Institute of Molecular Plant Sciences, Leiden University, Clusius Laboratory, Wassenaarseweg 64, Leiden, The Netherlands
*Author for correspondence (e-mail: offringa{at}rulbim.leidenuniv.nl)
Accepted July 25, 2001
![]() |
SUMMARY |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: Auxin, Signalling, Transport, Protein kinase, AtPIN, Efflux carrier, Arabidopsis thaliana
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Recent studies using molecular genetic approaches in Arabidopsis thaliana have shed new light on the molecular mechanisms behind PAT and auxin action. Important new insights were obtained through molecular characterisation of Arabidopsis pin-formed or pin1 mutants (Okada et al., 1991). These mutants develop a pin-like inflorescence, which is characteristic of wild-type plants grown in the presence of PAT inhibitors. Occasionally, flowers are produced on the inflorescence of pin1 mutant plants that have less sepals, more petals, no stamens and abnormal carpels. Some of these flowers consist only of carpelloid structures (Okada et al., 1991). Moreover, pin1 mutant embryos show defects in cotyledon number and position, a phenotype that can be mimicked by culturing plant embryos with PAT inhibitors (Liu et al., 1993). The AtPIN1 gene was cloned through transposon tagging and appeared to encode a transmembrane protein with similarity to bacterial-type transporters (Gälweiler et al., 1998). This suggested that the AtPIN1 protein represented the elusive AEC. In agreement with its proposed function as an AEC, the AtPIN1 protein was found to be localised to the basal end of xylem parenchyma and cambial cell files in the Arabidopsis inflorescence axis (Gälweiler et al., 1998).
AtPIN1 was found to be part of a multigene family in Arabidopsis comprising 8 members (Friml, 2000). Allelic loss-of-function mutants in another member of this gene family, AtPIN2, were independently isolated based on root agravitropism or ethylene resistance phenotypes (Chen et al., 1998; Luschnig et al., 1998; Müller et al., 1998; Utsuno et al., 1998). Immunolocalisation showed that the AtPIN2 protein is present at the anti- and periclinal sides of cortical and epidermal cells in the root tip (Müller et al., 1998). The distinct expression and cellular localisation of AtPIN1 and AtPIN2 combined with the phenotypes of the respective loss-of-function mutants suggested that the different members of the AtPIN family each direct distinct processes in plant development, which involve PAT.
The recent finding that lateral organs can be induced on pin1 inflorescences by exogenous application of IAA (Reinhardt et al., 2000) indicated that the IAA content in the pin1 inflorescence apex is sub-optimal for organ formation. This result suggested that continuous supply of IAA is essential for proper positioning and development of organs from the inflorescence meristem. The expression of AtPIN1 in floral organ primordia (Christensen et al., 2000), even at very young stages (Vernoux et al., 2000), suggested that polar transport of IAA is required to guarantee this supply.
Two Arabidopsis mutants that share several of the phenotypic characteristics of the pin1 mutant are monopteros (mp) and pinoid (pid) (Berleth and Jürgens, 1993; Bennett et al., 1995). mp mutants differ from pin1 and pid in that their vascular strands are disconnected and the root meristem is not formed in the embryo. However, like pin1 and pinoid, cotyledon positioning in the mp embryo is aberrant and the inflorescence carries few flowers and terminates prematurely (Przemeck et al., 1996). Flowers have fewer outer whorl organs and have abnormal carpels. In one mp allele PAT was found to be reduced, possibly through absence of a continuous vascular strand (Przemeck et al., 1996). The MONOPTEROS gene encodes ARF5, a protein with homology to the auxin responsive element binding factor ARF1 (Hardtke and Berleth, 1998). MP/ARF5 was found to be a positive regulator of auxin induced gene expression (Ulmasov et al., 1999) and likely has a function in cell axialisation and vascular development during plant development in response to auxin gradients. The phenotype of pid mutants closely resembles that of pin1 mutant plants, but is less severe. A cross between pid and the auxin resistant mutant axr1 suggested that AXR1 and PID have overlapping functions and that PID plays some role in an auxin-related process (Bennett et al., 1995). The PID gene was recently cloned and found to encode a protein-serine/threonine kinase (Christensen et al., 2000). Based on the phenotypes induced by constitutive expression of PID, Christensen and co-workers (Christensen et al., 2000) concluded that the protein kinase is a negative regulator of auxin signalling.
