Molecular Plant Advance Access originally published online on November 13, 2008
Molecular Plant 2008 1(6):1077-1087; doi:10.1093/mp/ssn071
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Membrane Steroid Binding Protein 1 (MSBP1) Stimulates Tropism by Regulating Vesicle Trafficking and Auxin Redistribution
National Key Laboratory of Plant Molecular Genetics, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, 300 Fenglin Road, 200032 Shanghai, China
1 To whom correspondence should be addressed. E-mail hwxue{at}sibs.ac.cn, fax +86-21-54924060, tel. +86-21-54924059.
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Abstract |
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 TOP Abstract INTRODUCTIONRESULTSDISCUSSIONMETHODSSUPPLEMENTARY DATAFUNDING |
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Overexpression of membrane steroid binding protein 1 (MSBP1) stimulates the root gravitropism and anti-gravitropism of hypocotyl, which is mainly due to the enhanced auxin redistribution in the bending regions of hypocotyls and root tips. The inhibitory effects by 1-N-naphthylphthalamic acid (NPA), an inhibitor of polar auxin transport, are suppressed under the MSBP1 overexpression, suggesting the positive effects of MSBP1 on polar auxin transport. Interestingly, sub-cellular localization studies showed that MSBP1 is also localized in endosomes and observations of the membrane-selective dye FM4-64 revealed the enhanced vesicle trafficking under MSBP1 overexpression. MSBP1-overexpressing seedlings are less sensitive to brefeldin A (BFA) treatment, whereas the vesicle trafficking was evidently reduced by suppressed MSBP1 expression. Enhanced MSBP1 does not affect the polar localization of PIN2, but stimulates the PIN2 cycling and enhances the asymmetric PIN2 redistribution under gravi-stimulation. These results suggest that MSBP1 could enhance the cycling of PIN2-containing vesicles to stimulate the auxin redistribution under gravi-stimulation, providing informative hints on interactions between auxin and steroid binding protein.
Key Words: MSBP1 • polar auxin transport • auxin response • gravitropism • vesicle trafficking
Received for publication August 10, 2008. Accepted for publication October 8, 2008.
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INTRODUCTION |
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TOPAbstractINTRODUCTIONRESULTSDISCUSSIONMETHODSSUPPLEMENTARY DATAFUNDING |
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Steroid hormones are low-molecular-weight compounds derived from isoprenoid, and have diverse roles in embryonic and postembryonic developments and adult homeostasis in animals (Clouse, 2002). The steroid binding proteins (SBPs), which are crucial in the effects of steroid hormones and mainly function as ligand-activated transcriptional regulators, are classified as membrane or nuclear SBPs based on the sub-cellular localizations in animal cells (Evans, 1988; Glass, 1994; Beato et al., 1995; Mangelsdorf et al., 1995). In the brassinosteroid (BR) signaling of higher plants, the membrane receptor is the predominant form of steroid signal perception (Wang et al., 2001). We recently identified a plant membrane-localized SBP (MSBP1) from Arabidopsis, which is homologous to the porcine membrane progesterone binding protein. The MSBP1 can bind to multiple steroid molecules including BR with different affinities (the highest affinity to progesterone) and negatively regulates cell elongation, and may play a role in plant photomorphogenesis (Yang et al., 2005).
Light induces phototropism, photomorphogenesis, chloroplast differentiation, and affects circadian rhythm and various developmental processes, and hence plays critical roles in plant growth. Plant cells can sense the direction, quality (wavelength), intensity, and periodicity of light, and adapt to the changed lights. Gravitropism is a process that dictates the growth of plant organs relative to gravity and ensures the roots will grow down into the soil, whereas shoots will grow upwards into the air (Perrin et al., 2005). Plant biologists have worked over a century on the mechanisms underlying the plant gravitropic response including perception of the gravity, transduction of the physiological and/or biochemical signals resulting in the differential growth and curvature formation.
There are two hypotheses on the mechanism of gravitropism: the starch–statolith hypothesis and the Cholodney–Went hypothesis (Blancaflor and Msaaon, 2003). Recently, analysis of the transcriptional responses to gravi-stimulation on genome level suggest the existence of the inositol 1,4,5-trisphospated (IP3)-dependent and -independent regulatory pathways (Perera et al., 2001), and Ca2+ is involved in the responses to gravi-stimulation in roots (Plieth and Trewavas, 2002).
