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First published online July 29, 2005; 10.1105/tpc.105.034637 © 2005 American Society of Plant Biologists
Maintenance of Embryonic Auxin Distribution for Apical-Basal Patterning by PIN-FORMED–Dependent Auxin Transport in Arabidopsis
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMETHODSREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMETHODSREFERENCES |
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; Jürgens and Mayer, 1994) that mark patterning events and stages and allow easy detection of defects in this process. First, the zygote divides asymmetrically to yield a smaller apical cell that generates most of the embryo and a larger basal cell that generates a filamentous supporting structure, the suspensor. At the globular stage of embryo development, cells in the center of the proembryo elongate along the future shoot–root axis. Simultaneously, the uppermost suspensor cell (hypophysis) switches from extraembryonic to embryonic fate and contributes to the establishment of the root meristem. Later, localized cell division activity at the apical flanks of the globular embryo initiates the cotyledons and transforms the embryo into a heart shape. At this stage, all embryo organs have been initiated, and later steps involve elongation growth and maturation of the embryo.
Despite its fundamental importance in shaping the future plant, regulatory molecules and mechanisms in plant embryo pattern formation have been identified only recently. Through genetics, several genes required for normal embryo patterning have been defined. Elucidating the function of these genes has not led to a unified model for embryo development, but the activity of some of the encoded proteins implicates the plant signaling molecule auxin (Weijers and Jürgens, 2005).
Auxin is a central regulator in many processes during plant growth and development. An important aspect of auxin action is its directional transport through the plant (Friml, 2003). This polar auxin transport (PAT) is important for many auxin-regulated processes and requires the activity of polarly localized efflux regulators, represented by members of the PIN-FORMED family. PAT can be inhibited by naphthylphthalamic acid (NPA) and other drugs. Treatment of immature embryos with such PAT inhibitors leads to embryo patterning defects in several plant species (Schiavone and Cooke, 1987; Liu et al., 1993; Hadfi et al., 1998; Friml et al., 2003). The fact that the same patterning defects are observed in Arabidopsis mutants in members of the PIN gene family, such as pin1 and the pin4 pin7 double mutant (Liu et al., 1993; Friml et al., 2002, 2003), supports the requirement of PIN-dependent PAT for normal embryo patterning.
Expression of the auxin-dependent reporter DR5rev green fluorescent protein (DR5rev-GFP) indicates dynamic changes in auxin distribution during specific patterning events in the embryo (Friml et al., 2003). Initially, DR5rev-GFP activity localizes to the proembryo, and later it shifts basally to the hypophysis. This dynamic distribution of auxin activity in the embryo, for simplicity here referred to as auxin gradients, depends on PIN-mediated auxin transport. Although it is now relatively well understood how auxin gradients are established and how they might be translated into gene expression patterns by the auxin response protein MONOPTEROS/AUXIN RESPONSE FACTOR5 (Hardtke and Berleth, 1998) and its inhibitor BODENLOS/INDOLEACETIC ACID12 (Hamann et al., 2002), it is yet unknown how robust auxin gradients are and by which mechanisms they are maintained. This could involve local auxin biosynthesis and degradation, auxin transport, or a combination of both.
Here, we show that neither enhancing the rate of auxin biosynthesis nor manipulation of auxin conjugation rates changed auxin gradients or embryo patterning, revealing a robust buffering mechanism. This buffering capacity depends critically on PIN-dependent PAT, suggesting that in addition to establishing auxin gradients, auxin transport also maintains these gradients during embryogenesis.
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; Romano et al., 1995). The Pseudomonas syringae indoleacetic acid-lysine synthetase (iaaL) gene encodes an enzyme that converts IAA and Lys into the biologically inactive IAA-Lys conjugate, and its expression results in a decrease of the levels of free IAA in plant cells (Romano et al., 1991). iaaM or iaaL expression generally causes cellular auxin concentration changes of 2- to 10-fold (Klee et al., 1987; Romano et al., 1991, 1995), well within the physiological range.
