was performed with 1 µg BglII/XbaI digested genomic DNA as template, 0.6 µM primer LXrevI, 200 µM dNTPs, and 5 U Taq polymerase (High fidelity, Roche Diagnostics). The single-strand DNA product synthesized over 40 cycles (annealing temperature 60 °C) was purified using magnetic streptavidin particles according to the 5'-RACE protocol (Gibco BRL). An anchor priming site at the 3'-end of the particle-bound single-strand DNA (dC nucleotide tail) was created using 150 U terminal deoxynucleotidyl transferase, 100 pmol dCTP, 0.75 mM CoCl2 (Roche Diagnostics) at 37 °C for 15 min. After purification of particles with PCR buffer, the complementary second strand was synthesized using the anchor primer complementary to the newly added tail. Afterwards, magnetic particles were removed. The last amplification step consisted of 10 cycles at 60 °C and 20 cycles at 65 °C annealing temperature. The reaction mixture contained 10 mM TRIS-HCl, pH 8.4, 1.5 mM MgCl2, 0.4 µM primer LXrevII and anchor primer, 200 µM dNTPs, 2.5 U Taq DNA polymerase. PCR products were ligated in the cloning vector pCR2.1 (Invitrogen). Positive clones were identified by colony hybridization with a 5'-cDNA probe. Both strands of the clone pPromLX were completely sequenced. The nucleotide sequence data (RNaseLX promoter) appeared in the EMBL Sequence Databases under the accession number AM295794.
RNA extraction and analyses
Isolation of total RNA from tomato plants was carried out using a phenol/chloroform/LiCl precipitation method according to Köck et al. (1998). Twenty µg total RNA per lane were loaded on 1.2% agarose gels, blotted on nylon membranes, and hybridized with 
-32P-dCTP-labelled cDNA probes using standard methods. Tomato TPSI1 and RSI-1 cDNA were cloned from mRNA via reverse transcription and PCR amplification using primers derived from the respective gene sequences (Taylor and Scheuring, 1994; Liu et al., 1997). Equal loading of lanes was controlled by rRNA pattern or hybridizing with a tomato ubiquitin3 probe (Köck et al., 1995). Before reusing the blots, radioactive probes were removed by stripping. Autoradiograms were exposed to X-ray film or analysed with a BAS Imager 1500 (Fuji/Raytest).
Generation of reporter gene construct and transgenic tomato plants
To construct the chimeric RNaseLX promoter-GUS gene (PromLX::uidA), a PCR fragment was amplified on the clone pPromLX using a reverse primer that creates a SmaI restriction endonuclease site at the ATG translation initiation codon and was subcloned into pCR2.1. A XbaI/SmaI restriction fragment containing 814 bp 5'-flanking sequence was ligated blunt end in the SmaI restriction site of the binary vector pBI 101. Correct amplification of promoter sequences was proven by DNA sequencing. Transformation of binary plasmid constructs into A. tumefaciens strain LBA 4404, Agrobacterium-mediated transformation of tomato cotyledons, and the regeneration of transgenic plants was done as described by Köck et al. (2004). Resistant plants were transferred into soil and grown in the greenhouse for seed production. T2 progeny of two lines was used for further experiments.
GUS activity, GUS histochemistry and microscopic analysis
Quantitative measurement of GUS activity was done according to Groß et al. (2004). GUS activity was localized histochemically by incubating plant material in appropriate staining buffers containing 0.5 mM X-Gluc (5-bromo-4-chloro-3-indolyl-ß-D-glucuronide) in the dark at 37 °C for 1–16 h. Tissue sections were fixed in 50 mM sodium phosphate, pH 7.0, 0.3% formaldehyde, 1 mM EDTA for 30 min, and washed with 50 mM sodium phosphate, pH 7.0 prior to staining. Fixation and staining solutions were infiltrated under reduced pressure for 2–5 min. Staining solutions were adapted to different tissues thereby following the protocol of Blume and Grierson (1997). After staining, tissues were fixed in a solution consisting of 45% ethanol, 5% formaldehyde, 5% acetic acid for 2–10 h at 4 °C and stored in 70% ethanol at 4 °C. For further microscopic analyses, sections were dehydrated and embedded in Paraplast (Köck et al., 2004). Eight µm sections were viewed after deparaffinization. Photographs were taken using a high quality digital camera (Canon), a Stemi SV 11 stereomicroscope, or an Axioscop microscope (Zeiss, Jena Germany) equipped with a CCD3 video camera (Sony, Japan).