In addition, polar auxin transport (PAT), which is mediated by the auxin influx and efflux facilitators and is responsible for the basipetal, long-distance movement of auxin from the apical site(s) of auxin synthesis, plays central roles in the gravity responses (Palme et al., 2006). Among the auxin efflux carriers, which are designed as PIN proteins, several of them are essential for root gravitropism. In Arabidopsis, PIN1 protein is required for basipetal auxin movement and crucial for gravitropic responses and photomorphogenesis (Rashotte et al., 2000; Friml, 2003); PIN2, which is localized to the upper membrane in epidermis/lateral root cap, is responsible for auxin basipetal transport towards the elongation zone (Muller et al., 1998). Overexpressed PIN2 results in the enhanced tropistic response, while pin2 mutant seedlings are agravitropistic. Moreover, gravi-stimulation would modulate PIN3 trafficking and target it preferentially to lower membrane of root cap columella to contribute the establishment of lateral auxin gradient across the root cap (Friml et al., 2002).
Recent studies showed that PIN1 protein cycles between the plasma membrane and endosomal compartments in a vesicle trafficking-dependent manner, which is of great significance to its polar localization and subsequent auxin efflux (Geldner et al., 2001). In addition, PIN2 and PIN3 also have a constitutive intracellular cycling (Dhonukshe et al., 2007; Friml et al., 2002). There are at least two distinct endosomal routes for the recycling or trafficking of auxin transport components: one is involved in GNOM-containing endosomes (by PIN1 and partially by PIN2), and the second uses AtSNX1-containing endosomes (by PIN2, but not PIN1 or AUX1). This dual option of regulating polar auxin trafficking ensures a fine control of auxin levels and auxin carriers at the sub-cellular level (Jaillais et al., 2006, 2008).
The common physiological effects including stimulating cell division and elongation by auxin and BR have been known for many years and uncovering the underlying mechanism has become an exciting research area. Although it has been shown that BR and auxin can cross-talk at different levels, including biosynthesis, traffic, and signal transduction (Bao et al., 2004; Goda et al., 2004; Mouchel et al., 2006; Hardtke, 2007; Vert et al., 2008), there are still many unclear questions owing to the complexity of the temporal and spatial regulation of them. Recently, Li et al. (2005) reported that BR is involved in the root gravitropic response regulation of Arabidopsis by modulating polar auxin transport.
We here show that MSBP1 stimulates the root gravitropism through enhancing the polar auxin transport. MSBP1 is localized in vesicle and overexpression of which increases the vesicle trafficking to stimulate the auxin asymmetric distribution under gravi-stimulation. The study will provide new insights into the function of plant SBPs, especially the cross-talk with auxin.
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RESULTS |
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Enhanced MSBP1 Stimulates Gravitropic Response and Auxin Redistribution under Gravi-Stimulation
Based on the facts that BR can enhance the gravity response and MSBP1 binds to steroid molecules, we firstly detected the effects of MSBP1 in gravitropism through testing the gravitropic response of seedlings with altered expression of MSBP1. Due to the lack of MSBP1-defective insertion mutant, transgenic seedlings with antisense MSBP1 expression, which results in the defective MSBP1 expression, were used (Supplemental Figure 1). The observations showed that, compared with controls, MSBP1-overexpressing or -deficient seedlings had enhanced or reduced root gravitropism and hypocotyl anti-gravitropism, respectively (Figure 1A). Detailed measurement on the curvatures of roots and hypocotyls showed that, after 90° re-orientation for 9 h, MSBP1-overexpressing seedlings showed 64.0° (L18) and 60.8° (L6) of root curvatures and 60.7° (L18) and 59.8° (L6) of hypocotyl curvatures, while the control plants curved only 51.3° in roots and 48.0° in hypocotyls. By contrast, the MSBP1-deficient seedlings bent more slowly, 45.9° (L4) and 47.7° (L2) in roots, 40.2° (L4) and 41.9° (L2) in hypocotyls, respectively.
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It has been shown that the plant curved growth is mainly due to the asymmetric distribution of auxin between the two sides of the curving organ (Went, 1937). The altered gravitropism suggested that MSBP1 may boost the formation or the perception of auxin gradient in the root tip. Indeed, through genetic crossing with the DR5-GUS marker line (a synthetic promoter that is sensitive to auxin in a dosage-dependent manner and its activity is thought to reflect the endogenous auxin levels, Ulmasov et al., 1997), the observations of the cross-progenies revealed the altered auxin distribution in root tip after gravi-stimulation under MSBP1 overexpression or deficiency.