The iaaM and iaaL genes were introduced into a GAL4 transcription factor/Upstream (GAL4/UAS) two-component gene expression system optimized for use in Arabidopsis (Weijers et al., 2003). A UAS-driven GFP-ß-glucuronidase (UAS-GFP:GUS) reporter gene was linked to the UAS-iaaM or UAS-iaaL genes to monitor expression of iaaM or iaaL. We have previously shown that this transactivation system is reliable and reproducible for domain-specific expression in Arabidopsis seeds (Weijers et al., 2003). As expected, UAS-iaaM;UAS-GFP:GUS (EF iaaM) and UAS-iaaL;UAS-GFP:GUS (EF iaaL) lines did not express the iaaM or iaaL mRNAs (see Supplemental Figure 1 online) and were wild-type in appearance.
To first assess whether this approach allows changing cellular auxin concentrations, we crossed EF iaaM and EF iaaL lines with an ACT LIPID TRANSFER PROTEIN1 (proLTP1) line that expresses GAL4 in the epidermis (Weijers et al., 2003) and analyzed postembryonic development.
The effects of iaaM expression on postembryonic development have been described in detail for Arabidopsis (Romano et al., 1995), tobacco (Nicotiana tabacum; Sitbon et al., 1992), and petunia (Petunia hybrida; Klee et al., 1987). In accordance with these studies, hypocotyl elongation was strongly enhanced in proLTP1>>iaaM seedlings (the notation proX>>Y describes transactivation of gene Y by promoter X; average hypocotyl length 1.66 ± 0.24 mm [n = 26] in the wild type, 4.61 ± 0.86 mm [n = 30] in proLTP1>>iaaM; Figure 1A). Such seedlings showed several other hallmarks of auxin overproduction, such as epinastic cotyledons and long petioles (Figure 1A). Upon rosette leaf formation, proLTP1>>iaaM phenotypes became more extreme, with leaves becoming epinastic and narrow (Figure 1B). Inflorescences were less branched, produced few flowers, and often terminated into a pin-like structure bearing only a few flower buds (Figure 1B; see Supplemental Figure 2 online). Measurement of free auxin levels in proLTP1>>iaaM plants showed that concentrations were elevated severalfold (Figure 1B).
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). The mechanism behind this phenomenon is not clear, but nonetheless we exploited this variability to correlate variations in phenotypes to auxin concentrations and to justify the use of the GFP:GUS reporter gene as marker for iaaM activity. The phenotypic strength clearly correlated with the free auxin levels, the strongest phenotypic class of plants showing as much as a sixfold increase in auxin concentration (Figure 1B). This increase is considerable since the LTP1 promoter is only active in epidermal cells of growing aerial organs. GUS activity also increased with auxin levels and phenotypic strength, thereby reinforcing that iaaM activity is reflected by activity of the coactivated GFP:GUS reporter gene.
As expected from previous reports (Gray et al., 1998; Zhao et al., 2001), proLTP1>>iaaL expression had the opposite effect on seedling and plant phenotype as proLTP1>>iaaM expression. Cotyledon expansion was increased, and hypocotyl length and root growth were decreased (Figure 1C). Furthermore, proLTP1>>iaaL seedlings showed altered response to gravity (Figure 1D), and flowering plants displayed a decrease of apical dominance (see Supplemental Figure 2 online). Taken together, postembryonic phenotypes and auxin concentration measurements show that GAL4/UAS-mediated expression of the iaaM and iaaL genes allows manipulating auxin homeostasis and that the induced changes are sufficient for altering postembryonic plant development.
Embryo Patterning Is Not Affected by Enhanced Auxin Biosynthesis or Conjugation
To assess the effects of local changes in auxin homeostasis on embryo pattern formation, preselected EF iaaM and EF iaaL lines (Table 1) were crossed to a range of lines, each expressing GAL4 in a subset of embryonic cells.
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, 2003) at stages ranging from two-cell to torpedo stage (Figures 2A, 2B, 2G, and 2H, Table 2).