Here we show that PID is an auxin-responsive gene and that the main site of PID expression is the vascular tissue in young developing organs. Based on these results, and on detailed analyses of 35S::PID overexpression phenotypes, we propose that PID functions as a positive regulator of polar auxin transport.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
RNA expression analysis
Poly(A) enriched RNA was isolated from root tips using the Quick Prep Micro mRNA Purification Kit (Pharmacia). Total RNA isolation and RNA blot analysis were performed as described previously (Memelink et al., 1994) and signal detection was performed by Phosphor-Image analysis (Molecular Dynamics). Whole-mount in situ localisation of PID mRNA was performed as described previously (de Almeida Engler, 1998; Friml, 2000), using a 337 bp 5' fragment of the PID cDNA and T3- and T7-polymerase (Promega) for digoxigenin-labelled sense and anti-sense probe synthesis, respectively.
identification of En1 transposon-induced pid alleles and detection of the pid-2 allele
The pid::En197 and pid::En310 mutants were identified from a collection of En-1 transposon mutagenized lines by a PCR-based screen using the En-1 specific primers En205 and En8130 (Wisman et al., 1998) and PINOID specific primers PKV (5'-TCCTTTCTCTCAAACCTCACCGATCC-3') and PKIII (5'-CGTAGAGAAACACTCCAAAGGCCCAC-3'). The presence of the pid-2 allele was detected by amplification of the PID locus using primers 1D.3 (5'-CATGCATTGACTCTGTTCAC-3') and 1D.4 (5'-TAACATTATCTATCGTTACAGTG-3') and digestion of the PCR product with DdeI.
Bacterial strains, DNA libraries and cloning procedures
General cloning and molecular biology procedures were performed as described previously (Sambrook et al., 1989) using E. coli strain DH5. For plant transformation, binary vectors were transferred to Agrobacterium strain LBA1115 by tri-parental mating (Ditta et al., 1980) or through electroporation (Den Dulk-Ras and Hooykaas, 1995).
The PID cDNA was isolated from a cDNA library of auxin-treated root cultures of Arabidopsis ecotype C24 (Neuteboom et al., 1999). DNA sequencing was performed by Eurogentec (Belgium).
The fusion between PID and gusA was created by cloning the SphI-MspAI genomic fragment containing 3.6 kb of 5' untranslated region and the complete PID gene, excluding the last six codons, in-frame with the gusA gene in pCAMBIA1381Xb (McElroy et al., 1995). For the sense overexpression constructs, the PINOID cDNA was cloned into the expression cassette of pART7, which was subsequently introduced as a NotI fragment onto binary vector pART27 (Gleave, 1992).
The pACT and pEF constructs containing the mGAL4:VP16 gene and UAS promoter, respectively, will be described in detail elsewhere (D. W., J. Haseloff, E. van Ryn, P. H. and R. O., unpublished). The DR5::GUS reporter was obtained by cloning a synthetic fragment containing 7 copies of the CCTTTTGTCTC sequence (Ulmasov et al., 1997) upstream of the 47 35S promoter and fusing the resulting promoter to the GFP::GUSA reporter gene (Quaedvlieg et al., 1998).
Histochemical staining and microscopy
Starch granule staining was performed as described previously (Sabatini et al., 1999). To detect gusA expression, plant tissues were fixed in 90% acetone for 1 hour at 20°C, washed three times in 10 mM EDTA, 50 mM sodium phosphate (pH 7.0), 2 mM K3Fe(CN)6, and subsequently stained for up to 16 hours in 10 mM EDTA, 50 mM sodium phosphate (pH 7.0), 1 mM K3Fe(CN)6, 1 mM K4Fe(CN)6 containing 1 mg/ml 5-bromo-4-chloro-3-indolyl-D-glucuronide (AG Biosynth). Tissue was cleared using chloral hydrate after fixation in ethanol:acetate (3:1). GUS-stained tissues were embedded in Technovit 7100 (Heraeus, Germany) and 5 µm sections were stained with Safranin (0.05% in water) for 10 seconds, washed with excess water and mounted in Epon. GUS expression and starch staining were visualised using a Zeiss Axioplan2 imaging microscope with DIC optics. For confocal laser scanning microscopy (CLSM) roots were stained for 10 minutes in 10 mg/l propidium iodide and visualised using a Zeiss Axioplan microscope equipped with a BioRad MRC 1024 confocal laser. Microscopic images were recorded using a Sony DKC 5000 3CCD digital camera and Adobe PhotoShop software. Angles of hypocotyls and root tips towards the horizontal axis were determined using Adobe PhotoShop. Root and hypocotyl lengths were measured using NIH Image.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
By screening En-1 transposon mutagenized lines (Wisman et al., 1998) for insertion mutations in the protein kinase gene, we obtained two new pid alleles in which a transposon disrupted the region encoding the conserved catalytic domain of the protein kinase. We named the mutants pid::En197 and pid::En310 according to the codon that was disrupted by the En-1 insertion (Fig. 1A). Mutant plants develop an inflorescence that ends in a pin-like structure and carries only a few aberrant flowers. Flowers generally contain few or no sepals and stamens, more petals, have a trumpet shaped pistil and produce no or only few seeds (Fig. 1C,F). Occasionally flowers develop with only carpelloid structures (Fig. 1D). Approximately 50% of the mutant seedlings showed abnormal cotyledons, with three cotyledons being the most common phenotype (Fig. 1E). The penetrance of the abnormal cotyledon phenotype indicated that the pid::En mutants represent strong loss-of-function alleles (Bennett et al., 1995; Christensen et al., 2000).