Comparing to the normal situation (Figure 1B, 1), after 4 h gravi-stimulation, most MSBP1-overexpressing seedlings have a streak of auxin redistribution extending basipetally from the tip along the lower side of the root (Figure 1B, 3, 4), whereas only 
15% DR5-GUS control plants showed the asymmetric auxin distribution (Figure 1B, 2) and that was almost undetectable in the MSBP1-deficiency plants. After 6 h gravi-stimulation, 47% DR5-GUS control plants had extending auxin distribution basipetally along the lower side, and a small proportion of MSBP1-deficient plants were found with asymmetric auxin distribution, implying that MSBP1 stimulates asymmetric auxin distribution under gravi-stimulation.
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MSBP1 Localizes in Vesicle and Positively Regulates Vesicle Trafficking
Based on the observation that auxin distribution was modulated under altered MSBP1, whether polar auxin transport was affected was further assessed. Analysis on the root growth under NPA treatment showed that, compared to the significantly inhibited primary root growth of control seedlings (in a dose-dependent manner), the inhibitory effects of NPA are much suppressed in MSBP1-overexpressing seedlings and MSBP1-deficient lines are much more sensitive to NPA inhibition (Figure 2A). In addition, MSBP1-overexpressing seedlings showed much faster gravitropic response than control plants when grown in the presence of NPA (Figure 2B). Therefore, it is probably prudent to assume that MSBP1 influences gravitropsim through affecting polar auxin transport.
Our previous studies showed that MSBP1 is located at plasma membrane. The detailed studies on the fluorescence of fused MSBP1–EGFP revealed that MSBP1 is located at both plasma membrane and endosomes (Figure 3A, 1, 4), which was confirmed by observing the internalization (Figure 3A, 2, 5) and merging (Figure 3A, 3, 6) with endocytosis and vesicle-selective dye FM4-64 (a powerful experimental water-soluble and membrane selective fluorescent dye that has been frequently used as a vesicle trafficking marker to monitor the endocytosis in mammalian, fungal, and plant cells, Bolte et al., 2004). To date, four classes of endocytosis signals targeting surface proteins to clathrincoated vesicles have been identified, and analysis of the MSBP1 protein revealed the presence of multiple Tyrosine-based signals of NPXY or YXXØ sequence (Figure 3B; X can be any amino acid, Ø is an amino acid with a bulky hydrophobic group), which were initially identified in LDLR and transferrin receptor, respectively (Li et al., 2000), being consistent with the localization of MSBP1 in vesicles.
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Observation of the internalization of FM4-64 revealed that vesicle trafficking in Arabidopsis root cells was evidently increased under MSBP1 overexpression (Figure 4A, 2, 3 and 7, 8 respectively) and was suppressed in MSBP1-deficient plants (less fluorescently labeled vesicles, Figure 4A, 4, 5, 9, and 10), compared to that in control seedlings (Figure 4A, 1, 6). This suggests that MSBP1 positively regulates vesicle trafficking in root cells.
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MSBP1 Suppresses BFA Effects
As auxin efflux carrier PIN proteins cycled between internal compartments and the plasma membrane, the localization and stimulating roles of MSBP1 in vesicles suggest that MSBP1 may regulate polar auxin transport through modulating vesicle trafficking. To test this, the fungal macrocyclic lactone brefeldin A (BFA), an inhibitor of protein trafficking in the endomembrane system of mammalian cells (Sciaky et al., 1997) and has been extensively used to study the vesicle trafficking-related processes in plant cells (Geldner et al., 2003), was used.
BFA specifically blocks cell exocytosis but allows endocytosis, resulting in the internalization and accumulation of recycling plasma membrane proteins in BFA compartments (Nebenfuehr et al., 2002). Auxin efflux carriers including PIN1 and PIN2 (Geldner et al., 2003; Grebe et al., 2003) have been detected in BFA compartments. Studies on the BFA compartment formation showed that, after BFA treatment (50 µM) for 2 h, two or three BFA compartments were formed in almost every root cell of control (Figure 4B, panel 1) and MSBP1-deficient seedlings (the size was much smaller, Figure 4B, panels 4 and 5); however, the BFA compartments were much less formed in root cells of MSBP1-overexpressing seedlings (Figure 4B, panels 2 and 3).