Protein extraction, immunodetection and RNase activity assays
Following homogenization of cultivated cells (suspension culture; Köck et al., 1998) or roots under liquid N2, total proteins were extracted with 150 mM Na-acetate buffer, pH 5.6, containing 1 mM PMSF and 2 mM EDTA. Soluble proteins were quantified using the Bradford reagent Bioquant (Merck). SDS-PAGE (12.5%) and immunodetection of RNaseLX protein was done according to Lehmann et al. (2001). Quantitative analysis of total RNase activity and in-gel activity assay was done as described in Groß et al. (2004). One RNase unit (U) is defined as the amount of enzyme causing an increase in A260 of 1.0 min–1 cm–1 ml–1 (Abel and Köck, 2001).
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Results |
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RNaseLX is part of the phosphate starvation response in tomato
In cultured tomato cells, the expression of RNaseLX is modulated by availability of inorganic phosphate (Köck et al., 1995). To identify the expression profile in tomato plants, seedlings were grown for 14 d in phosphate-free (–Pi) or phosphate-containing (+Pi) mineral medium. The RNaseLX transcript level increased significantly in roots, hypocotyls, and cotyledons (Fig. 1A). As a control, expression of other Pi-responsive genes from tomato was tested. The putative riboregulator TPSI1 (tomato phosphate starvation induced 1; Liu et al., 1997) and the intracellular phosphatase PS2 (Baldwin et al., 2001; Stenzel et al., 2003) were also expressed both in roots and aerial parts of Pi-starved plants. Phosphate starvation-induced expression of RNaseLE, a paralogue of RNaseLX (Köck et al., 1995), was only detectable in roots and hypocotyls. The down-regulation of transcript levels in phosphate-starved seedlings after replenishment of inorganic phosphate demonstrates the tight regulation of RNaseLX and RNaseLE by phosphate availability (Fig. 1B).
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Isolation and structural analysis of the RNaseLX gene
For functional analysis of the gene, RNaseLX promoter sequences were cloned using a PCR assisted approach adopted from the 5'-RACE technique as described in the Materials and methods. The clone pPromLX has a total insert length of 1209 bp (Fig. 2) and contains a 713 bp putative promoter sequence followed by a 101 bp 5'-untranslated sequence as identified by RNase protection assay. Accordingly, a TATA box was found 5'-upstream, at nucleotides –130 through –124. The clone also comprises 395 bp coding and intron1 sequences (Fig. 2) which are in accordance with the corresponding sequences of the PCR-derived clone pPCRLX and the cDNA clone pRLX01.
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The promoter sequence of the RNaseLX gene was analysed for the presence of putative regulatory elements. Interestingly, the RNaseLX promoter contains a sequence perfectly matching the GNATATNC motif (GCATATTC, –163 through –156, Fig. 2) which is recognized by the transcription factor PHR1 (Rubio et al., 2001).
To complete the structural characterization of the gene, a PCR product was amplified on genomic tomato DNA with primers derived from the 5'- and 3'-ends of the coding sequence of RNaseLX (pRLX01, EMBL Accession No. X79338). Both strands of the resulting clone pPCRLX (EMBL Accession No. Y17446) were completely sequenced showing identity to the nucleotide sequence of the cDNA clone pRLX01 and providing information on the exon–intron structure. Three introns with length of 279 bp, 1451 bp, and 318 bp, respectively, interrupt the coding sequence at nucleotides 108, 264, and 457, respectively, after the translational start (+1).
Tissue-specific RNaseLX promoter activity in roots is regulated by phosphate availability
To investigate the expression of RNaseLX in tomato plants and the impact of Pi starvation on the expression profile in more detail, an RNaseLX promoter–GUS fusion consisting of 814 bp 5'-flanking sequence of the RNaseLX gene ligated in front of the reporter gene uidA (PromLX::uidA) was constructed. The chimeric gene was introduced into the homologous tomato cultivar Lukullus by Agrobacterium-mediated gene transfer. Eighteen independent tomato lines were regenerated and 14 lines showed differential GUS expression in response to phosphate starvation. For further experiments two lines were chosen (T2 generation).
