JXB Advance Access originally published online on October 20, 2007
Journal of Experimental Botany 2007 58(13):3797-3810; doi:10.1093/jxb/erm236
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RESEARCH PAPER |
Involvement of hormones and KNOXI genes in early Arabidopsis seedling development
emysl Sou
ek1,2etislav Brzobohat
1,2,*
1Institute of Biophysics, Academy of Sciences of the Czech Republic v.v.i., Královopolská 135, CZ-61265 Brno, Czech Republic
2Department of Molecular Biology and Radiobiology, Faculty of Agronomy, Mendel University of Agriculture and Forestry, Zem
dlská 1, CZ-61300 Brno, Czech Republic
* To whom correspondence should be addressed. E-mail: brzoboha{at}ibp.cz
Received 18 May 2007; Revised 22 August 2007 Accepted 30 August 2007
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Abstract |
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Plant hormones control plant development by modulating the expression of regulatory genes, including homeobox-containing KNOXI genes. However, much remains to be elucidated about the interactions involved. Therefore, hormonal regulation of KNOXI gene expression was investigated using hormone applications and an inducible transgenic ipt expression system to increase endogenous cytokinin (CK) levels. Treatments with auxin, abscisic acid (ABA), cytokinins, ethylene, and gibberellin (GA) did not result in ectopic expression of the BP (BREVIPEDICELLUS) gene. However, BP expression was strongly reduced by ABA, increased by auxin treatment (correlating with the initiation of lateral root meristems, which strongly express BP), and did not significantly respond to short-term treatments with the other hormones in whole seedlings. Following short-term ipt activation, organ-specific differential regulation of KNOXI gene expression was observed. While several KNOXI genes were transiently up-regulated to low levels, STM was selectively repressed (especially at low light) in hypocotyls. In cotyledons, activation of CK-responsive genes preceded ipt induction, suggesting that CKs are transported more rapidly than the inducing agent (dexamethasone). Long-term increases in CK levels induced raised levels of several KNOXI transcripts in hypocotyls, correlating with the radial expansion of vascular tissues, the main domains of KNOXI gene expression, suggesting that CKs had little effect on KNOXI promoter activity. No alterations in hormone sensitivity were observed in a bp null mutant. Constitutive BP overexpression caused reductions in the length and number of lateral roots, while the primary root remained unaffected. The transgenic seedlings displayed weak, but significant, alterations in sensitivity to ABA, CK, and 1-amino-cyclopropane-1-carboxylic acid.
Key words: Arabidopsis thaliana, KNOX genes, plant hormones, pOp/LhGR system, regulation of gene expression
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionSupplementary materialReferences |
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Homeodomain proteins were first discovered in Drosophila (Gehring et al., 1994) during investigations of homeotic transformations of the fly's body plan. Since then, a number of homeodomain proteins have been found in animals, plants, and yeasts. Plant homeodomain proteins participate as transcription factors in the regulation of a number of developmental processes by activating and/or repressing sets of target genes (Chan et al., 1998; Reiser et al., 2000). The first homeobox gene identified in plants was KNOTTED1 (KN1) from maize (Vollbrecht et al., 1991). In Arabidopsis thaliana, the KNOTTED1-like homeobox (KNOX) gene family consists of eight KN1 homologues, of which STM (SHOOT MERISTEMLESS), BP (BREVIPEDICELLUS), KNAT2, and KNAT6 are KNOXI genes, while KNAT3, KNAT4, KNAT5, and KNAT7 are KNOXII genes. KNOXI genes are expressed mainly in the shoot apical meristem (SAM; Lincoln et al., 1994; Long et al., 1996; Laufs et al., 1998; Nishimura et al., 1999) and are required for SAM maintenance, and thus for the establishment of shoot architecture, as inferred mainly from analyses of loss-of-function mutants. In Arabidopsis, stm mutants have displayed complete or partial absence of the SAM, and consequent reductions in leaf and flower numbers, together with fused cotyledons (Barton and Poethig, 1993; Clark et al., 1996; Endrizzi et al., 1996). The roles of other KNOXI genes in the SAM have not been fully characterized. However, the roles of BP and STM seem to overlap at least partly, since an active BP allele can restore SAM maintenance in stm as1 (asymmetric leaves1) plants (Byrne et al., 2002). Failure to down-regulate KNOXI gene expression in leaf primordia and mature leaves can cause abnormal leaf development (Ori et al., 2000; Byrne et al., 2003). In addition to their function(s) in the SAM, there is also growing evidence implicating the involvement of KNOXI genes in defining inflorescence architecture (Lincoln et al., 1994; Douglas et al., 2002; Venglat et al., 2002).
Recently, several indications that KNOXI genes have functions in roots have emerged. Notably, it has been reported that BP (Truernit et al., 2006), maize RS1 and KNOX4 (Kerstetter et al., 1994), and Arabidopsis KNAT2 (Hamant et al., 2002) and KNAT6 (Dean et al., 2004) are all expressed in roots. KNAT6 expression appears to be localized in the phloem of the primary root and the bases of lateral roots, while BP expression has been detected mainly at the bases of lateral root primordia. Potential functions of KNOXI genes in lateral root initiation have also been inferred from increases in the numbers of lateral root primordia caused by the down-regulation of KNAT6 transcript levels (Dean et al., 2004).
Interest in interactions between KNOXI genes and plant hormones has been strongly stimulated by recent research on their co-ordinated involvement in balancing SAM maintenance and organ production. For instance, KNOXI genes have been found to be down-regulated in a SAM region in which highly localized increases in auxin levels occur that are essential for initiation of leaf primordia. Perturbations of the auxin pattern caused by either auxin transport inhibitors (Scanlon, 2003) or mutations in genes encoding the auxin transport facilitators PIN-FORMED1 and PINOID (Furutani et al., 2004) resulted in defects in lateral organ formation. A possible feedback relationship between KNOX proteins and auxin is indicated by data suggesting that KNOX proteins may also inhibit auxin transport (Tsiantis et al., 1999; Treml et al., 2005). Kuijt et al. (2004) suggested that auxin may regulate the distribution of KNOXI proteins between the cytoplasm and nucleus.