The suppressed BFA effects under MSBP1 overexpression were further confirmed by physiological studies. High concentrations of BFA lead to growth arrest of cells, probably due to the severe block of intracellular trafficking, which inhibits the fundamental processes such as cell-plate expansion during cytokinesis (Yasuhara and Shibaoka, 2000); in contrast, low concentrations BFA have surprisingly specific effects on auxin transport-related processes such as root hair cell polarity, root and hypocotyl elongation, gravitropism, or initiation of lateral root primordial (Geldner et al., 2001). Measurement on the lengths of roots and hypocotyls of seedlings grown on the vertical agar plates supplemented with low concentration of BFA (10 µM, dark condition) showed that, compared with control, MSBP1-overexpressing seedlings were less sensitive, while the MSBP1-deficient seedlings were significantly more sensitive to BFA inhibition, respectively (Figure 5).
Enhanced MSBP1 Stimulates PIN2 Cycling and Distribution
Effects of MSBP1 on auxin efflux carriers, especially PN2, were further studied. To achieve this, PIN2-enhanced green fluorescent protein (PIN2::PIN2–EGFP, simplified as PIN2–EGFP) expression cassette (Xu and Scheres, 2005) was individually transferred into MSBP1-overexpressing or -deficient lines through genetic crossing. Semi-quantitative RT–PCR analysis showed that PIN2 expression was not changed under altered MSBP1 expression (Figure 6A, left panel), and there was no difference on the polar localization of PIN2 by microscopic observation (Figure 6A, right panel).
In roots, within 1–2 h of gravi-stimulation, asymmetric PIN2 distribution can be detected, namely PIN2-specific signals were weaker at the upper side than the lower side of horizontally positioned roots, and the difference was most pronounced in epidermal cells (Abas et al., 2006). Microscopic observation showed that the differential distribution of PIN2 at upper or lower sides was not distinct within 60 min gravi-stimulation in control seedlings (Figure 6B, 1), while that was much pronounced in epidermal cells of MSBP1-overexpressing ones (Figure 6B, 2, arrows), and there was no difference in MSBP1-deficient lines (Figure 6B, 3).
It has been shown that the cycling of PIN1 and PIN2 is vesicle-dependent. Compared to the appearance of two or three large BFA compartments after BFA treatment (2 h, Figure 6C, 1), the BFA-induced cytoplasmic aggregation of PIN2–EGFP was evidently suppressed under MSBP1 overexpression (Figure 6C, 2, 3). In MSBP1-deficient lines, PIN2 coalesced into patches similar to control, though the compartments were smaller in size and fewer in number (Figure 6C, 4, 5). In addition, most BFA compartments disappeared and polar localization of PIN2 proteins was recovered after BFA washout for 1.5 h in control lines (Figure 6C, 6), while the PIN2 proteins resumed the polar localization in MSBP1-overexpressing lines was enhanced, namely within 1–1.5 h (Figure 6C, 7, 8). In contrast, accumulated PIN2–EGFP in MSBP1-deficient lines could not be resumed efficiently (Figure 6C, 9, 10), confirming the MSBP1 function in stimulating vesicle trafficking.
MSBP1 Stimulates IAA Response
Whether MSBP1 affects the auxin response was further studied. When grown in the light at high temperature (29°C), Arabidopsis seedlings exhibit dramatic hypocotyl elongation compared with those grown at 20°C (Gray et al., 1998). This auxin-dependent response was suppressed in the MSBP1-deficient seedlings (although not as strongly as tir1-1), and much enhanced under MSBP1 overexpression (Figure 7A). Previous studies indicated that the temperature-induced hypocotyl elongation correlates with an increase in IAA levels that depends on the auxin transport (Gray et al., 1998). These results further suggest that MSBP1 is involved in auxin response by regulating auxin transport.
Measurement of roots growth under exogenous IAA showed that the inhibitory effects of auxin on root growth were enhanced in MSBP1-overexpressing seedlings and suppressed in MSBP1-deficient ones. At 0.1 nM IAA treatment, the root length of MSBP1-overexpressing seedlings was suppressed while that of control seedlings was still promoted. At 0.1 µM IAA, MSBP1-deficient seedlings showed slight inhibition of root growth, 91% (L4) of the untreated ones, while the control is inhibited to 72% and the MSBP1-overexpressing one is 55% (L6). These provided further evidence for the involvement of MSBP1 in auxin responses, besides the polar transport.