Transgenic tomato seedlings grown in Pi-rich mineral medium for 14 d were histochemically analysed for ß-glucuronidase (GUS) activity. GUS staining, however, was virtually undetectable (Fig. 3A). By contrast, very strong GUS staining was observed in primary root tips and almost all lateral root tips of those plants that were subjected to Pi starvation (Fig. 3B). Adventitious root primordia (Fig. 3B, arrow), early lateral root primordia (Fig. 3D) and outgrowing lateral roots (Fig. 3F, G) expressed high GUS activity. Microscopic analysis of cross-sections through outgrowing lateral roots elucidated that RNaseLX promoter activity was present in epidermal cells (Fig. 3I). GUS activity was highest in the meristematic zone of roots as exemplified in Fig. 3E, but was not detectable directly in the root cap (columella) as revealed by the inspection of numerous roots (see also Fig. 5). GUS activity was present in the vascular tissue of the emerging root as well as at the initiation sites in the main root (Fig. 3F, G, arrows). Microscopic analysis also revealed that RNaseLX promoter activity appeared preferentially in the cell layers of the lateral root being close to the vascular tissue and pericycle cells of the main root (Fig. 3J). Taken together, these data suggest a spatial RNaseLX expression pattern in primary and lateral roots during plant growth under Pi deficiency. Fluorometric GUS assay was used to quantify promoter activity in total roots. GUS activity increased from 0.2 pmol µg–1 protein min–1 in Pi-supplied root to 3–7 pmol µg–1 protein min–1 depending on the transgenic line used, suggesting transcriptional activation of the RNaseLX gene by Pi starvation.
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In aerial plant parts, GUS staining was found in emerging leaves of Pi-starved seedlings, in the vascular tissue of hypocotyls and cotyledons, and especially in the region where the vascular strands turned into leaves (Fig. 3C). Prolonged Pi starvation, however, resulted in the activation of RNaseLX promoter activity in the abscission zones of cotyledons and leaves (Fig 3C, arrow), where GUS staining was detectable in the parenchyma cells of cotyledons and leaf petioles (Fig. 3H, arrows).
RNaseLX expression during Pi starvation correlated with accelerated root growth and was not modulated by auxin and ethylene treatment
Phosphate limitation affects RSA and is known to impact on many aspects of root growth and development. In order to test a possible link between spatial expression of RNaseLX and the Pi starvation-induced response of RSA in tomato, several root-specific growth parameters were determined. Tomato seedlings responded to Pi starvation with increased root growth and reduced shoot growth resulting in a higher root-to-shoot ratio (Table 1). Notably, primary roots (PR) of Pi-starved plants were significantly longer than those of Pi-supplied plants. This effect of Pi starvation was also illustrated by the significantly higher distance between the root tip and the first visible lateral root (LR). Although the total number of LR per –Pi plant was also higher in comparison with +Pi plants, LR frequencies were similar in both variants (Table 1). Accordingly, LR induction was only slightly favored by Pi starvation. The youngest LRs were short and were not able to contribute much to the increased total root mass observed under Pi limitation. These results indicate that Pi starvation of tomato plants influences root elongation rather than LR initiation. Pi starvation-induced RNaseLX expression, especially in primary and lateral root tips, correlated well with accelerated root elongation of Pi-starved tomato plants.
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Analysis of RNaseLX promoter activity revealed activation by Pi starvation and preferential RNaseLX expression in the root tips. To substantiate this result further RNaseLX was analysed at the protein and enzyme activity levels. In Pi-supplied roots, RNaseLX protein was neither detectable in total roots nor in isolated root tips, whereas RNaseLX protein was clearly found in Pi-starved roots (Fig. 4A). RNaseLX protein amount in root tips was conspicuously higher than in total roots (Fig. 4A), indicating preferential accumulation in the root tips. Total RNase activity increased from a low level (0.16 U mg–1 protein min–1) in Pi-supplied roots to 5 U mg–1 protein min–1 in Pi-starved roots. A more detailed analysis using an in-gel RNase activity assay, which differentiates between distinct RNA-degrading enzyme activities, confirmed strong induction of RNaseLX activity in roots during Pi starvation which was accompanied by an increase of RNaseLE activity (Fig. 4B).