PINOID is expressed in young vascular tissue and aerial organ primordia
PID mRNA is most abundant in young flower buds (not shown). Expression in both roots and shoots of seedlings is low but can be induced by auxin and cycloheximide treatment (Fig. 1B and not shown). To obtain a reliable impression of the spatial and temporal distribution of PID gene expression and the cellular localisation of the PID protein, we fused the complete gene, including the 3.8 kb 5' untranslated region, but excluding the last six codons, in-frame to the gusA reporter gene on pCAMBIA1381Xb (PID:GUS). Multiple lines were generated in ecotype Columbia (Col) that expressed the 4 kb PID:GUS transcript and showed the same ß-glucuronidase (GUS) expression pattern. Transformation of the empty pCAMBIA1381Xb vector did not result in a detectable GUS expression. One representative line, PID:GUS-18, was selected for further analysis.
The pid-2 mutant (Bennett et al., 1995) was crossed with the PID:GUS-18 line and F2 progeny were tested for complementation of the intermediate pid-2 allele by the PID:GUS gene. Among 301 F2 seedlings only 5 developed abnormal cotyledons, a phenotype that showed 20% penetrance in the pid-2 mutant. The data were significant for goodness of fit (2=0.4, P>0.5) with the 1:80 ratio expected for complementation of the pid-2 mutation by the PID:GUS transgene. After transfer to soil, F2 individuals were checked by PCR for the presence of the pid-2 allele and the PID:GUS construct. Plants homozygous for the pid allele developed the typical pid inflorescence, whereas plants that were homozygous for the pid allele but also contained the PID:GUS construct developed a wild-type inflorescence (not shown). These results proved that the PID:GUS fusion protein restores normal growth to pid mutant plants and therefore has wild-type PINOID function. More importantly, the complementation of pid-2 by PID:GUS showed that the GUS activity in line PID:GUS-18 is likely to reveal the spatial and temporal expression and cellular localisation of the endogenous PID protein kinase.
GUS expression in PID:GUS-18 seedlings was mainly localised to the vascular tissue and was strongest in regions of vascular differentiation proximal to the meristems and lateral root primordia (Fig. 2A,C,E). A cross section of the hypocotyl just below the shoot apical meristem showed that GUS activity is present in the xylem parenchyma cells and in the endodermis around the vasculature (Fig. 2G). Moreover, closer examination of the sub-cellular localisation of the GUS signal in untreated and auxin-treated seedlings suggested that PINOID does not accumulate in the nucleus (Fig. 2H and data not shown). Treatment of seedlings with 5 µM IAA induced a significant increase of expression in vascular tissue and leaf primordia (Fig. 2B,D,F). In the inflorescence, expression was detected in anther primordia, in the vasculature of the growing flower stalk, of young pedicels and bracts (Fig. 2I,J,K,L) and of developing sepals, but not in petals (Fig. 2M). In pistils, PID was transiently expressed in the vasculature of the style and the septum (Fig. 2M), in the integuments and funiculus of the developing ovule (Fig. 2N) and in the cotyledon primordia of embryos (Fig. 2O).