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DISCUSSION |
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TOPAbstractINTRODUCTIONRESULTSDISCUSSIONMETHODSSUPPLEMENTARY DATAFUNDING |
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SBPs are essential for growth and development in animals, and play critical roles in steroid hormone signal transduction through interacting with the specific receptors on the plasma membrane of target cells (Rosner et al., 1999; Breuner and Orchinik, 2002). In higher plants, the SBPs have been recently annotated but only limited knowledge on their functional and biochemical characters has been obtained. It has been shown that one of them, MSBP1, can bind steroid molecules and is involved in plant growth regulation as a negative regulator of cell elongation (Yang et al., 2005). We here illustrate that MSBP1 is involved in the regulation of vesicle trafficking, PIN2 cycling, and plant gravitropic response, providing hints of cross-talk between SBPs and polar auxin transport.
MSBP1 Locates in Vesicles
The first membrane progesterone receptor complex 1 (PGRMC1), homologue of MSBP1 in animals, has altered sub-cellular locations under certain circumstances. PGRMC1 contains the endocytosis signal YXXØ motif (Cahill, 2007), and existence of YXXØ motif in MSBP1 suggested its localization in vesicles. Indeed, co-localization analysis with FM4-64 dye confirmed the localization of MSBP1 in vesicles, suggesting the possible link between MSBP1 and vesicle trafficking. The enhanced vesicle trafficking and resistance to BFA under MSBP1 overexpression indicate that MSBP1 may be involved in multiple vesicle trafficking-related processes, including polar auxin transport, through regulating vesicle trafficking, providing new insights into the functional mechanism of steroid binding proteins.
MSBP1 Regulates PIN2 Cycling and Gravitropic Response through Stimulating Vesicle Trafficking
The enhanced or suppressed gravitropic responses under altered MSBP1 expression suggest a functional link of SBPs with plant tropism. Indeed, MSBP1 enhanced auxin asymmetric distribution under gravi-stimulation, and observation on the up-ground of cross-progenies with DR5–GUS seedlings showed that GUS activities were increased (occurring at cotyledon edges, even the entire cotyledon, and slightly in the root elongation region) under MSBP1 overexpression (Supplemental Figure 2), confirming that MSBP1 plays a positive role in modulating auxin transport.
The polar auxin transport is mediated by plasma membrane-localized auxin efflux carrier proteins (PINs, Muday and Delong, 2001, 2002), and the cycling of PIN proteins (PIN1 and PIN2) between membrane and endosomal compartments, which is dependent on vesicle trafficking (Geldner et al., 2001, 2003; Grebe et al., 2003; Muday et al., 2003; Jaillais et al., 2006), is important for their functions. It was proposed that a positive role of MSBP1 in vesicle trafficking is at least partially correlated with its beneficial effect on the rapid cycling of PIN2, enhanced auxin transport/distribution and gravitropic responses.
BR stimulates root gravitropic response through enhancing polar auxin transport (Li et al., 2005), and MSBP1 could bind BR in vitro and be involved in BR signaling (Yang et al., 2005). However, considering the negative role of MSBP1 in BR signaling and the low affinity of MSBP1 to BR, it was supposed that MSBP1 stimulates the polar auxin transport mainly through enhancing the vesicle trafficking, rather than the BR signaling.
Besides PIN2, whether MSBP1 affects other auxin efflux carriers needs to be further investigated, to provide more information on the effects of steroid binding protein on auxin transport and auxin-mediated growth regulation.
MSBP1 Is Involved in Auxin Response Regulation
Besides the effects on polar auxin transport, whether MSBP1 is involved in auxin signal transduction needs to be considered before making the appropriate decision. Modulated auxin responses (Figure 7), as well as the altered expression of auxin-responsive genes, revealed by chip hybridization (Supplemental Table 1), suggest this cross-talk is intricate. However, there is no evidence showing that MSBP1 is directly involved in auxin response. Further studies to examine whether other SBPs are involved in auxin responses will provide more information.
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Recent studies showed that chemical endosidin 1 (ES1) blocked the endocytosis of PIN2 and AUX1, which traffic through a TGN/endosome compartment containing the proteins SYP61 and VHA-a1. The BRI1 receptor also traffics through this SYP61/VHA-a1 endosomal compartment, indicating that at least three plasma membrane (PM) proteins share overlapping endocytic pathways (Robert et al., 2008), illustrating a possible relationship between BR signaling and auxin transport, which enlightens us to explore the cross-talk of MSBP1 with auxin.