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; Vroemen et al., 1996; Figures 2C and 2I). Expression in the suspensor was achieved by crossing with a line expressing GAL4 from a suspensor-specific promoter fragment (Figures 2D and 2J; D. Weijers and R. Offringa, unpublished data) of the SHOOT MERISTEMLESS (STM) gene (Long et al., 1996) or from GAL4 enhancer trap lines J3281 and M0167 (Figures 2F and 2L; www.plantsci.cam.ac.uk). iaaM and iaaL were expressed in the central embryo domain using GAL4 enhancer trap line Q0990 (www.plantsci.cam.ac.uk; Figures 2E and 2K) and in an auxin-dependent fashion using the DR5(7x) promoter (Ulmasov et al., 1997). Finally, the genes were expressed in endosperm using GAL4 enhancer trap line KS22I (Boisnard-Lorig et al., 2001). In each of the crosses of the selected GAL4 lines and iaaM or iaaL lines, the UAS-dependent GUS or GFP reporter genes were correctly expressed (Figure 2). None of these genotypes, however, caused changes in embryo pattern formation (Figure 1, Table 2; for the genotypes that are not listed in Table 2, at least 100 embryos were analyzed). These results indicate that in contrast with the strong effects on postembryonic development, manipulation of auxin biosynthesis or conjugation activity do not alter embryo patterning, suggesting the existence of mechanisms that buffer the changes in auxin homeostasis in the embryo.
Enhanced Auxin Biosynthesis or Conjugation Rates Leave Embryonic Auxin Gradients Unaffected
The absence of iaaM- or iaaL-induced embryo phenotypes could mean that the genes, despite their postembryonic activity, are not functional or lack substrates during embryogenesis. Alternatively, the enzymes are active and auxin levels are changed in transgenic embryos, but the patterning mechanism is buffered against changes in auxin levels. As direct auxin concentration measurements on young embryos are not yet feasible, and immunolocalization of auxin is not quantitative, we used a combination of other approaches to discriminate between these possibilities. First, we analyzed the activity of the auxin-dependent DR5rev-GFP reporter gene (Friml et al., 2003) in embryos expressing iaaM or iaaL. The DR5rev-GFP reporter was crossed into ACT proRPS5A, ACT proLTP1, and EF iaaM and EF iaaL lines, and plants carrying both transgenes were selected and used in crosses.
Consistent with the reported activity of the iaaM enzyme, proRPS5A>>iaaM embryos and seedling roots showed enhanced DR5rev-GFP signals (Figures 3A, 3B, 3G, and 3H). However, the enhanced signals were restricted to cells that normally express the marker in control embryos. To test whether other cells in proRPS5A>>iaaM embryos also contain elevated auxin concentrations that might not be sufficient to activate the DR5rev-GFP reporter, embryos were treated with 10 µM of the synthetic auxin 2,4-D. Whereas 2,4-D treatment enhanced the DR5rev-GFP activity peak only in the basal region in wild-type embryos (Figure 3C), in proRPS5A>>iaaM embryos, the signal was increased in both the basal region and the provascular tissues (Figure 3I). The lower threshold auxin concentration for induction of the DR5rev promoter in proRPS5A>>iaaM embryos indicates that iaaM is also active in these cells and its substrate is not limiting for enhanced auxin biosynthesis. Exogenous application of high concentrations (1 mM) of Trp to developing seeds enhanced the DR5rev-GFP signal in iaaM-expressing embryos (Figure 3J) but not in wild-type embryos (Figure 3D). This corroborates our previous conclusion that the iaaM embryos express an active iaaM enzyme that is able to convert the Trp substrate into the auxin precursor indolacetamide. Although expression of the coregulated GUS gene in proDR5(7x)>>iaaL embryos (Table 2) was strongly reduced compared with proDR5(7x)>>GUS control embryos (Table 2), this reduced auxin activity could not be confirmed using the DR5rev-GFP reporter, even when embryos were cultured in medium containing Lys, auxin, or auxin transport inhibitors (see Supplemental Figure 3 online). It can therefore not be directly shown that iaaL is effective in embryos.
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). Surprisingly, the expression of iaaM from the strong ubiquitous RPS5A promoter caused enhanced auxin levels/activity only in those cells that normally express the marker; consequently, iaaM-expressing embryos are normal in phenotype. This suggests that embryos can regulate the activity or distribution of excess auxin to maintain the existing auxin gradients.