|
|
|
|
PID overexpression phenotypes are rescued by polar auxin transport inhibitors
The phenotype of pid loss-of-function mutants most closely resembles that of the pin1 mutants, which are proposed to be blocked in PAT owing to the absence of a functional AEC (Gälweiler et al., 1998). This led us to hypothesise that the PID protein kinase acts by regulating PAT. To further examine this possible role for PID, we studied the effect of PAT inhibitors on root meristem collapse. Seeds of wild-type Arabidopsis and the 35S::PID lines Col-10 and Col-21 were germinated on medium containing 0.1 or 0.3 µM naphthylphtalamic acid (NPA). These specific concentrations were used because such treatments exerted only mild effects on wild-type root development, as observed by root growth, lateral root initiation (Fig. 6A,B) and patterning of the root meristem (Fig. 6E,F). Moreover, the expression of an auxin responsive DR5::GUS reporter indicated that only minor changes occurred with regard to auxin distribution, or sensitivity, in roots treated with these NPA concentrations (Fig. 6I,J). Growth of 35S::PID seedlings on NPA significantly increased root elongation (Fig. 6C,D) and prevented collapse of the primary root meristem (Fig. 6G,H). Similar results were obtained when seedlings were grown on TIBA, which belongs to a different class of PAT inhibitors. Clearly, suppression of PAT by inhibitor concentrations that only mildly interfere with the development of wild-type seedlings was sufficient to rescue the organisation of the primary root meristem in 35S::PID lines.
|
More detailed analysis showed that root meristem collapse in the strong overexpression line 35S::PID Col-21 was observed by 3 days after germination in some seedlings, and after 7 days in most (Fig. 6M). Meristem disintegration also occurred in line 35S::PID Col-10, which expresses an intermediate level of the 35S::PID transgene, but in only up to 50% of the seedlings and with significantly delayed timing. Primary root meristems of the majority of the 35S::PID Col-21 seedlings were rescued with 0.3 µM NPA. This concentration was critical, since at a slightly lower concentration (0.1 µM) the rescue was only partial for high expressing line 35S::PID Col-21, whereas rescue was almost complete for the intermediate expressing line 35S::PID Col-10.
The p35S-driven PID overexpression typically inhibits lateral root development in young seedlings, but the inhibition is overcome by collapse of the primary root meristem. Since root meristem disintegration occurs earlier and more frequently in the high expressing 35S::PID lines, seedlings of these lines generally develop a more vigorous root system. In the presence of NPA however, the meristem is rescued and lateral root formation remains suppressed. To quantify this prolonged suppression by NPA, we grew wild-type and 35S::PID Col-10 and Col-21 seedlings for 14 days on 0.3 µM NPA and determined the length and the number of lateral roots per primary root. NPA-rescued primary roots of both 35S::PID lines were similar in length, but significantly shorter than those of wild-type seedlings (P<0.001; Fig. 6N). Although the inhibition of lateral root formation was maintained for both 35S::PID lines in the presence of NPA, it was apparent that Col-21 seedlings developed significantly more lateral roots than those of line Col-10 (P<0.001; Fig. 6N). Apparently, high, as opposed to intermediate, levels of p35S-driven PID expression promote branching of primary roots. These findings clearly do not fit with a role of PID as a negative regulator of auxin signalling (Christensen et al., 2000), which implies a negative correlation between root branching and the level of 35S::PID expression. Instead, our observations are more readily explained by general enhancement of auxin efflux due to ectopic PID expression, which results in dynamic changes in auxin distribution during development of the 35S::PID seedlings.
Ectopic PINOID expression in the shoot results in more lateral roots
Participation of PID in the regulation of PAT implies that PID does not only act locally in tissues where it is expressed, but also exerts its effect over a distance. To test this, we analysed the effects of tissue-specific PID expression using a GAL4-based transactivation-reporter system (D. W., J. Haseloff, E. van Ryn, P. H. and R. O., unpublished). This system enabled us to study the effects of the ectopic expression of PID and to simultaneously localise PID expression through the observation of GFP:GUS reporter transactivation. An activator plant line ACT-LTP1 containing the epidermis-specific promoter of the Arabidopsis thaliana Lipid Transfer Protein 1 (LTP1) gene fused to the GAL4:VP16 gene was crossed with an effector line (EF-PID) harbouring both the PID cDNA and the GFP:GUS reporter under control of the GAL4-dependent UAS promoter. F2 seedlings were germinated without selection and 11- to 12-day-old seedlings were stained for GUS expression. Segregating at the expected 9:7 ratio for GUS-positive and GUS-negative seedlings, the F2 population clearly displayed significantly enhanced lateral root formation by LTP1 promoter-driven PID expression (Table 1, exp. 1). A striking observation, especially since LTP1 promoter activity is confined to the aerial parts of seedlings (Fig. 7A), except for expression in the young epidermis of lateral roots. Since pLTP1 activity in lateral roots is not detectable before a stage VI primordium (Malamy and Benfey, 1997) (Fig. 7B,C), it is unlikely that this local pLTP1-driven PID expression is the cause of the enhanced lateral root formation.