No data on interactions between KNOXI genes and abscisic acid (ABA) have been reported. However, KNAT2 and ethylene have been shown to act antagonistically in the regulation of leaf structure and SAM architecture in Arabidopsis (Hamant et al., 2002), and numerous, often conflicting, reports on interactions between KNOXI genes and cytokinins (CKs) have accumulated over the last 16 years. The overexpression of ipt, a bacterial gene encoding a key enzyme of CK biosynthesis—isopentenyltransferase—confers a phenotypic syndrome very similar to that of KNOXI overexpression in tobacco, including ectopic shoot meristem formation on leaves (Estruch et al., 1991; Sinha et al., 1993). On the other hand, alterations in levels of several plant hormones, including elevation of CK levels, have been reported in transgenic plants constitutively and ectopically expressing KN1 homologues from tobacco and rice (Tamaoki et al., 1997; Kusaba et al., 1998). These findings do not provide conclusive proof that CK levels are regulated by KNOX genes, due to the constitutive nature of KN1 expression. However, two independent groups have recently obtained direct experimental evidence that CKs are involved in mediating control of the SAM by KNOXI genes (Jasinski et al., 2005; Yanai et al., 2005). Activation of several different KNOXI proteins using an inducible system resulted in increases in CKs due to selective up-regulation of AtIPT7, a gene encoding a genuine Arabidopsis isopentenyltransferase (Kakimoto, 2001; Takei et al., 2001a). Furthermore, KNOXI proteins have been shown to suppress directly the expression of a key gene of gibberellin (GA) biosynthesis, in the tobacco SAM (Sakamoto et al., 2001). In addition, work by Hay et al. (2002) led to a suggestion that repression of GA activity by KNOXI proteins is a key component of SAM function.
Overall, the published evidence has prompted the hypothesis that KNOXI proteins act as general orchestrators of growth regulator homeostasis at the shoot apex of Arabidopsis by simultaneously activating CK and repressing GA biosynthesis, thus promoting meristem activity (Jasinski et al., 2005; Yanai et al., 2005). However, an opposite link between CKs and KNOXI genes has been proposed based on the finding that steady-state mRNA levels of STM and BP increased in response to activation of ipt expression in Arabidopsis seedlings (Rupp et al., 1999). The model proposed by the cited authors positioned CKs upstream of BP and STM in the regulation of SAM establishment and/or maintenance. Taken together, the data obtained from experiments in which KNOXI protein activity and ipt expression have been induced indicate that expression of KNOX gene family members can lead to increases in CK biosynthesis and, in turn, that these genes are up-regulated by CKs, suggesting a positive interdependence between CK and the KNOX family of meristem maintenance genes. However, the proposed feedback loop has been questioned recently based on the finding that BP and STM were not significantly increased within 48 h of ipt induction in 14-d-old Arabidopsis seedlings (Craft et al., 2005) and, in genome-wide analysis, neither BP nor STM was induced in young seedlings that were either expressing ipt (Hoth et al., 2003) or treated with exogenous cytokinin for 24 h (Rashotte et al., 2003). Nevertheless, transient increases were observed in BP and KNAT4 transcripts 6 h after ipt induction in young Arabidopsis seedlings by Hoth et al. (2003). This left open the possibility that KNOX gene expression may be highly specifically temporally and spatially regulated by CKs. Further, subtle changes in KNOX gene transcript levels may not have been detected if they did not exceed the thresholds applied in the genome-wide analysis.
In the study presented here, changes in steady-state levels of BP, KNAT2, KNAT6, and STM transcripts in response to both short- and long-term CK treatments were re-examined by quantitative (Q) RT-PCR (reverse transcription-PCR) in individual organs and apical regions of Arabidopsis seedlings cultivated at various light intensities. Combinations of several CK and light treatments were included since some aspects of plant development are influenced by both CKs and light (Su and Howell, 1995; A Reková et al., personal communication), and evidence of a link between light and KNOXII expression has been presented (Serikawa et al., 1997). The spatial regulation of BP expression by CKs was analysed in detail using a BP promoter–uidA fusion construction and in situ mRNA localization, and the effects of CKs were compared with those of other plant hormones. The sensitivity of the Arabidopsis bp mutant and a transgenic line constitutively overexpressing BP to CKs and other plant hormones was also examined. In addition, novel information was obtained on the kinetics of transgene activation in individual Arabidopsis organs using a dexamethasone (DEX)-inducible gene expression system, pOp/LhGR (Craft et al., 2005; 
ámalová et al., 2005), together with evidence supporting the view that CK is a long-range rather than a paracrine signal.
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Materials and methods |
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Plant material and growth conditions
Arabidopsis thaliana ecotype Col-0 was obtained from the European Arabidopsis Stock centre. pBP-GUS (β-glucuronidase) transgenic plants (lines K3, K4, K9, K14, and K15) and CaMV35S>GR>ipt plants expressing agrobacterial ipt via the glucocorticoid-inducible system (Craft et al., 2005; lines 11 and 13) were kindly provided by Sarah Hake and Ian Moore, respectively. Both of the glucocorticoid-inducible lines (11 and 13) were used to analyse phenotypic changes following DEX applications, and the stronger line (11) was used for determination of transcript levels. 35S-BP (ecotype Nossen), brevipedicellus (bp) mutant (ecotype Ler), and control lines were obtained from the European Arabidopsis Stock centre. Plants were grown in vertically oriented square Petri dishes on Murashige and Skoog (MS) medium (Duchefa) supplemented with 1% sucrose and 1.2% agar at a constant distance from a fluorescent lamp, in a growth chamber (Percival CU36L5, Percival Scientific) under long day conditions (16 h light, 21 °C/8 h dark, 19 °C). Unless stated otherwise, the light intensity was 82 µmol m–2 s–1. Hormones and DEX were added directly to the cultivation medium at 50°C in appropriate concentrations in an identical amount of solvent to final concentrations of 0.01% dimethylsulphoxide (DMSO; for hormones and hormone precursors) or 0.0025% ethanol (for DEX). Control MS medium contained only solvents at equal concentrations. From 30 to 60 seedlings were used for measurements of morphological characteristics and statistical analysis. The significance of morphological differences between different plants was evaluated by t-tests (deeming differences to be significant if P <0.05).