Maintenance of such gradients in the seedling root requires PAT activity (Friml et al., 2002). To test whether an auxin transport-dependent mechanism also operates to maintain embryonic auxin gradients, immature seeds containing either wild-type or proRPS5A>>iaaM embryos were cultured on growth media containing different concentrations of the PAT inhibitor NPA. If PAT is responsible for maintaining auxin levels in the proembryo at concentrations that allow normal pattern formation, treatment of proRPS5A>>iaaM F1 seeds with NPA is expected to lead to increased accumulation of auxin and thus to embryo phenotypes at lower NPA concentrations compared with the wild type. Culturing with 5 µM NPA enhanced the DR5rev-GFP signal in wild-type embryos (Figure 3E) but did not alter the embryo pattern, whereas only higher (up to 20 µM) concentrations caused fusion of cotyledons (Friml et al., 2003; Figure 3F). By contrast, treatment of proRPS5A>>iaaM F1 seeds with 5 µM NPA led to nearly complete fusion of cotyledons (Figures 3K and 3L), indicating that pattern formation in proRPS5A>>iaaM embryos is sensitized to inhibition of PAT. This result indicates that PAT activity buffers the normal distribution of auxin when auxin biosynthesis rates are changed.
PINs Maintain an Auxin Distribution Required for Apical and Basal Embryo Patterning
Four PIN proteins are active in the Arabidopsis embryo (Friml et al., 2003; Blilou et al., 2005). PIN7 is required for establishment of the preglobular auxin gradient, while PIN1 is involved in its reversal during the globular stage. PIN4 is expressed immediately after reversal of the gradient, and PIN3 is expressed even later (Friml et al., 2003).
Hence, the PINs most likely to be involved in maintenance of auxin gradients during the stages analyzed above are PIN1 and PIN4. To test whether these proteins are indeed involved in maintenance of auxin gradients, the sensitivity of pin mutant embryos to exogenously applied 1-naphthylacetic acid (1-NAA) was compared with that of wild-type embryos. Immature pin1-3 or pin4-3 mutant seeds, as well as seeds from wild-type plants, were cultured in the presence of 1-NAA or on control medium. Approximately half of the embryos homozygous for the pin1-3 mutation show weak fusion of cotyledons (Aida et al., 2002). A similar frequency of weak cotyledon fusion was observed when pin1-3 embryos were cultured on control media (Figure 4A). When cultured on 1-NAA, however, these apical defects were strongly enhanced and included complete fusion of cotyledons (Figure 4B). Similarly, whereas pin4-3 embryos have no apical embryonic defects (Figures 4C and 4D), culturing on 1-NAA induced apical asymmetry and cotyledon fusion (Figure 4E). These results suggest that both PIN1 and PIN4 are involved in maintaining the embryonic auxin gradient.
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), thus facilitating the interpretation of possible iaaM- or iaaL-induced pattern defects. Strikingly, while all controls showed <1% abnormal embryos (Table 1, Figure 4F), 9% of the pin4-3;proRPS5A>>iaaM embryos showed fusion of cotyledons (Figures 4G and 4H; Table 2). This result indicates that PIN4-mediated auxin transport is required to control auxin concentrations in the proembryo to allow apical patterning. Development of the basal embryo pole was not affected by proRPS5A>>iaaM expression in pin4-3 embryos (Figures 4G and 4H; Table 2). Likewise, the basal embryo pole developed normally when pin4-3 embryos were cultured in the presence of 1-NAA (Figure 4E). By contrast, 25% of the pin4-3;proRPS5A>>iaaL embryos showed deformations of the root pole at the late globular stage (Figures 4I and 4J). This result provides evidence that the iaaL gene is active in the embryo and more importantly that free auxin is required for regulation of division patterns in the basal region of the globular Arabidopsis embryo. In addition, these results show that PIN4 activity in the embryo maintains a differential distribution of auxin between the proembryo and hypophysis, which on one hand allows apical patterning and on the other hand regulates root pole patterning. |
DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMETHODSREFERENCES |
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), the general uniformity of the final shape suggests a robust and tightly controlled underlying regulatory mechanism. Pharmacological studies on embryos of several plant species and genetic studies in Arabidopsis have suggested an important role for auxin in the regulation of plant embryogenesis. Auxin activity, as measured by the expression of an auxin-dependent reporter gene, is highly dynamic during Arabidopsis embryogenesis, and the establishment of auxin gradients requires an intact polar transport system (Friml et al., 2003). Here, we have studied how these gradients are maintained. Our findings show that auxin biosynthesis and inactivation rates can be modified without an effect on auxin gradients or on pattern formation. However, altering auxin biosynthesis or conjugation rates under conditions where auxin transport is compromised inflicts changes in auxin gradients and embryo patterning. Our results show that auxin transport is crucial for maintenance of embryonic auxin gradients during pattern formation.