Thinking back to the fundamental role of steroid binding proteins, the more we know about the SBPs in plants, the more we probably know about the steroids, as most of the animal steroid hormones were found in plants, though lots of them were poorly understood. In rats, astrocyte-derived estrogenic enhanced synaptic transmission was at least mediated by strengthening presynaptic vesicle release (Hu et al., 2006), which generates some ideas for further work on the roles of steroid hormones in higher plants.
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TOPAbstractINTRODUCTIONRESULTSDISCUSSIONMETHODSSUPPLEMENTARY DATAFUNDING |
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Chemical Reagents
All enzymes used for DNA manipulation were purchased from Fermentas. DNA primers were synthesized commercially (Sangon). IAA (I-2886) and Brefeldin A (BFA, B-7651) were purchased from Sigma–Aldrich (Missouri, USA). 1-N-naphthylphthalamic acid (NPA, N0926) was purchased from Duchefa Biochemie (Haarlem, The Netherlands). FM4-64 was purchased from Molecular Probes (Oregon, USA).
Constructs and Transformation
The whole MSBP1 cDNA was amplified by primers MSBP1-1 (5'-GCTCTAGAATGGCGTTAGAACTATGGC-3', added XbaI site underlined) and MSBP1-2 (5'-ATTTGCGGCCGCCTACTCCTCCTTCTTCAAC-3', added NotI site underlined), and sub-cloned into pATC940 (p35S::MSBP1). Constructs pCAMBIA1302–MSBP1–GFP and pCAMBIA1301–MSBP1 (p35S::A-MSBP1), which are used for sub-cellular localization analysis and generating antisense transgenic plants, respectively, were described previously (Yang et al., 2005).
Bacterial, Plant Materials, and Growth Conditions
Escherichia coli strain XL1-Blue MRF was used for DNA amplification and sub-cloning. Agrobacterium tumefaciens strain GV3101 was used for plant transformation. The Arabidopsis Columbia ecotype was used as control and for transformation in the study. Seeds were sterilized and germinated on MS medium supplemented with sucrose (2%, w/v) and bacto-agar (1%, w/v). Plates were stored at 4°C in darkness for 48 h for stratification, and then transferred to phytotron (seedlings were grown vertically) under 22°C with a 16-h light/8-h dark cycle.
For IAA assay, seeds were sown on MS medium with gradients of IAA (0.01, 0.1, 10, 100, 1000 nM), and 8-day-old vertically grown seedlings in light were used for measuring the primary root length. For BFA treatment, 3-day-old seedlings were transferred onto medium supplemented with 10 µM BFA and grew vertically for another 6 d, then lengths of primary roots were measured and calculated. For hypocotyl measurement, after stratification, seeds were germinated under a 16-h light/8-h dark cycle for 1 d, followed by growing in dark for another 7 d (supplemented with 10 µM BFA in the medium), and then lengths of hypocotyls were measured and calculated. For NPA treatment, seeds were sown on MS medium with gradients of NPA (0.1, 1, 5, 10 µM), and 7-day-old vertically grown seedlings in light were used for measurement of the primary root lengths. At least 30 seedlings of each line were measured in these experiments with the aid of the image-analysis program ImageJ (version 1.34 for Windows; http://rsb.info.nih.gov/ij/) and statistical analysis was performed using a one-tailed student's t-test. For each experiment, three independent experiments were performed, which produced similar data and statistical analysis results.
For hypocotyl elongation under high temperature, control and MSBP1-overexpressing and -deficient seedlings were germinated and vertically grown at 22 or 29°C for 9 d, followed by measurement of hypocotyl lengths and statistical analysis (n > 30).
Genetic Cross
Genetic cross was carried out to transfer the DR5–GUS and PIN2–EGFP (PIN2::PIN2–EGFP) cassette individually into p35S::MSBP1 (L6, L18) and p35S::A-MSBP1 (L2, L4) plants. Homozygous cross offspring were used for further analysis.
Root Gravitropism Assay
Four-day-old vertically grown Arabidopsis seedlings with similar root lengths were selected and transferred to fresh MS medium to make sure their root tips were vertically downward. Seedlings were reoriented by 90° in darkness and photographed at an interval of 3 h with a Nikon Coolpix 4500 digital camera. The angles of root curvature were measured at 3, 6, 9, and 12 h, and hypocotyl curvature were measured at 6, 9, 12, and 24 h after reorientation with the aid of the image-analysis program Image J. Experiments were repeated three times (n > 30) and statistically analyzed.