|
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
PINOID expression reveals a role in organ development
PID expression was observed in the cotyledon primordia of embryos, in young leaves and in young floral organs (Christensen et al., 2000; our observations). This expression pattern corroborates the function of PID in the regulation of aerial organ development and cotyledon positioning and separation. PID expression in vascular tissues initially suggested that PID might direct vascular development in young developing organs. However, mild vascular defects were only observed in the aberrant flowers and not in other organs of pid loss-of-function mutants (Christensen et al., 2000) (our observations), indicating that the regulation of vascular development is not the primary task of PID. More likely, based on the interpretation of the loss-of-function mutant phenotype and gene expression, PID is probably involved in determining the position and outgrowth of cotyledon, leaf, flower and floral organ primordia. The importance of auxin and the involvement of PIN1 in positioning and outgrowth of lateral aerial organs was demonstrated recently (Reinhardt et al., 2000; Vernoux et al., 2000).
In contrast to the clear developmental defects observed in aerial organs, the effects of pid loss-of-function mutations on root development were not very obvious. However, we did observe an irregular root waving pattern in pid loss-of-function mutants (not shown), while 35S::PID overexpressors had strong alterations in root gravitropism and development. This suggests that some PID functions do indeed influence auxin-mediated processes in roots.
PINOID: a negative regulator of auxin signalling?
Based on two phenotypes of the 35S::PID lines, decreased expression of the auxin responsive DR5::GUS reporter and reduced lateral root initiation even upon exogenous application of auxin, Christensen et al. (Christensen et al., 2000) concluded that PID acts as a negative regulator of auxin signalling. However, the new observations that we have made argue against such a function for PID. DR5::GUS expression is reduced in young roots of the 35S::PID lines, but we found that expression of the reporter was clearly present in the root vasculature after collapse of the primary root meristem or in roots of older plants (not shown), suggesting that auxin signalling to this promoter is not impaired, but rather, prevented in young primary roots. Another auxin-dependent process that is initially perturbed in 35S::PID lines is lateral root formation. However, 14-day-old seedlings of the high expressing 35S::PID line, Col-21, developed more lateral roots than those of the intermediate expressing line, Col-10, indicating that auxin signalling leading to lateral root induction is not repressed by PID (over)expression. Moreover, roots of 35S:PID lines are clearly as sensitive to exogenously applied auxins as wild-type roots (Christensen et al., 2000) (our observations). Reduced sensitivity of root elongation to exogenously applied auxin has been one of the major criteria for distinguishing mutations in components or regulators of auxin signalling (e.g. axr1). In conclusion, none of the observations provides sufficient evidence that auxin signalling in 35S::PID roots is perturbed and, although we do not exclude an involvement of the PID protein kinase in auxin signalling, we consider the available data indicating a role for the protein kinase as a positive regulator of PAT.
PINOID acts as a positive regulator of auxin efflux
The pin-shaped inflorescences and the aberrant cotyledons and flowers of the pid mutants closely resemble those of pin-formed mutants. The cotyledon and inflorescence phenotypes can be mimicked by treatment with auxin transport inhibitors of globular stage embryos or mature plants, respectively (Okada et al., 1991; Liu et al., 1993; Hadfi et al., 1998). Recently it was shown that some aspects of the aberrant flower development in pid and pin1 mutants can be mimicked by spraying flowers with the same inhibitors (Nemhauser et al., 2000). Moreover, expression studies show that PID co-localises with AtPIN1 in the xylem parenchyma cells of the vascular tissue (our observations) (Gälweiler et al., 1998). All these data strongly suggest that PID acts in concert with AtPIN1 in the vasculature, or with other AtPINs in other tissues, to positively regulate PAT. If so, loss-of-function pid mutants would be expected to show reduced levels of PAT and PID function would be sensitive to PAT inhibitors.
Indeed, auxin transport seemed reduced in the inflorescence stems of loss-of-function pid alleles (Bennet et al., 1995) (our observations). However, PAT levels in inflorescence stems are determined by many indirect factors, such as the presence and number of developing aerial organs (Bennett et al., 1995; Oka et al., 1998) and proper development of the vascular tissue (Carland and McHale, 1996; Przemeck et al., 1996), which indicates that conclusions from direct transport measurements should also be confirmed by other functional tests. Two important observations support the role of PID as a regulator of PAT: (i) the PAT inhibitor sensitivity of PID action and (ii) the fact that, spatially, PID expression and the resulting effects on plant development do not necessarily overlap.