Histochemical analysis of GUS activity
To minimize the possibility of positional effects of transgenic insertions influencing the obtained results, five independent lines of pBP-GUS, named K3, K4, K9, K14, and K15, were used. Intact plants were vacuum infiltrated with a substrate solution containing 0.1 M phosphate buffer (pH 7.0), 1% Triton X-100, 5 mM potassium ferrocyanide, 5 mM potassium ferricyanide, and 0.1% 5-bromo-4-chloro-3-indolyl-β-D-glucoronide (X-gluc), then incubated at 37 °C for 2, 5, or 20 h. Chlorophyll was removed from the plants' tissues using 75% ethanol and they were cleared by immersion in 0.25 M HCl and 20% methanol at 57 °C. The plants were then brought to 50% glycerol in a graded series, and their tissues were photographed using an Olympus BX61 microscope fitted with an Olympus DP50 digital camera. Consistent results were obtained for the five lines, and images obtained from plants of line K14 are presented.
In situ hybridization
The 644 bp long fragment from the 5' end of BP cDNA was used to synthesize sense and antisense probes. The cDNA clone was synthesized using the specific primers: fBPish (5'-CTC TAC TAG TGC CTT TTT GAT TTC ATA GAT-3') and rBPish (5'-GCA TAC TAG TTC AAT GAT TTT AGC CTT C-3'), and incorporated into the SpeI site of pBluescript SK–. The specificity and orientation of the inserts were verified by sequencing. The sense and antisense RNA probes were labelled with digoxigenin-11-UTP using T3 and T7 polymerase (Roche) and the hybridized probes were detected using anti-digoxigenin antibody conjugated to alkaline phosphatase (Roche). Sections 5 µm thick were cut after tissues had been fixed in 4% formaldehyde and embedded in ParaplastPlus (Fisher Scientific). Pre-treatment of the sections and in situ hybridization were performed according to Lincoln et al. (1994). Very slight non-specific staining was detected in leaves, with both sense and antisense probes.
Quantitative RT-PCR and data analysis
To perform RT-PCR, total RNA was isolated from plant tissues frozen in liquid nitrogen using TRIzol reagent (Invitrogen) according to the manufacturer's instructions. Duplicates of RNA samples were isolated and analysed in all but a few cases, including the following types of samples from seedlings in which ipt was activated throughout the whole cultivation period: hypocotyls (5 µmol m–2 s–1/200 nM DEX; 56 µmol m–2 s–1/200 nM DEX) and apices (5 µmol m–2 s–1/200 nM DEX and 5 µmol m–2 s–1/500 nM DEX). RNA samples were treated with DNase I (Top-Bio, Czech Republic) to digest contaminating DNA, and the efficiency of the DNase treatment was checked by PCR amplification of actin, using non-transcribed RNA as a template. First-strand cDNA was prepared using SuperScript II reverse transcriptase (Invitrogen) and the oligo(dT) (Hradilová et al., 2007) primer according to the manufacturer's instructions. Each cDNA preparation was subsequently subjected, in triplicate, to Q RT-PCR using a RotorGene real-time analyser (Corbett Research) with SYBR Green I as a fluorescent dye to quantify DNA. To amplify gene-specific products, the following primers were used: ACTfwd (5'-GGT GAT GGT GTG TCT-3'), ACTrev (5'-ACT GAG CAC AAT GTT AC-3') (Szyroki et al, 2001), fIPTrt (5'-ATC CTC CCT CAA GAA TAA GC-3'), rIPTrt (5'-CTG AAA GGA ACG ACG C-3'), fARR5rt (5'-GCT GAT AGA ACC AAG ACT GA-3'), rARR5rt (5'-CTT CCA AAA TAA CAC ACC AC-3'), fKBPrt (5'-GAG AAT TGC TTC CGA TCT G-3'), rBPrt (5'-ATG GCT TCA ACA TCG CTT AC-3'), fKNAT2rt (5'-GGG CGT AAC GAG ACA-3'), rKNAT2rt (5'-TCT ACC CGA ATG GAT AAT GA-3'), fKNAT3rt (5'-AGG TGA CAA TAG CGG AAG A-3'), rKNAT3rt (5'-GGG CAT AAT CAC ATA ACC AA-3'), fKNAT4rt (5'-CCA GTA CAT GGT ATG GCT TT-3'), rKNAT4rt (5'-TGG CAG TGA AGA CTG ACG AT-3'), fKNAT6rt (5'-TGT TTC CTT CCG ATT ACC AA-3'), rKNAT6rt (5'-GCG GAG ACG GCT ACT GAG-3'), fSTMrt2 (5'-GGA TAA TAG TGA TGG TCC GA-3'), and rSTMrt2 (5'-GGG GAG GAG CTA GTG ATT GA-3'). The steady-state levels of transcripts were quantified using standard curve quantitation, and all expression levels were normalized to actin. Results are shown as means of biological replicates and the corresponding standard deviations, except for the non-duplicated samples specified above. The significance of differences between activated and non-activated plants was evaluated by one-way ANOVA (P <0.05). Correlations between BP expression and morphological data were evaluated by calculating Pearson correlation coefficients.