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The altered RNaseLX expression and changed root growth parameter following phosphate starvation prompted an investigation into whether Pi-dependent RNaseLX expression in roots can be modulated by treatment with auxin and ethylene which are known to affect root growth and architecture. The tomato RSI-1 gene (root system inducible-1) was used as an auxin-dependent marker gene since (i) treatment with 5 µM naphthyl acetic acid (NAA) led to its rapid induction and (ii) RSI-1 promoter driven GUS activity was detected both in lateral and adventitious root initials (Taylor and Scheuring, 1994) being very similar to the spatial expression of the RNaseLX gene. RSI-1 was expressed at low levels in roots of 14-d-old tomato plants in a Pi starvation-independent manner (Fig. 4C). Auxin (NAA) treatment, however, increased RSI-1 transcript levels both in Pi-supplied and in Pi-starved roots indicating hormone-dependent expression. Consistent with previous results, RNaseLX transcripts were not detectable in Pi-supplied roots. RNaseLX expression was also not found in NAA-treated Pi-supplied roots indicating that auxin is unable to increase RNaseLX transcript levels under normal growth conditions. In addition, treatment with high auxin concentrations did not markedly change Pi starvation-induced RNaseLX and RNaseLE transcript levels (Fig. 4C). Accordingly, application of auxin transport inhibitor TIBA (2,3,5-triiodobenzoic acid) did not influence Pi starvation-induced RNaseLX transcript levels. Application of the ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC) or inhibition of endogenous ethylene production by 2-aminoethoxyvinyl glycine (AVG) neither induced expression under normal condition nor influenced Pi starvation-induced accumulation of RNaseLX transcripts (Fig. 4D). Similarly, the strong increase of phosphatase PS2 transcript levels was not modulated by auxin and ethylene in Pi-starved roots.
RNaseLX expression in mature tomato plants
To determine the impact of developmental factors on the expression of RNaseLX in soil-grown mature tomato plants several PromLX::uidA transgenic lines were analysed for GUS activity. RNaseLX was expressed in lateral root tips, in emerging lateral root initials, and in adventitious root initials (Fig. 5A, C, D) supporting results obtained with 2-week-old plants (Fig. 3). Cross and longitudinal sections through lateral root tips (Fig. 5B) and adventitious root primordia (Fig. 5E), respectively, elucidated that GUS activity was restricted to epidermal cell layers of the emerging root and to the meristematic and elongation zone at the root tip. According to these observations in roots of 14-d-old plants GUS activity could not be detected directly in the root cap. Microscopic inspection gave no indication for expression in root hair cells. Interestingly, promoter-driven GUS activity was found in the stigmas of different flower stages, beginning with small flower buds of about 6 mm, and in mature pollen grains (Fig. 5F, G). Whereas the RNaseLX promoter was not yet active in immature green fruits, GUS activity became detectable in the vasculature during ripening (Fig. 5H, I). Although these results do not rule out other sites of RNaseLX promoter activity, it clearly showed a spatial and temporal expression pattern during plant ontogenesis.
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Discussion |
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It is demonstrated here that ribonuclease LX is part of the Pi starvation response of tomato plants. Down-regulation of RNaseLX expression after Pi replenishment supports previous results obtained with tomato cell cultures (Köck et al., 1998). The increase of transcript levels in roots during Pi starvation correlates with high promoter activity in main and lateral roots as well as LR primordia and points to transcriptional control of RNaseLX expression. The RNaseLX promoter contains an incomplete palindrome which is identical with the PHR1 binding motif in the AtIPS1 promoter (Rubio et al., 2001) suggesting that a tomato transcription factor orthologous to AtPHR1 is involved in the regulation of RNaseLX expression following Pi deficiency. PHR1 acts as a transcriptional activator modulating expression of several PSI genes such as IPS1, At4, and RNS1 (Rubio et al., 2001). Accordingly, the promoter of RNaseLE, the putative AtRNS1 homologue in tomato, carries the motif GAATATAC matching the PHR1 core sequence. So far, the TPSI-1 gene has been the only known gene with a PHR1 motif in tomato. The presence of identical promoter elements in PSI genes from Arabidopsis, Medicago, rice, and tomato (Franco-Zorrilla et al., 2004) are indicative of a common regulation of these genes during phosphate starvation. Recently, it has been reported that PHR1 is a target of AtSIZ1, a SUMO E3 ligase (Miura et al., 2005). AtSIZ1 appears to function in Pi signalling and acquisition upstream of all presently identified modulators, thereby acting negatively as in the case of PHR1 or positively on other Pi deficiency responses (Miura et al., 2005).