The NPA/TIBA-sensitivity of PID action was first demonstrated by the rescue of root growth of 35S::PID seedlings by low doses of these PAT inhibitors. We propose a model to explain the 35S::PID phenotype (Fig. 8), in which the 35S promoter-mediated PID expression preferentially enhances downward-directed PAT through the axis of the seedlings. This canalisation of auxin depletes the more peripheral tissue layers of auxin, which leads to reduced elongation and agravitropy of the hypocotyl and delays lateral root formation. Indeed, increasing endogenous auxin levels by growing the seedlings at 28°C (Gray et al., 1998) resulted in enhanced hypocotyl elongation of 35S:PID seedlings (not shown), thereby confirming that IAA concentrations are sub-optimal for hypocotyl elongation. In our model, ectopic PID expression results in enhanced efflux of auxin from the root meristem (Fig. 8), thereby resolving the proposed auxin maximum as organiser of the root meristem (Sabatini et al., 1999) and thus leading to collapse of the meristem. The sub-optimal IAA levels in the root tip explain the reduced pDR5::GUS expression in 35S::PID background roots and the slow and agravitropic growth of these roots. The collapse of the primary root meristem in 35S::PID seedlings alleviates auxin depletion of the root and thereby releases the initial delay in lateral root formation. This is in line with previous findings that removal of the root meristem or the root cap enhances the formation of lateral root primordia and the emergence of lateral roots (Torrey, 1950; Reed et al., 1998; Tsugeki and Fedoroff, 1999). In the presence of NPA at sub-micromolar concentrations, elevated PAT is slowed down in roots of 35S::PID seedlings, allowing for maintenance of the organising auxin maximum (Sabatini et al., 1999), rescue of the root meristem and partial rescue of the gravitropy and growth of the root. The recent finding that PAT inhibitor treatments induce an accumulation of IAA in the root tip (Casimiro et al., 2001) is consistent with this model.
|
Conclusion
Based on the data presented above we propose that the protein kinase PID is a positive regulator of PAT. The inducibility of the PID gene by auxin suggests that PAT is enhanced by accumulation of PID in the cell in response to an increase in local IAA levels. Indications for regulation of PAT by auxin are provided by the fact that PAT levels in inflorescence stems are significantly reduced when developing flower buds or siliques, the presumed source tissues of IAA, are removed or absent (Bennett et al., 1995; Oka et al., 1998), by the report that auxin efflux is enhanced in the auxin overproducing sur1 mutant (Delarue et al., 1999) and from the observed twofold increase of acropetal [3H]IAA transport through the root in the presence of cold IAA (Rashotte et al., 2000). The phenotypic characteristics of the pid loss-of-function mutants suggest that the PID function is essential for proper cotyledon positioning and development, for maintenance of the inflorescence meristem, for whorl definition during flower development and it is important for wild-type root growth. Enhancement of PAT may be necessary to prevent feed-back regulation of auxin biosynthesis due to accumulation of auxin, and may possibly guarantee a continuous source-to-sink transport of auxin that is essential for the organisation of, and differentiation in, these developing organs. One important step in elucidating the mechanism of the PID-mediated enhancement of PAT will be to investigate interactions between PID and the putative AECs.
![]() |
ACKNOWLEDGMENTS |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Bennett, S. R. M., Alvarez, J., Bossinger, G. and Smyth, D. R. (1995). Morphogenesis in pinoid mutants of Arabidopsis thaliana. Plant J. 8, 505-520.
Berleth, T. and Jürgens, G. (1993). The role of the MONOPTEROS gene in organising the basal body region of the Arabidopsis embryo. Development 118, 575-587.
Carland, F. M. and McHale, N. A. (1996). LOP1: a gene involved in auxin transport and vascular patterning in Arabidopsis. Development 122, 1811-1819.
Casimiro, I., Marchant, A., Bhalerao, R. P., Beeckman, T., Dhooge, S., Swarup, R., Graham, N., Inzé, D., Sandberg, G., Casero, P., Bennett, M. (2001). Auxin transport promotes Arabidopsis lateral root initiation. Plant Cell 13, 843-852.
Chen, R., Hilson, P., Sedbrook, J., Rosen, E., Caspar, T. and Masson, P. H. (1998). The Arabidopsis thaliana AGRAVITROPIC 1 gene encodes a component of the polar-auxin-transport efflux carrier. Proc. Natl. Acad. Sci. USA 95, 15112-15117.
Christensen, S. K., Dagenais, N., Chory, J. and Weigel, D. (2000). Regulation of auxin response by the protein kinase PINOID. Cell 100, 469-478.[Medline]
Clough, S. J. and Bent, A. F. (1998). Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J. 16, 735-743.[Medline]
de Almeida Engler, J., van Montagu, M. and Engler, G. (1998). Whole-mount in situ hybridization in plants. Methods Mol. Biol. 82, 373-384[Medline]
Delarue, M., Muller, P., Bellini, C. and Delbarre, A. (1999). Increased auxin efflux in the IAA-overproducing sur1 mutant of Arabidopsis thaliana: A mechanism of reducing auxin levels? Physiol. Plant. 107, 120-127.