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Results |
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BP expression patterns in Arabidopsis seedlings
Transgenic lines harbouring the uidA gene driven by the BP promoter (pBP-GUS; Ori et al., 2000) were used to obtain the first insights into the hormonal regulation of BP expression in Arabidopsis seedlings. Whole-mount histochemical analysis of young seedlings grown on MS indicated that BP was strongly expressed in the shoot apex. GUS staining and in situ mRNA hybridization of longitudinal sections of the shoot apex visualized BP expression in the subapical region of the shoot and the bases of mature leaves as well as leaf primordia (Fig. 1). GUS staining was found in vascular tissues of hypocotyls and roots in whole-mount preparations and hypocotyl cross-sections (Fig. 1D, M, Q). Interestingly, strong BP expression was also observed throughout the course of lateral root development. At the one-cell layer stage, GUS staining was detected in all cells of developing primordia and the nearest cells of the pericycle within 2 h of staining. In two- and three-cell layer primordia, the staining was distributed in all cells, while at later stages it became restricted to the lateral root base. In fully developed lateral roots, BP expression was restricted to a peripheral ring of cells at their bases or, in some cases, was not detected at all (Supplementary Fig. S1 available at JXB online). The expression pattern revealed by histochemical analysis was confirmed by in situ mRNA hybridization (Fig. 1O, P, and Supplementary Fig. S1E, F at JXB online).
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Hormonal regulation of BP expression patterns
To assess the effects of key plant hormones on BP expression, 6-d-old pBP-GUS seedlings were transferred to liquid medium supplemented with: the gibberellin GA3 (10 µM and 100 µM); the auxins 2,4-diphenoxyacetic acid (2,4-D; 1 µM and 10 µM) and naphthalene acetic acid (NAA; 1 µM and 10 µM); the ethylene precursor 1-amino-cyclopropane-1-carboxylic acid (ACC; 1 µM); the cytokinins benzyladenine (BA; 10 µM and 100 µM), trans-zeatin (t-Z; 10 µM and 100 µM), and abscisic acid (ABA; 10 µM and 100 µM); incubated for 24 h and subsequently analysed for GUS activity (Fig. 2). Auxin treatment induced no major changes in BP expression in the aerial parts of the seedlings, but a dramatic increase in BP expression was observed in their roots (Fig. 2C, D). This increase was due to BP expression in the developing lateral root primordia and became apparent concomitantly with their initiation. Treatment with ABA at both tested concentrations resulted in dramatic reductions in GUS staining in the shoots and roots (Fig. 2E, F). GA, CKs, and ethylene had no apparent effects on GUS staining at the end of the 24 h treatments, and thus presumably no effects on BP expression patterns (not shown). These hormonal effects on BP expression were confirmed by Q RT-PCR; no statistically significant changes in steady-state levels of BP transcripts compared with untreated seedlings were found following CK and ethylene treatments (not shown), but their levels increased by 35% and decreased by 70%, respectively, following treatments with 1 µM 2,4-D and 100 µM ABA (Fig. 2G).
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Short-term effects of CKs and light on KNOXI transcript levels
The glucocorticoid-inducible transgene expression system pOp/LhGR (Craft et al., 2005;
ámalová et al., 2005) was employed to study changes in expression of KNOXI genes in response to increased levels of endogenous CKs in Arabidopsis seedlings. To investigate short-term effects of increased CK levels, seedlings of CaMV35S>GR>ipt line 11 (Craft et al., 2005) were grown on MS plates under the 82 µmol m–2 s–1 light intensity regime, At appropriate time points, seedlings were transferred to plates containing MS supplemented with 500 nM DEX to induce ipt expression for 3, 6, 10, 15, and 24 h by the end of a 10 d growth period. The apical part (hereafter referred to as the apex), cotyledons, hypocotyls, and leaves were dissected out from the harvested seedlings, and the abundance of the ipt, ARR5, BP, KNAT2, KNAT6, and STM transcripts in them was analysed by Q RT-PCR. Pronounced increases in ipt transcript levels were observed, initially in roots, in which 
30% of saturation response levels were reached after 3 h, and saturation levels after 6 h of induction. Ipt levels increased in the other tissues after further lag periods, reaching levels close to saturation in hypocotyls after 10 h, and in the apex and cotyledons after 15 h (Fig. 3). Levels of ARR5 transcripts, which are rapidly induced by CKs (D'Agostino et al., 2000), and have been shown to be proportional to CK levels in many cases, were used to monitor increases in CK levels. DEX treatment increased the abundance of ARR5 transcripts in all organs investigated, regardless of site-specific ipt activation, within 6 h of DEX application, although the level of induction was relatively low in roots. In cotyledons, ARR5 transcript levels rose prior to those of ipt transcripts, suggesting that CKs are transported or translocated much more rapidly than DEX into cotyledons. Hoth et al. (2003) reported KNAT4 to be transiently up-regulated after 6 h of ipt activation. Therefore, KNAT4 and KNAT3, another member of the KNOXII gene family, were included as additional reporters of increased CK levels in cotyledons. KNAT3 and KNAT4 transcripts peaked in cotyledons after 6 h and 10 h of DEX treatment, respectively (Fig. 3D). Thus, their increases clearly preceded increases in ipt transcript levels, in a similar manner to those of the ARR5 transcript.
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In apices, BP transcript levels increased
2.0-fold from 6 h after induction. Slightly weaker increases (1.7-fold; Supplementary Table S1 at JXB online), with comparable kinetics, were observed for KNAT2 and KNAT6 transcripts. Slight increases in STM transcript levels became apparent within 10 h of DEX treatment (Fig. 3). No consistent, significant effects of DEX treatment on the expression of KNOXI genes were observed in either cotyledons or roots (Supplementary Table S1 at JXB online).