PSI phosphate transporters of the Pht1 gene family have been the only genes analysed for their spatial expression in Pi-starved tomato roots. Pht1 expression was detected preferentially in root epidermis, in root hairs or in vascular tissue (Daram et al., 1998; Liu et al., 1998). Therefore, Pi starvation-induced expression of RNaseLX in lateral and adventitious root primordia before emergence of LRs from parent tissue was unexpected and has, to our knowledge, not yet been reported for any other PSI gene. RNaseLX shares spatial expression in the main root elongation zone with the PHR1-regulated AtIPS1 gene (Martin et al., 2000). Whether both genes are functionally related has to be analysed further.
Although RNaseLX was expressed in root tip epidermis directly at the root–soil interface, its intracellular accumulation (Lehmann et al., 2001) argues against a role in Pi rescue from the rhizosphere. On the other hand, spatial RNaseLX expression in Pi-starved root tips correlates with induced root growth. It may indicate that the role of RNaseLX is connected with specific RNA turnover or metabolite recycling supporting root growth. Root apices being metabolically very active are dependent at least to some degree on local nutrient supply. Due to lack of maturity of the xylem in this zone they are isolated from the rest of the root (Melchior and Steudle, 1993).
Comparison of Pi deprivation-triggered RSA responses of different plant species indicated that nutrient utilization differs between them. Unlike tomato, common bean responded to Pi deficiency with reduced root mass and lateral root number but left main root length unchanged (Borch et al., 1999). Nacry et al. (2005) identified two different temporal phases in the Pi starvation response of the Arabidopsis root system, changing from increased LR formation and elongation in response to low Pi accompanied by inhibition of PR elongation (Williamson et al., 2001; López-Bucio et al., 2002) to negative effects on all root parameters in later stages. Pi starvation in tomato acts on the existing roots by accelerating PR growth and LR elongation which explains at least partly the auxin insensitivity of PSI expression. Using PSI GUS activity as a marker, it was possible to detect many lateral primordia in Pi-deficient tomato roots which were arrested in an early developmental stage. Similarly, low Pi availability also had contrasting effects on various stages of LR development in Arabidopsis, with a marked inhibition of primordia initiation but a strong stimulation of initiated primordia (Nacry et al., 2005).
Results on RNaseLX promoter activity in the vasculature of tomato fruits are consistent with data on RNaseLX expression during xylem differentiation (Lehmann et al., 2001). Both phloem tissue and xylem vessels exist throughout the fruit reflecting the important role in feeding embryos and the expanding fruit (Gillaspy et al., 1993). The prompt increase of RNaseLX promoter activity at the start of fruit ripening indicates that ethylene could be involved in RNaseLX expression. Interestingly, the sequence motif AATTCAAA in the RNaseLX promoter (sequence –678 through –670; Fig. 2) has similarity to an ethylene responsive element involved in the expression of the carnation GSTI gene (Itzhaki et al., 1994) and the tomato E4 gene (Montgomery et al., 1993). RNaseLX transcript levels increased during natural senescence and were induced in non-senescent leaves by ethylene (Lers et al., 1998) supporting the importance of an ethylene-responsive element in the RNaseLX promoter. In addition to its involvement in senescence, germination, and xylem differentiation, local GUS expression in leaf and cotyledon abscission zones (Fig. 3C, H) points out to a role of RNaseLX in another cell death-related process (van Doorn and Stead, 1997). Nucleic acids and various cellular materials are degraded during PCD processes and metabolites are reabsorbed by adjoining cells and tissues or are transported to sink organs. Studies on knock-out plants will help to find out to what extent RNaseLX contributes to the recycling of nutrients.
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Acknowledgements |
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We thank Karin Lehmann for GUS analysis of mature plants and Karin Klar for technical assistance. We are grateful to Claus Wasternack for critical reading and helpful comments on the manuscript. The work was supported by a grant to Margret Köck from Deutsche Forschungsgemeinschaft (SFB 363).