Embryonic Auxin Gradients and Pattern Formation Are Robustly Buffered
Postembryonic manipulation of auxin homeostasis using bacterial enzymes effectively alters auxin-mediated plant development, including elongation of hypopcotyl and petioles, outgrowth of lateral buds, and initiation of flowers (Romano et al., 1991, 1995; this report). It has recently been established that pattern formation in plant embryos also involves auxin activity. Nonetheless, strong expression of the same bacterial enzymes that interfere with postembryonic development does not interfere with embryo patterning. Because both the iaaM auxin biosynthesis enzyme and the iaaL auxin conjugation enzyme use an amino acid as a substrate, a trivial explanation would be that the substrate for these enzymes is lacking in embryos, whereas it is available postembryonically. Because of the fast growth rate of embryos and the associated requirement for de novo protein synthesis (Weijers et al., 2001), amino acids should be available in abundance. Yet it is conceivable that despite abundance, there is no excess and that most amino acids are efficiently shuttled into the protein biosynthesis pathway. Visualizing auxin distribution indirectly, using an auxin responsive reporter gene, showed that iaaM expression does increase cellular auxin concentrations. Additional Trp feeding experiments showed that Trp is used as a substrate and that endogenous Trp concentrations are not saturating for maximal iaaM activity. Nonetheless, the absence of iaaM-induced embryo defects is not due to unavailability of the Trp substrate but rather involves a mechanism that buffers the patterning process from alterations in cellular auxin concentration. For the iaaL enzyme, which uses Lys to inactivate IAA, we obtained indirect evidence for its activity in embryos. iaaL expression only affects auxin levels (as measured by altered auxin-dependent pattern formation) if auxin transport activity is compromised, and under those conditions, Lys concentrations in the embryo are clearly not limiting to its activity.
A buffering mechanism for auxin activity could involve flexible adaptation of de novo auxin biosynthesis, auxin inactivation, or auxin transport. The first two mechanisms are not well understood at the molecular level, and identification and functional analysis of critical components for auxin catabolism and biosynthesis in the embryo will be required to assess the exact contribution of these pathways. We found that PAT activity is crucial for maintaining embryonic auxin gradients when cellular auxin concentrations are changed.
Particularly, we observed that at least two of the embryonically active PIN proteins, PIN1 and PIN4, act in maintaining such gradients. This suggests that differential auxin distribution requires the continuous activity of PIN proteins in order to compensate for fluctuations in cellular auxin concentrations. Several interpretations could account for the flexible regulation of auxin transport activity, one being that differences in auxin concentration between adjacent cells are sensed and feed back on auxin efflux activity. Alternatively, cell-autonomous auxin-dependent regulation of PIN expression or activity could account for the observed flexibility.
It is likely that, in addition to auxin transport, other cellular homeostasis mechanisms contribute to maintenance of auxin gradients. When more auxin is transported to the maximum of the gradient, more auxin has to be inactivated there. Thus, robustness in auxin gradient maintenance will also require flexibility in auxin biosynthesis and degradation machinery, whose local activity is adjusted to the amount of auxin flowing through the gradient. Although this has not experimentally been tested, embryos may have considerable auxin biosynthesis and conjugation capacity.