In hypocotyls, BP, KNAT2, and KNAT6 transcript levels fluctuated within relatively narrow ranges without any clear decreasing or increasing trends. In contrast, STM transcript levels declined after 10 h of DEX treatment, to only 50% of those found in non-treated seedlings after 24 h treatment (Fig. 3B; Supplementary Table S1 at JXB online). The repression was dramatically enhanced (STM transcript levels falling to only 8% of untreated control levels) when the treatment was carried out under a low light intensity regime (5 µmol m–2 s–1). Interestingly, no similar effect of decreased light intensity on transcript levels was found for the other members of the KNOXI gene family (Fig. 3B; Supplementary Table S1 at JXB online). According to A Reková et al. (personal communication), ipt induction in seedlings grown under low light intensity conditions does not result in greater increases in CK levels compared with induction under standard light intensities. Thus, this effect cannot be explained by CK levels being higher in seedlings grown under low light intensity conditions.
Effects of long-term CK treatments on BP expression patterns
To investigate long-term changes in BP expression patterns in response to treatment with exogenous CKs and constitutive increases in endogenous CK levels via induction of ipt expression, the resulting patterns were visualized histochemically and by in situ RNA hybridization of BP transcripts, respectively. The histochemical analysis was performed with pBP-GUS seedlings grown on MS medium supplemented with BA (0.1–10 µM) for 14 d (Fig. 1), and for the in situ RNA analysis CaMV35S>GR>ipt (line 11) seedlings grown on MS medium supplemented with 500 nM DEX for up to 12 d were used. Both approaches gave essentially identical results. In apices, no differences in the BP expression patterns between treated and non-treated seedlings were observed. Thus, BP expression remained restricted to the subapical region of the shoot apex, and the bases of leaf primordia and mature leaves with no expansion or contraction of BP expression in the SAM (Fig. 1K, L, O, P). At the hypocotyl–root junction, no staining associated with developing lateral root primordia was detected in the CK-treated seedlings, in accordance with the suppression of lateral root primordium formation in the presence of CKs. In contrast, staining associated with vascular tissues expanded in BA-treated seedlings in a concentration-dependent manner, without any noticeable change in staining intensity. Thus, increases in the expansion of GUS staining appeared to be correlated with the periclinal expansion of vascular tissues observed in CK-treated seedlings (Fig. 1D–F). In roots, GUS staining decreased in CK-treated seedlings co-ordinately with the suppression of lateral root formation. No ectopic BP expression was observed as a consequence of CK treatment in leaves (Fig. 1J).
Morphological alterations of CaMV35S>GR>ipt seedlings in response to increased CK levels at various light intensities
To modulate endogenous CKs to various levels, as judged by the severity of morphological alterations, CaMV35S>GR>ipt (line 11) seedlings were grown on media supplemented with DEX at concentrations of 70, 200, and 500 nM for 14 d under the 82 µmol m–2 s–1 light intensity regime. Weak, but consistent, morphological alterations typical of increased CK levels were observed in seedlings grown on media supplemented with 70 nM DEX, including slight reductions of their root system and biomass of their aerial parts, and slight widening of their hypocotyls. However, neither deposition of anthocyanins nor chlorosis was observed. In contrast, strong reductions in the root system, cotyledons, and leaf rosette, accompanied by pronounced widening of the apex base and hypocotyls, and anthocyanin accumulation and achlorosis, were found in seedlings grown on media supplemented with 500 nM DEX. Seedlings grown on media supplemented with 200 nM DEX displayed an intermediate phenotype.
The hypocotyl widening was reminiscent of the effects of KNAT2 overexpression in Arabidopsis (Hamant et al., 2002). To investigate possible interactions between CKs, KNOXI genes, and light in hypocotyls, the CK-overproducing seedlings were cultivated at four different light intensities, which resulted in distinct morphological responses in the hypocotyls and apices. In general, the extent of the morphological alterations found at a particular DEX concentration decreased with decreasing light intensity, as did its dose dependence (Supplementary Fig. S2 at JXB online).
Modulation of KNOXI transcript levels in response to long-term CK action at various light intensities
Steady-state transcript levels of the individual members of the KNOXI gene family determined in seedlings grown under the conditions outlined above are summarized in Supplementary Table S2 at JXB online, and exemplified (using BP expression data) in Fig. 4. The roots were excluded from this analysis because it was not feasible to collect sufficient root tissues for standard Q RT-PCR. BP transcripts were not detectable in cotyledons and leaves. In apices and hypocotyls, the strength of the CK effect on BP expression depended on the light intensity provided during their cultivation. In apices of seedlings grown under the 82 µmol m–2 s–1 and 56 µmol m–2 s–1 light intensity regimes, BP transcript levels increased with increases in the DEX concentration, but no significant increases in BP transcript levels, compared with uninduced controls, were observed in seedlings grown under the 16 µmol m–2 s–1 and 5 µmol m–2 s–1 regimes. Similarly, BP transcript levels increased significantly with increases in DEX concentration in hypocotyls of seedlings grown under the 16, 56, and 82 µmol m–2 s–1 regimes, but no significant differences in BP transcripts from control levels were observed in seedlings grown under the 5 µmol m–2 s–1 regime.
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The histochemical analysis of BP expression patterns in pBP-GUS seedlings grown for 14 d on medium supplemented with BA (Fig. 1) indicated that increases in BP expression may reflect increases in the size of the apex and hypertrophy of vascular tissues caused by CKs rather than increases in BP promoter activity. Therefore, the correlations between BP transcript levels and apex and hypocotyl width were investigated. As hypothesized, a strong correlation between the apex size (represented by the width of the apex base) and BP transcript levels was found in seedlings grown under the 82 µmol m–2 s–1 and 56 µmol m–2 s–1 regimes (correlation coefficients r=0.89 and r=0.82, respectively), and between hypocotyl width and BP transcript levels in seedlings grown under the 82, 56, and 16 µmol m–2 s–1 regimes (correlation coefficients r=0.94, r=0.98, and r=0.81, respectively).