For DR5–GUS, DR5–GUS/p35S::MSBP1, and DR5–GUS/p35S::A-MSBP1 seedlings, after reorientation of 90° for 4 and 6 h, the roots were subjected to GUS staining solution for 3 h. After clearing, roots were photographed by the Interference Discrepancy Contrast Microscope system (Leica) and observed. Each experiment was repeated three times.
For NPA assay, 4-day-old dark-grown Arabidopsis seedlings with similar root lengths were selected and transferred to fresh MS medium supplemented with 0.1 µM NPA and grown for another 1 d, then reoriented by 90° in dark and photographed at 7 or 10 h.
FM4-64 Staining and Confocal Microscopy Observation
Roots of 4-day-old vertically grown wild-type, MSBP1-deficient or -overexpressing plants were incubated with FM4-64 (5 µM) for 10 min at room temperature, followed by washing with water three times. The fluorescence was observed 15 or 30 min after washing with a confocal laser scanning microscope (FITC488, Zeiss LSM500).
FM4-64 fluorescence was excited with a 543-nm argon ion laser and a 600-nm long-pass emission filter. All images within a single experiment were captured with the same settings. For BFA treatment, the roots were treated with BFA (50 µM) for 2 h, then incubated with solution containing BFA (50 µM) and FM4-64 (5 µM) for 30 min, followed by examination with a confocal laser scanning microscope. Each experiment was repeated three times.
Visualization of PIN2 Distribution and Cycling
The homozygous progenies of cross-lines were used for observation. Visualization was performed with a confocal laser scanning microscope at a wavelength of 488 nm (EGFP) and 4-day-old seedlings were observed after re-orientation by 90° for 1 h. For BFA treatment, seedlings were pretreated with BFA (50 µM) for 2 h, and observed immediately or after washout for 90 min.
Semi-Quantitative RT–PCR Analysis
Arabidopsis seedlings were grown on MS medium for 7 d under a 16-h light/8-h dark cycle. Total RNAs were extracted with TRIzolR reagent (Invitrogen, Carlsbad, CA, USA) and reverse-transcribed (Toyobo, Osaka, Japan). Equal amounts of first-strand cDNAs were used as templates for PCR amplification. The Arabidopsis ACTIN2 gene (At3g18780) was amplified using primers Actin-1 (5'-TCTTCTTCCGCTCTTTCTTTCC-3’) and Actin-2 (5'-TCTTACAATTTCCCGCTCTGC-3’), and used as internal positive control to quantify the relative amounts of cDNA. The primers used were as follows: MSBP1 (At5g52240, MSBP1-1 and 1-2, see above), PIN2 (At5g57090, 5'-CACGATCTGCACCATTAGGTTAC-3', and 5'-CACTTCCTCGCTGCTGATTCTC-3’).
Particle Bombardment-Mediated Transient Expression in Onion Epidermal Cells
Transient expression of proteins in onion epidermal cells was performed with a Model PDS-1000/He Biolistic Particle Delivery System (BioRad, CA, USA). The plasmids coated on gold particles were bombarded with the following parameters: 1100 psi rupture disc; 27 in. Hg vacuum; and 6 cm distance from the stopping screen to the target tissues. After bombardment, onion pieces were incubated on solid MS medium at 25°C under darkness for at least 24 h.
For FM4-64 staining, onion epidermis was incubated in distilled water containing FM4-64 (5 µM, final concentration) at room temperature. Stained samples were washed twice with water and then observed.
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SUPPLEMENTARY DATA |
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TOPAbstractINTRODUCTIONRESULTSDISCUSSIONMETHODSSUPPLEMENTARY DATAFUNDING |
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Supplementary Data are available at Molecular Plant Online.
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FUNDING |
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TOPAbstractINTRODUCTIONRESULTSDISCUSSIONMETHODSSUPPLEMENTARY DATAFUNDING |
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This work was supported by the National Natural Science Foundation of China (No. 90717001, 30721061, 30425029) and Science and Technology Commission of Shanghai Municipality (08XD14049).
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Acknowledgements |
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We thank Jian Xu (Utrecht University, Netherlands) for providing Arabidopsis seeds containing DR5–GUS and PIN2–EGFP expression cassettes. No conflict of interest declared.
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