PIN4 Fulfills a Similar Function in the Embryo and Seedling Root
Our results reveal a new function for PIN4 in the maintenance of embryonic auxin gradients. This function is not apparent during normal development of the pin4 mutant but is uncovered when mutant embryos are challenged with elevated or decreased auxin concentrations. It is difficult to judge what variations in auxin concentrations normally occur in plant development. Nonetheless, PIN4 function, in addition to that of PIN1, ensures that whatever variations exist, the shape of the embryonic auxin gradient is not altered.
There is an interesting parallel between the embryonic and the postembryonic PIN4 function. In postembryonic root tips, PIN4 is expressed in the quiescent center and the surrounding stem cells where it focuses auxin transport to form a DR5rev activity peak (Sabatini et al., 1999; Friml et al., 2002). In the pin4 loss-of-function mutant, DR5rev activity is still focused below the quiescent center, but it is generally enhanced and detectable in more distal cells, correlating with the elevated auxin concentrations in pin4 root tips (Friml et al., 2002). Likewise, in pin4 mutant embryos, DR5rev activity, which is normally restricted to the hypophysis derivatives in the wild type, is observed in the apical half of the embryo. Thus, in both situations, a localized activity of PIN4 at or adjacent to the maximum of the auxin gradient is required to prevent accumulation of auxin in distal cells. Although it has been suggested that auxin catabolism is induced in the auxin maximum in root tips (Jiang et al., 2003), at present it is unclear how such feedback control works, and better understanding will require the dissection of PIN protein activity and the identification of the regulators of these proteins.
Concluding Remarks
This work has identified that auxin-dependent pattern formation of plant embryos is highly flexible, in that it can accommodate changes in auxin levels without affecting the dynamic auxin gradients or pattern formation in the embryo. Our results show that the PIN auxin efflux facilitators not only play a central role in the establishment of these auxin gradients but that they are essential components in their robust maintenance as well. In view of our findings, there is a striking parallel between the responsiveness of embryonic and postembryonic auxin-dependent processes toward altered auxin concentrations and the degree to which the same processes can be modulated by environmental conditions. A future challenge will be to identify how other auxin homeostasis mechanisms are linked to the PIN auxin efflux network to create this robust buffering system.
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METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMETHODSREFERENCES |
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) and in vitro culturing of immature seeds (Friml et al., 2003) were performed as described. The pin4-3 mutant and DR5rev-GFP line were described previously (Friml et al., 2002, 2003). GAL4 enhancer trap lines Q0990, J3281, and M0167 were obtained from the Nottingham Arabidopsis Stock Centre. The KS22I enhancer trap line has been described previously (Boisnard-Lorig et al., 2001). For crossing experiments, T2 to T4 generation plants were used. Homozygotes for the T-DNA insertion locus were selected in the T3 and T4 generations. When T2 generation EF lines were used in crosses, a test cross between a single transgenic plant and a homozygous ACT RPS5A#5 line was performed to determine the frequency of GUS-positive embryos. The same plant was then used for subsequent crossing experiments. ACT lines and wild-type plants were used as female parents in all crosses, unless indicated otherwise.
Ovule culture experiments were performed as described (Sauer and Friml, 2004) using F1 seeds directly after crossing of ACT proRPS5A DR5rev-GFP and EF iaaM DR5rev-GFP plants or on a line homozygous for pin4-3 and DR5rev-GFP. Controls were parental lines used for crosses. Media were supplemented with 1-NAA (Duchefa; 5 or 10 µM), 2,4-D (Duchefa; 10 µM), NPA (Duchefa; 5 or 10 µM), or Trp (Roth; 1 mM).
T-DNA Constructs, Transgenic Plants, and Selection of Lines
All ACT lines except ACT proSTM and the EF GGi lines have been described (Weijers et al., 2003). For ACT proSTM, a 1.5-kb region upstream of the ATG start codon of the Arabidopsis STM gene (Long et al., 1996) was amplified with primers STM-5-HD1 (5'-GCAAGCTTCAGGGATAAACAGGTACAGG-3') and STM-3-BH1 (5'-CCGGATCCCTTCTCTTTCTCTCACTAG-3') with added HinDIII and BamHI sites (underlined), subcloned in pBluescript SK+, and cloned as a HinDIII-BamHI fragment upstream of mGAL4:VP16 in pSDM1600.