Similarly, increases in the steady-state levels of KNAT2 and KNAT6 transcripts appeared to correlate with increases in the proportions of vasculature in the cotyledons and leaves of seedlings cultivated in the presence of DEX. For instance, the proportions of vasculature to total cotyledon area were 2.7±0.14% in untreated seedlings of line 11, and 5.2±0.57% in seedlings of this line cultivated in the presence of 500 nM DEX (Supplementary Fig. S3 at JXB online). Similar results were observed in leaves (not shown).
In apices of the seedlings grown in the presence of DEX, there was no clear correlation between levels of transcripts of any of the other KNOXI genes and DEX concentrations. However, in hypocotyls, KNAT2 and KNAT6 transcript levels followed trends similar to those found for BP, except that they showed clear increases in DEX-induced seedlings relative to control levels, even in seedlings grown under the 5 µmol m–2 s–1 light intensity regime. Interestingly, a distinct pattern of gene regulation was observed for STM transcript levels, which were higher than control levels in DEX-induced seedlings under the 82 µmol m–2 s–1 and 56 µmol m–2 s–1 regimes, unaffected by DEX in seedlings under the 16 µmol m–2 s–1 regime, and progressively declined with increasing DEX concentrations in seedlings grown under the 5 µmol m–2 s–1 regime (Supplementary Table S2 at JXB online).
Effects of knock-out mutation and constitutive overexpression of BP on sensitivity to hormone treatments
To investigate further the possible role(s) of BP in hormone signalling, BP knock-out mutant (bp; Douglas et al., 2002; Venglat et al. 2002) and CaMV35S-BP transgenic seedlings (Lincoln et al., 1994) were grown on MS media supplemented with BA (5 nM to 10 µM), 2,4-D (1 nM to 10 µM), NAA (1 nM to 10 µM), ACC (5 nM to 20 µM), GA3 (50 nM to 50 µM), and ABA (50 nM to 1 µM) for 12 d. No significant developmental differences between bp and control seedlings grown on MS and hormone-supplemented media were observed (not shown). However, significant differences were found in development and responses to exogenous hormones between CaMV35S-BP and wild-type seedlings (Fig. 5). In 12-d-old CaMV35S-BP seedlings grown on MS medium, the hypocotyl length was reduced (2.3±0.3 cm compared with 3.8±0.7 cm in the wild type) while the primary root was slightly elongated (7.34±0.88 cm compared with 6.71±0.91 cm in the wild type). However, the numbers and lengths of lateral roots were reduced in CaMV35S-BP seedlings, which released 13.4±3.3 lateral roots compared with 17.4±2.6 in the wild type, and the longest lateral roots were 0.79±0.27 cm and 1.32±0.41 cm in CaMV35S-BP and the wild type, respectively. The numbers and lengths of the other lateral roots were also reduced in CaMV35S-BP seedlings (Fig. 5C).
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The roots of CaMV35S-BP and wild-type seedlings displayed distinct differences in sensitivity to exogenous CK treatment. The inhibition of primary root growth was more severe, and the severity of the inhibition increased more rapidly with increasing CK levels, in CaMV35S-BP seedlings. The lengths of the main root of CaMV35S-BP seedlings reached 70–80% of wild-type lengths in the presence of 25–100 nM BA, but only 35% in the presence of 10 µM BA (t-test, P <10–12; Fig. 5E). The main root of CaMV35S-BP seedlings was also found to be more sensitive to ACC, although the differences between the transgenic and wild-type seedlings were less pronounced, albeit still highly significant (t-test, P <10–12), at high ACC concentrations. The lengths of the main transgenic roots were 71% of wild-type lengths when the seedlings were grown in the presence of 10 µM ACC (Fig. 5F). Inhibition of lateral root development by BA and ACC was comparable in the transgenic and wild-type seedlings (not shown). ABA also had similar effects on transgenic and wild-type seedlings, except for their lateral root development. In transgenic seedlings, lateral roots were shorter and essentially insensitive to ABA while the growth of lateral roots was inhibited by ABA in wild-type plants in a dose-dependent manner, dropping to the length of the transgenic lateral roots at 1 µM ABA (Fig. 5D). No significant, consistent differences in transgenic and wild-type seedling development were observed on media supplemented with 2,4-D, NAA, or GA3 (not shown).
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Discussion |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionSupplementary materialReferences |
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Plant hormones regulate plant development, in conjunction with environmental signals, by modulating the expression of specific regulatory genes, including KNOXI genes, a family of homeobox-containing genes that have been found in all plant species examined to date. However, much remains to be elucidated about the interactions involved. Therefore, interactions between hormones (auxin, ABA, CK, ethylene, and GA) and KNOXI genes in early Arabidopsis seedling development were analysed both by applying exogenous hormone treatments and by using inducible expression of ipt, a CK biosynthesis gene. None of the short-term hormone treatments resulted in ectopic expression of the BP gene. However, BP expression was strongly down-regulated by ABA. Increases in BP transcript levels were observed following auxin treatment, correlating with the initiation of lateral root meristems (in which BP is strongly expressed). BP expression did not significantly respond to short-term treatments with the other hormones when assayed in whole seedlings. Following short-term ipt activation, organ-specific differential regulation of KNOXI gene expression was observed; several KNOXI genes were generally activated to low or moderate levels, but STM was specifically repressed in hypocotyls, and reducing the light intensity dramatically strengthened the repression. Long-term increases in CK levels induced increases in BP, KNAT2, and KNAT6 transcript levels, mainly in hypocotyls, that apparently correlated with the radial expansion of vascular tissues (the main domains of KNOXI gene expression). No changes in hormone sensitivity were observed in bp, a loss-of-BP-function mutant. Constitutive BP expression driven by the CaMV35S promoter resulted in reductions in the length and number of lateral roots, while the primary root remained unaffected. The transgenic seedlings displayed weak, but significant alterations in sensitivity to ABA, CK, and ACC treatments. Interestingly, in cotyledons, activation of CK-responsive genes preceded induction of ipt, suggesting that CKs were more rapidly transported than the inducing agent, DEX.