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Footnotes |
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Present address: University of Göttingen, Albrecht-von-Haller-Institute for Plant Sciences, Department for Plant Biochemistry, D-37077 Göttingen, Germany. 
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References |
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Abel S and Köck M. (2001) Secretory acid ribonucleases from tomato (Lycopersicon esculentum Mill.). In Nicholson AW (Ed.). Ribonucleases, methods in enzymology(Academic Press, New York) Vol. 341: pp. 351–368.
Abel S, Nürnberger T, Ahnert V, Krauss G-J, Glund K. (2000) Induction of an extracellular cyclic nucleotide phosphodiesterase as an accessory ribonucleolytic activity during phosphate starvation of cultured tomato cells. Plant Physiology 122:543–552.
Abel S, Ticconi CA, Delatorre CA. (2002) Phosphate sensing in higher plants. Physiologia Plantarum 115:1–8.[CrossRef][Medline]
Baldwin JC, Karthikeyan AS, Raghothama KG. (2001) LePS2, a phosphorus starvation-induced novel acid phosphatase from tomato. Plant Physiology 125:728–737.
Bariola PA and Green PJ. (1997) Plant ribonucleases. In D'Alessio G and Riordan JF (Eds.). Ribonucleases: structures and functions(Academic Press, New York) pp. 163–190.
Bariola PA, MacIntosh GC, Green PJ. (1999) Regulation of S-like ribonuclease levels in Arabidopsis. Antisense inhibition of RNS1 or RNS2 elevates anthocyanin accumulation. Plant Physiology 119:331–342.
Blume B and Grierson D. (1997) Expression of ACC oxidase promoter–GUS fusions in tomato and Nicotiana plumbaginifolia regulated by developmental and environmental stimuli. The Plant Journal 12:731–746.[CrossRef][Web of Science][Medline]
Borch K, Bouma TJ, Lynch JP, Brown KM. (1999) Ethylene: a regulator of root architectural responses to soil phosphorus availability. Plant, Cell and Environment 22:425–431.[CrossRef]
Bosse D and Köck M. (1998) Influence of phosphate starvation on phosphohydrolases during development of tomato seedlings. Plant, Cell and Environment 21:325–332.[CrossRef]
Casimiro I, Beeckman T, Graham N, Bhalerao R, Zhang H, Casero P, Sandberg G, Bennett MJ. (2003) Dissecting Arabidopsis lateral root development. Trends in Plant Science 8:165–171.[CrossRef][Web of Science][Medline]
Clark DG, Gubrium EK, Barrett JE, Nell TA, Klee HJ. (1999) Root formation in ethylene-insensitive plants. Plant Physiology 121:53–59.
Coenen C, Christian M, Lüthen H, Lomax TL. (2003) Cytokinin inhibits a subset of diageotropica-dependent primary auxin responses in tomato. Plant Physiology 131:1692–1704.
Daram P, Brunner S, Persson BL, Amrhein N, Bucher M. (1998) Functional analysis and cell-specific expression of a phosphate transporter from tomato. Planta 206:225–233.[CrossRef][Web of Science][Medline]
Essigmann B, Güler S, Narang RA, Linke D, Benning C. (1998) Phosphate availability affects the thylakoid lipid composition and the expression of SQD1, a gene required for sulfolipid biosynthesis in Arabidopsis thaliana. Proceedings of the National Academy of Sciences, USA 95:1950–1955.
Franco-Zorrilla JM, Martin AC, Solano R, Rubio V, Leyva A, Paz-Ares J. (2002) Mutations at CRE1 impair cytokinin-induced repression of phosphate starvation responses in Arabidopsis. The Plant Journal 32:353–360.[CrossRef][Web of Science][Medline]
Franco-Zorrilla JM, Martin AC, Leyva A, Paz-Ares J. (2005) Interaction between phosphate-starvation, sugar, and cytokinin signaling in Arabidopsis and the roles of cytokinin receptors CRE1/AHK4 and AHK3. Plant Physiology 138:847–857.
Franco-Zorrilla JM, González E, Bustos R, Linhares F, Leyva A, Paz-Ares J. (2004) The transcriptional control of plant responses to phosphate limitation. Journal of Experimental Botany 55:285–293.