Den Dulk-Ras, A. and Hooykaas, P. J. J. (1995). Electroporation of Agrobacterium tumefaciens. In Methods in Molecular Biology Vol. 55: Plant Cell Electroporation and Electrofusion Protocols. (ed. J. A. Nickoloff), pp. 63-72. Totowa, NJ: Humana Press Inc.
Ditta, G., Stanfield, S., Corbin, D. and Helinski, D. R. (1980). Broad host range DNA cloning system for gram-negative bacteria: construction of a gene bank of Rhizobium meliloti. Proc. Natl. Acad. Sci. USA 77, 7347-7351.[Abstract]
Friml, J. (2000) Isolation and characterisation of novel AtPIN genes from Arabidopsis thaliana L. PhD thesis, Cologne.
Gälweiler, L., Guan, C., Müller, A., Wisman, E., Mendgen, K., Yephremov, A. and Palme, K. (1998). Regulation of polar auxin transport by AtPIN1 in Arabidopsis vascular tissue. Science 282, 2226-2230.
Gleave, A. P. (1992). A versatile binary vector system with a T-DNA organisational structure conducive to efficient integration of cloned DNA into the plant genome. Plant Mol. Biol. 20, 1203-1207.[Medline]
Gray, W. M., Ostin, A., Sandberg, G., Romano, C. P. and Estelle, M. (1998). High temperature promotes auxin-mediated hypocotyl elongation in Arabidopsis. Proc. Natl. Acad. Sci. USA 95, 7197-7202.
Hadfi, K., Speth, V. and Neuhaus, G. (1998). Auxin-induced developmental patterns in Brassica juncea embryos. Development 125, 879-887.
Hanks, S. K. and Hunter, T. (1995). Protein Kinases 6 The eukaryotic protein kinase superfamily: kinase (catalytic) domain structure and classification. FASEB J. 9, 576-596.
Hardtke, C. S. and Berleth, T. (1998). The Arabidopsis gene MONOPTEROS encodes a transcription factor mediating embryo axis formation and vascular development. EMBO J. 17, 1405-1411.
Liu, C.-M., Xu, Z.-H. and Chua, N.-H. (1993). Auxin polar transport is essential for the establishment of bilateral symmetry during early plant embryogenesis. Plant Cell 5, 621-630.
Lomax, T. L., Muday, G. K. and Rubery, P. H. (1995). Auxin transport. In Plant Hormones, Physiology, Biochemistry and Molecular Biology (ed. P. J. Davies), pp. 509-530. Dordrecht: Kluwer Academic Publishers.
Luschnig, C., Gaxiola, R. A., Grisafi, P. and Fink, G. R. (1998). EIR1, a root-specific protein involved in auxin transport, is required for gravitropism in Arabidopsis thaliana. Genes Dev. 12, 2175-2187.
Malamy, J. E. and Benfey, P. N. (1997). Organisation and cell differentiation in lateral roots of Arabidopsis thaliana. Development 124, 33-44.
Masson, J. and Paszkowski, J. (1992). The culture response of Arabidopsis thaliana protoplasts is determined by the growth conditions of donor plants. Plant J. 2, 829-833.
McElroy, D., Chamberlain, D. A., Moon, E. and Wilson, K. J. (1995). Development of gusA reporter gene constructs for cereal transformation: Availability of plant transformation vectors from the CAMBIA molecular genetic resource service. Mol. Breed. 1, 27-37.
Memelink, J., Swords, K. M. M., Staehelin, L. A. and Hoge, J. H. C. (1994). Southern, northern and western blot analysis. In Plant Molecular Biology Manual (ed. S. B. Gelvin, R. A. Schilperoort and D. P. S. Verma). Dordrecht, NL: Kluwer Academic Publishers.
Müller, A., Guan, C., Gälweiler, L., Tanzler, P., Huijser, P., Marchant, A., Parry, G., Bennett, M., Wisman, E. and Palme, K. (1998). AtPIN2 defines a locus of Arabidopsis for root gravitropism control. EMBO J. 17, 6903-6911.
Nemhauser, J. L., Feldman, L. J. and Zambryski, P. C. (2000). Auxin and ETTIN in Arabidopsis gynoecium morphogenesis. Development 127, 3877-3888.