Effects of short-term hormone treatments on BP expression patterns
BP is expressed in various tissues during both vegetative and generative development (Lincoln et al., 1994). According to these authors, in the vegetative shoot apex BP expression is preferentially localized in the peripheral and rib meristem zones, and does not occur at detectable levels in the central zone. During the course of seedling development, BP expression extends to the subapical region of the shoot apex and the bases of leaf primordia and mature leaves, where it accumulates in cells adjacent to vascular tissues, and the bases of stipules. BP expression is associated with vascular tissues in hypocotyls and roots. The presented results confirm the BP expression pattern found in aerial parts of seedlings by Lincoln et al. (1994). In lateral root primordia, BP was expressed in all of the cells at the one-cell layer stage, up to the time of lateral root emergence, after which its expression was restricted to the bases of the roots (Supplementary Fig. S1 at JXB online). This expression pattern in roots is similar to that of KNAT6 (in roots, KNAT2 and KNAT6 expression has been detected in phloem and at the base of the primary root; Dean et al., 2004), indicating that these KNOXI genes may have overlapping functions in the regulation of lateral root development. The BP expression pattern was not affected by short-term treatments with CK, ethylene, or GA (Fig. 2). Increased expression levels following auxin treatment were detected in roots, localized in newly initiated lateral root primordia, but this was apparent only after their initiation. Interestingly, ABA treatment resulted in strong reductions in BP expression in apices and roots, even though lateral root primordia were formed in ABA-treated seedlings, indicating that BP expression is not a key prerequisite for lateral root development. Thus, none of the short-term hormone treatments resulted in ectopic expression of the BP gene that would have led to the formation of new structures.
The pOp/LhGR system as a tool to regulate ipt expression in Arabidopsis seedlings
The ability of the pOp/LhGR system to control expression of the ipt gene has been demonstrated in Arabidopsis and tobacco recently (Craft et al., 2005; ámalová et al., 2005). In the two cited studies, the absence of root growth inhibition indicated that ipt expression in CaMV35S>GR>ipt lines was stringently repressed in the absence of exogenous inducer, while strong phenotypic changes typical of CK overexpression indicated that ipt was efficiently activated in the presence of DEX. Q RT-PCR enabled levels of ipt transcripts in individual Arabidopsis organs to be quantified before and after DEX treatment. In untreated CaMV35S>GR>ipt seedlings, clearly detectable degrees of ‘leakiness’ of the pOp/LhGR system (Supplementary Table S1 at JXB online) were found, but the leakiness is insufficient to cause consistent increases in the pools of endogenous CK in uninduced seedlings, according to Hradilová et al. (2007). DEX treatment resulted in the rapid induction of ipt expression in the roots of seedlings growing on solid medium (Fig. 3). Similar kinetics have been reported for CaMV35S>GR>uidA seedlings treated in liquid medium (Craft et al., 2005). Ipt induction was delayed in the aerial part of the seedlings (Fig. 3). Elevated levels of transcripts of the CK-responsive gene ARR5 were observed, indicating that the levels of ipt induction achieved effectively elevated CK activity. Interestingly, in cotyledons and apices, increases in ARR5 transcript levels substantially preceded increases in ipt transcript levels (Fig. 3). Furthermore, increases in KNAT3 and KNAT4 transcripts followed shortly after increases in ARR5 transcript levels, but their levels had already declined to basal levels again when ipt transcript levels peaked. Previously, the KNAT4 gene has been reported to be transiently up-regulated after 6 h, and to return to basal levels after 24 h of ipt induction in Arabidopsis seedlings (Hoth et al., 2003).
The striking correlation between the induction kinetics of ipt in roots and ARR5 in cotyledons suggests that ARR5, KNAT3, and KNAT4 were activated in cotyledons by CKs originating in roots, which are known to be transported to shoots via xylem vessels at regulated rates (Takei et al., 2001b, 2002). The translocation of CK bases and nucleosides can be mediated by certain members of the purine permease and equilibrative nucleoside transporter families, respectively (Bürkle et al., 2003; Hirose et al., 2005), but seedlings do not appear to possess a transport system that can be recruited to transport DEX at rates comparable with those observed for CKs. This view is consistent with the classical concept of CKs as carriers of information from roots to shoots rather than the restriction of CK functions to paracrine signalling as proposed by Faiss et al. (1997). Low but significant increases in ARR5 transcript levels in response to ipt activation in roots (Fig. 3) are consistent with the finding reported by Werner et al. (2003) that the responsiveness of ARR5 to CKs is lower in the root than in the shoot.
Modulation of KNOXI transcript levels by short- and long-term elevation of endogenous CK levels
No dramatic increases in the expression of KNOXI genes have been detected in genome-wide screenings for CK-responsive genes, either in young Arabidopsis seedlings expressing ipt (Hoth et al., 2003) or in those treated with exogenous CKs for periods ranging from 15 min to 24 h (Rashotte et al., 2003; Brenner et al., 2005), although slight transient increases in BP and KNAT4 have been observed after 6 h of ipt induction (Hoth et al., 2003). In the present study, slight, but clearly detectable, organ-specific increases were found in several KNOXI gene transcripts in response to short-term elevations of endogenous CKs (Fig. 3; Supplementary Table S1 at JXB online). Interestingly, the responses of STM expression to increases in CK levels appear to differ sharply in apices (where they increased) and hypocotyls (where they fell). The reduction in expression in the hypocotyls was dramatically strengthened in seedlings cultivated under low light, but changes in light intensity do not strongly affect changes in CK levels following ipt activation, according to A Reková et al. (unpublished results), suggesting that CKs and light may have synergistic effects on STM expression.