Frentzen M. (2004) Phosphatidylglycerol and sulfoquinovosyldiacylglycerol: anionic membrane lipids and phosphate regulation. Current Opinion in Plant Biology 7:270–276.[CrossRef][Web of Science][Medline]
Gillaspy G, Ben-David H, Gruissem W. (1993) Fruits: a developmental perspective. The Plant Cell 5:1439–1451.
Groß N, Wasternack C, Köck M. (2004) Wound-induced RNaseLE expression is jasmonate and systemin independent and occurs only locally in tomato (Lycopersicon esculentum cv. Lukullus). Phytochemistry 65:1343–1350.[CrossRef][Web of Science][Medline]
Harrison MJ. (1999) Molecular and cellular aspects of the arbuscular mycorrhizal symbiosis. Annual Review of Plant Physiology and Molecular Plant Physiology 50:361–389.
Irie M. (1999) Structure–function relationship of acid ribonucleases: lysosomal, vacuolar, and periplasmic enzymes. Pharmacology and Therapeutics 81:77–89.
Itzhaki H, Maxson JM, Woodson WR. (1994) An ethylene-responsive enhancer element is involved in the senescence-related expression of the carnation glutathione-S-transferase (GSTI) gene. Proceedings of the National Academy of Sciences, USA 90:8925–8929.
Kaletta K, Kunze I, Kunze G, Köck M. (1998) The peptide HDEF as a new retention signal is necessary and sufficient to direct proteins to the endoplasmic reticulum. FEBS Letters 434:377–381.[CrossRef][Web of Science][Medline]
Karandashov V and Bucher M. (2005) Symbiotic phosphate transport in arbuscular mycorrhizas. Trends in Plant Science 10:22–29.[CrossRef][Web of Science][Medline]
Köck M, Löffler A, Abel S, Glund K. (1995) Structural and regulatory properties of a family of starvation induced ribonucleases from tomato. Plant Molecular Biology 27:477–485.[CrossRef][Web of Science][Medline]
Köck M, Theierl K, Stenzel I, Glund K. (1998) Extracellular administration of phosphate sequestering metabolites induces ribonucleases in cultured tomato cells. Planta 204:404–407.[CrossRef][Web of Science]
Köck M, Groß N, Stenzel I, Hause G. (2004) Phloem-specific expression of the wound-inducible ribonuclease LE from tomato (Lycopersicon esculentum cv. Lukullus). Planta 219:233–242.[CrossRef][Web of Science][Medline]
Lehmann K, Hause B, Altmann D, Köck M. (2001) Tomato ribonuclease LX with the functional ER retention motif HDEF is expressed during physiological cell death processes including xylem differentiation, germination and senescence. Plant Physiology 127:436–449.
Lers A, Khalchitski A, Lomaniec E, Burd S, Green PJ. (1998) Senescence-induced RNases in tomato. Plant Molecular Biology 36:439–449.[CrossRef][Web of Science][Medline]
Linkohr BI, Williamson LC, Fitter AH, Leyser OHM. (2002) Nitrate and phosphate availability and distribution have different effects on root system architecture of Arabidopsis. The Plant Journal 29:751–760.[CrossRef][Web of Science][Medline]
Liu C, Muchhal US, Raghothama KG. (1997) Differential expression of TPSI1, a phosphate starvation-induced gene in tomato. Plant Molecular Biology 33:867–874.[CrossRef][Web of Science][Medline]
Liu C, Muchhal US, Uthappa M, Kononowicz AK, Raghothama KG. (1998) Tomato phosphate transporter genes are differentially regulated in plant tissues by phosphorus. Plant Physiology 116:91–99.
Löffler A, Glund K, Irie M. (1993) Amino acid sequence of an intracellular, phosphate-starvation-induced ribonuclease from cultured tomato (Lycopersicon esculentum) cells. European Journal of Biochemistry 214:627–633.[Web of Science][Medline]
López-Bucio J, Hernández-Abreu E, Sánchez-Calderón L, Nieto-Jacobo MF, Simpson J, Herrera-Estrella L. (2002) Phosphate availability alters architecture and causes changes in hormone sensitivity in the Arabidopsis root system. Plant Physiology 129:244–256.