The iaaM coding region (position 5762 to 8076 according to Barker et al., 1983) was amplified by PCR from Ti plasmid pTi15955using Expand Taq polymerase (Roche Molecular Biochemicals) and the primers iaaM-F (5'-CGGTTGATGTGGTTATTTATCTACAC-3') and iaaMSacR (5'-ACGAGCTCCTAATTTCTAGTGCGGTAG-3'). The 2.3-kb PCR fragment was sequenced, digested with SacI, and cloned into the EcoRV-SacI sites of pSDM7022 (Weijers et al., 2003) to yield pIC proUAS-iaaM-tNOS (pSDM7009). A 2.7-kb HindIII fragment containing proUAS-iaaM-tNOS was cloned into pSDM7006 (Weijers et al., 2003) to yield pEF iaaM (pSDM7010). The iaaL coding region was isolated as part of a 1.8-kb BglII-BamHI iaaL-tNOS fragment from pMON690 (Romano et al., 1991) and fused to proUAS in pSDM7000 to yield pSDM7011 (Weijers et al., 2003). Next, the UAS-iaaL-tNOS cassette was isolated as a HindIII fragment and ligated into pSDM7006 to yield EF iaaL (pSDM7012). (Unfortunately, due to the license policy of Monsanto, no iaaL derivative constructs or plant lines can be distributed.) Constructs were electroporated into Agrobacterium tumefaciens strain LBA1115 as described previously (Weijers et al., 2001). Transgenic lines were analyzed in the T2 generation. To estimate the number of T-DNA copies in EF lines, DNA gel blot analysis was performed as described (Weijers et al., 2003) using gene-specific probes and a GFP probe. RT-PCR was performed on RNA isolated from young siliques using primer sets specific for iaaM or iaaL. The lines used for experiments (unless indicated otherwise) are as follows: ACT proRPS5A#5, ACT proLTP1#8, ACT proSTM#6, ACT proDR5(7X)#3, EF GFP:GUS#15, EF iaaM#5, and EF iaaL#18.
Auxin Quantification
proLTP1#8>>iaaM#20 F1 seeds were germinated on soft agar medium and transferred to fresh medium after 2 weeks. After another 2 weeks, plants were classified as wild-type (I), moderate (II), or severe (III) phenotypes (see Figure 1B). The plants within each class were pooled, and the auxin concentration was measured in five samples from collected leaves and apical regions as described (Edlund et al., 1995).
Fluorometric GUS Assays
For quantification of GUS activity in proLTP1#8>>iaaM#20 F1 plants, seeds were germinated on medium containing kanamycin and PPT. Resistant seedlings were transferred to soil after 2 weeks, and after another 2 weeks, GUS activity was quantified in triplicate in two parallel protein extracts from the aboveground portion of the pooled plants of three defined classes (see above and Figure 1B) according to Jefferson et al. (1987).
Phenotypic Analysis and Histochemistry
Upon crossing, embryo phenotypes and GUS activity were analyzed by differential interference contrast microscopy in cleared ovules as described (Weijers et al., 2001). Siliques in which the majority of embryos were younger than the transition stage were abandoned from the analysis. Consistently, no obvious phenotypes were observed at preglobular stages.
Microscopy and Photography
Embryos were viewed on a Zeiss Axioplan II microscope equipped with differential interference contrast optics. Images were acquired in Adobe Photoshop using a Sony DKC5000 digital camera. Seedlings were photographed using a stereomicroscope equipped with a Sony DKC5000 digital camera. All image compositions and contrast enhancements were performed using Adobe Photoshop 6.0 and Adobe Illustrator 10.
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Acknowledgments |
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Footnotes |
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Online version contains Web-only data. 
Article, publication date, and citation information can be found at www.plantcell.org/cgi/doi/10.1105/tpc.105.034637.
Received May 27, 2005; Revision received May 27, 2005. accepted June 25, 2005.
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REFERENCES |
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