It has been hypothesized that CKs act upstream of BP and STM, and the effects of CKs on homeobox genes provide links between the hormones and developmental genes, implying that CKs play roles in the development of the SAM. Evidence for this hypothesis comes from the observation that Arabidopsis plants subjected to daily heat shock-induced ipt expression for 14 d exhibited substantial increases in the steady-state abundance of transcripts of the KNOXI genes STM and BP (Rupp et al., 1999). However, more recently, Craft et al. (2005) observed no significant increases in either STM or BP transcript levels in CaMV35S>GR>ipt seedlings or leaf tissues treated with DEX. To explain these differences, Craft et al. (2005) suggested that the heat-treated 4-week-old seedlings studied by Rupp et al. (1999) may have possessed substantially more active axillary meristems (released by increased CKs at this developmental stage) than the controls, thus increasing the amount of meristem tissue in the samples and, consequently, STM and BP transcript levels. The presented results support and extend those reported by Craft et al. (2005) and Hoth et al. (2003). In addition to effects caused by short-term increases in CKs (see above), changes as a result of long-term CK action in KNOXI transcript abundance were observed. However, these changes correlate with morphological alterations, largely increases in the proportions of vascular tissues, caused by CKs and light (Supplementary Fig. S3 at JXB online). Expression of the KNOXI genes has been found to be associated with vascular tissues in cotyledons and leaves (Dean et al., 2004). Therefore, the observed increases in the transcript levels may merely reflect increases in the proportions of cells expressing KNOXI in seedlings with elevated levels of endogenous CKs rather than increases in the copy numbers of the transcripts per cell.
BP has been shown to induce the production of lobed leaves with ectopic meristems when overexpressed in Arabidopsis (Chuck et al., 1996), which were presumed to be extensions of serrations. Since CKs can cause leaf serration in Arabidopsis (Rupp et al., 1999), a possible explanation of this phenomenon was that it could have been mediated by BP in response to CKs, if CKs promote the expansion of BP expression from the peripheral zone of the SAM to adjacent regions, especially to leaf primordia and leaves. However, it is demonstrated here that ectopic expression of BP in leaf primordia and leaves is not induced either in CK-treated Arabidopsis seedlings or in Arabidopsis seedlings overexpressing ipt (Fig. 1), indicating that BP is not a downstream agent in establishment of the serrated leaf phenotype in response to CKs in Arabidopsis.
Responses to hormonal treatments in BP knock-out and overexpressing Arabidopsis seedlings
Changes in the sensitivity to CKs of seedlings or tissues over- or underexpressing a gene or (more rigorously) its product are commonly used as indicators that the gene is involved in a CK signalling chain, as illustrated by the following examples. The CK receptor CRE1 was identified in a screening experiment for mutants with impaired CK responses, including rapid cell proliferation and shoot formation in tissue culture (Inoue et al., 2001). Plants with a mutation affecting the CK signalling inhibitor AHP6 displayed increased sensitivity to exogenous CKs in an adventitious root formation assay (Mähönen et al., 2006). Silencing of a CrHPt1 gene encoding a histidine-containing phosphotransmitter in Catharanthus roseus abolished the inductive effect of CK on transcription of CrRR1, a gene encoding a type-A response regulator in C. roseus (Papon et al., 2004). ARR1-overexpressing plants display hypersensitive responses to exogenous CKs, while a loss-of-function mutation in ARR1 results in CK hyposensitivity (Sakai et al., 2001). In addition, double and higher order mutants in type-A ARRs show progressively increasing sensitivity to CK in various CK assays, indicating that these genes act as negative regulators of CK responses (To et al., 2004).
A link between KNOXI genes and ethylene has been demonstrated by Hamant et al. (2002), who found that ACC treatment repressed KNAT2 expression in wild-type Arabidopsis seedlings and suppressed some phenotypic alterations (including hypocotyl elongation, lobed leaves, and epinastic cotyledons) in KNAT2-overexpressing transgenic seedlings. No significant differences in sensitivity to exogenous hormone treatments between bp and wild-type seedlings were detected. This might reflect partial functional redundancy with other KNOXI genes, as indicated by the partial overlaps observed in their expression patterns. Increases in main root sensitivity to CK and ACC in seedlings overexpressing BP (Fig. 5) are consistent with the hypotheses that BP is involved in the mediation of CK and ethylene responses. However, the differences in the increases indicate that CK and ethylene responses may be mediated by at least two partly independent pathways. Interestingly, the increases were not paralleled in lateral roots. On the contrary, in seedlings overexpressing BP, the lateral roots lost sensitivity to ABA while the sensitivity of the main root remained unaltered. Thus, BP might represent an integrator of plant hormone stimuli, and may play distinct roles in primary and lateral root development. Analysis of multiple knock-out mutants and conditional overexpressors of KNOXI genes is likely to yield more detailed insights into interactions between KNOXI genes and plant hormones involved in the regulation of plant development.
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Supplementary material |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionSupplementary materialReferences |
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The following supplementary data are available at JXB online. (i) Figure S1 showing BP expression in roots; (ii) Figure S2 showing effects of endogenously overproduced CKs on seedling morphology; (iii) Figure S3 showing the morphology of cotyledons in seedlings overproducing CKs; (iv) Table S1 summarizing levels of ipt, ARR5, KNAT3, KNAT4 and KNOXI transcripts following short-term DEX treatment; (v) Table S2 listing levels of KNOXI and ARR5 transcripts following long-term DEX treatment.
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Acknowledgements |
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We thank Drs Miltos Tsiantis and Nagavalli S Kiran for critical reading and valuable discussions on the manuscript, and Professor Sarah Hake and Dr Ian Moore for pBP-GUS and CaMV35S>GR>ipt seeds, respectively. This work was supported by grants IAA600380507 and IAA600040612 from the Grant Agency of the Academy of Sciences of the Czech Republic, LC06034 from the Ministry of Education of the Czech Republic, and AVOZ50040507 from the Academy of Sciences of the Czech Republic.
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