López-Bucio J, Cruz-Ramírez A, Herrera-Estrella L. (2003) The role of nutrient availability in regulating root architecture. Current Opinion in Plant Biology 6:280–287.[CrossRef][Web of Science][Medline]
Ma Z, Baskin TI, Brown KM, Lynch JP. (2003) Regulation of root elongation under phosphorus stress involves changes in ethylene responsiveness. Plant Physiology 131:1381–1390.
Martin AC, del Pozo JC, Iglesias J, Rubio V, Solano R, de la Pena A, Leyva A, Paz-Ares J. (2000) Influence of cytokinins on the expression of phosphate starvation responsive genes in Arabidopsis. The Plant Journal 24:559–567.[CrossRef][Web of Science][Medline]
Melchior W and Steudle E. (1993) Water transport in onion (Allium cepa L.) roots (changes of axial and radial hydraulic conductivities during root development). Plant Physiology 101:1305–1315.[Abstract]
Mimura T. (1999) Regulation of phosphate transport and homeostasis in plant cells. International Review of Cytology 191:149–200.
Miura K, Rus A, Sharkhuu A, et al. (2005) The Arabidopsis SUMO E3 ligase SIZ1 controls phosphate deficiency responses. Proceedings of the National Academy of Sciences, USA 102:7760–7765.
Montgomery J, Goldman S, Deikman J, Margossian L, Fischer RL. (1993) Identification of an ethylene-responsive region in the promoter of a fruit ripening gene. Proceedings of the National Academy of Sciences, USA 90:5939–5943.
Nacry P, Canivenc G, Muller B, Azmi A, Van Onckelen H, Rossignol M, Doumas P. (2005) A role for auxin redistribution in the responses of the root system architecture to phosphate starvation in Arabidopsis. Plant Physiology 138:2061–2074.
Peralta IE, Knapp S, Spooner DM. (2005) New species of wild tomatoes (Solanum section Lycopersicon: Solanaceae) from Northern Peru. Systematic Botany 30:424–434.[CrossRef][Web of Science]
Plaxton WC and Carswell CM. (1999) Metabolic aspects of the phosphate starvation response in plants. In Lerner H (Ed.). Plant responses to environmental stresses: from phytohormones to genome reorganization(Marcel Dekker, New York) pp. 349–372.
Raghothama KG. (1999) Phosphate acquisition. Annual Review of Plant Physiology and Plant Molecular Biology 50:665–693.[CrossRef][Web of Science]
Rubio V, Linhares F, Solano R, Martin AC, Iglesias J, Leyva A, Paz-Ares J. (2001) A conserved MYB transcription factor involved in phosphate starvation signaling both in vascular plants and in unicellular algae. Genes and Development 15:2122–2133.
Schikora A and Schmidt W. (2002) Formation of transfer cells and H+-ATPase expression in tomato roots under P and Fe deficiency. Planta 215:304–311.[CrossRef][Web of Science][Medline]
Stenzel I, Ziethe K, Schurath J, Hertel SC, Bosse D, Köck M. (2003) Differential expression of the LePS2 phosphatase gene family in response to phosphate availability, pathogen infection and during development. Physiologia Plantarum 118:138–146.[CrossRef][Medline]
Taylor BH and Scheuring CF. (1994) A molecular marker for lateral root initiation: the RSI-1 gene of tomato (Lycopersicon esculentum Mill.) is activated in early lateral root primordia. Molecular and General Genetics 243:148–157.
Ticconi CA and Abel S. (2004) Short on phosphate: plant surveillance and countermeasures. Trends in Plant Science 9:548–555.[CrossRef][Web of Science][Medline]
van Doorn WG and Stead AD. (1997) Abscission of flowers and floral parts. Journal of Experimental Botany 48:821–837.
Vance CP, Uhde-Stone C, Allan DL. (2003) Phosphorus acquisition and use: critical adaptations by plants for securing a nonrenewable resource. New Phytologist 157:423–447.[CrossRef][Web of Science]
Williamson L, Ribrioux S, Fitter A, Leyser O. (2001) Phosphate availability regulates root system architecture in Arabidopsis. Plant Physiology 126:1–8.
Wykoff DD, Grossman AR, Weeks DP, Usuda H, Shimogawara K. (1999) Psr1, a nuclear localized protein that regulates phosphorus metabolism in Chlamydomonas. Proceedings of the National Academy of Sciences, USA 96:15336–15341.
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