Neuteboom, L. W., Ng, J. M., Kuyper, M., Clijdesdale, O. R., Hooykaas, P. J. and van-der-Zaal, B. J. (1999). Isolation and characterisation of cDNA clones corresponding with mRNAs that accumulate during auxin-induced lateral root formation. Plant Mol. Biol. 39, 273-287.[Medline]
Oka, M., Ueda, J., Miyamoto, K. and Okada, K. (1998). Activities of auxin polar transport in inflorescence axes of flower mutants of Arabidopsis thaliana: Relevance to flower formation and growth. J. Plant Res. 111, 407-410.
Okada, K., Ueda, J., Komaki, M. K., Bell, C. J. and Shimura, Y. (1991). Requirement of the auxin polar transport system in early stages of Arabidopsis flower bud formation. Plant Cell 3, 677-684.
Quaedvlieg, N. E. M., Schlaman, H. R. M., Admiraal, P. C., Wijting, S. E., Stougaard, J. and Spaink, H. P. (1998). Fusions between green fluorescent protein and beta-glucuronidase as sensitive and vital bifunctional reporters in plants. Plant Mol. Biol. 38, 861-873.[Medline]
Przemeck, G. K., Mattsson, J., Hardtke, C. S., Sung, Z. R. and Berleth, T. (1996). Studies on the role of the Arabidopsis gene MONOPTEROS in vascular development and plant cell axialization. Planta 200, 229-237.[Medline]
Rashotte, A. M., Brady, S. R., Reed, R. C., Ante, S. J. and Muday, G. K. (2000). Basipetal auxin transport is required for gravitropism in roots of Arabidopsis. Plant Physiol. 122, 481-490.
Reed, R. C., Brady, S. R. and Muday, G. K. (1998). Inhibition of auxin movement from the shoot into the root inhibits lateral root development in Arabidopsis. Plant Physiol. 118, 1369-1378.
Reinhardt, D., Mandel, T. and Kuhlemeier, C. (2000). Auxin regulates the initiation and radial position of plant lateral organs. Plant Cell 12, 507-518.
Sabatini, S., Beis, D., Wolkenfelt, H., Murfett, J., Guilfoyle, T., Malamy, J., Benfey, P., Leyser, O., Bechtold, O., Weisbeek, P. and Scheres, B. (1999). An auxin-dependent distal organizer of pattern and polarity in the Arabidopsis root. Cell 99, 463-472.[Medline]
Sambrook, J., Fritsch, E. F. and Maniatis, T. (1989). Molecular Cloning, A laboratory manual. (ed. C. Nolan), Cold Spring Harbor, NY, USA: Cold Spring Harbor Laboratory Press.
Thoma, S., Hecht, U., Kippers, A., Botella, J., de Vries, S. and Somerville, C. (1994). Tissue-sepcfic expression of a gene encoding a cell wall-localised lipid transfer protein from Arabidopsis. Plant Physiol. 105, 35-45.
Torrey, J. G. (1950). The induction of lateral roots by indoleactetic acid and root decapitation. Am. J. Bot. 37, 257-264.
Tsugeki, R. and Fedoroff, N. V. (1999). Genetic ablation of root cap cells in Arabidopsis. Proc. Natl. Acad. Sci. USA 96, 12941-12946.
Ulmasov, T., Murfett, J., Hagen, G. and Guilfoyle, T. J. (1997) Aux/IAA proteins repress expression of reporter genes containing natural and highly active synthetic auxin response elements. Plant Cell 9, 1963-1971.
Ulmasov, T., Hagen, G. and Guilfoyle, T. J. (1999). Activation and repression of transcription by auxin-response factors. Proc. Natl. Acad. Sci. USA 96, 5844-5849.
Utsuno, K., Shikanai, T., Yamada, Y. and Hashimoto, T. (1998). Agr, an agravitropic locus of Arabidopsis thaliana, encodes a novel membrane-protein family member. Plant Cell Physiol. 39, 1111-1118.[Medline]
Vernoux, T., Kronenberger, J., Grandjean, O., Laufs, P., Traas, J. (2000). PIN-FORMED 1 regulates cell fate at the periphery of the shoot apical meristem. Development 127, 5157-5165.
Wisman, E., Hartmann, U., Sagasser, M., Baumann, E., Palme, K., Hahlbrock, K., Saedler, H. and Weisshaar, B. (1998). Knock-out mutants from an En-1 mutagenized Arabidopsis thaliana population generate phenylpropanoid biosynthesis phenotypes. Proc. Natl. Acad. Sci. USA 95, 12432-12437.