Plant shoots do not respond when they are reoriented relative to gravity at 4 °C. However, when returned to vertical at room temperature, these organs bend in response to the previous cold gravistimulation. The inflorescence stem of the Arabidopsis thaliana gravity persistent signal (gps) mutants respond abnormally after the cold gravistimulation: gps1 does not bend when returned to room temperature, gps2 bends the wrong way and gps3 over-responds, curving past the predicted angle. In wild type and the mutants, basipetal auxin transport in the inflorescence stem was abolished at 4 °C but restored when plants were returned to room temperature. In gps1, auxin transport was increased; in both gps2 and gps3, no significant difference was found when compared to wild type. Expression of the auxin-inducible PIAA2::GUS reporter gene, indicated that auxin-induced gene expression was redistributed to the lower side of the inflorescence stem in wild type after gravistimulation at 4 °C. In gps1, no asymmetries in PIAA2::GUS expression were seen. In gps2, PIAA2::GUS expression was localized to the upper side of the stem and in gps3, asymmetric PIAA2::GUS expression was extended throughout the elongation zone of the inflorescence stem. These results are consistent with altered lateral Indole-3-acetic-acid (IAA) gradients being responsible for the phenotype of each mutant.
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Gravity plays a major role in the life and development of all living organisms. In response to environmental and developmental factors, plant organs orient their position with respect to the gravity vector (Digby & Firn 1995). The gravity response involves perception of a biophysical stimulus and transformation of that perception into physiological signals that result in differential growth across plant organs. Plants perceive gravity through sedimentation of dense organelles, usually amyloplasts (reviewed by Kiss 2000). In roots, perception occurs in the innermost columella cells of the root cap (Blancaflor, Fasano & Gilroy 1998). In shoots, the inner layer of the cortex (the starch sheath or endodermal layer) is responsible for gravity perception (Fukaki et al. 1998; Fukaki & Tasaka 1999). Unlike roots, where perception is restricted to the root cap, perception in shoots occurs throughout the elongation zone (Fukaki, Fujisawa & Tasaka 1996; Weise & Kiss 1999). The sedimentation of amyloplasts is proposed to trigger the biochemical signal transduction events that induce differential growth.
The plant growth regulator auxin is believed to be responsible for the differential growth that occurs in the elongation zone of both shoots and roots in response to a gravitropic stimulus (reviewed by Muday 2001; Blancaflor & Masson 2003). The Cholodny-Went hypothesis states that auxin is redistributed to the lower side of gravistimulated organs, causing an increase in auxin concentration on the lower side, resulting in increased elongation in the shoot and inhibition of elongation in the root; this asymmetric growth results in the bending of the gravistimulated organ (reviewed by Muday 2001). Indole-3-acetic acid (IAA), the predominant naturally occurring auxin, is transported in a basipetal manner from cell to cell in plants (Lomax, Muday & Rubery 1995). The movement of auxin from the shoot apex to the rest of the plant is brought about by the auxin efflux and influx facilitators, which allow for polar movement of auxin from cell to cell (Muday & DeLong 2001). In roots, the auxin from the shoot moves acropetally through the vascular cylinder to the root tip, where it is redirected basepitally through the cortex and epidermis (Muday & DeLong 2001). This basipetal auxin transport is necessary for root gravitropic response (Rashotte et al. 2000; Muday 2001) and provides a mechanism for the establishment of the auxin gradient seen across the elongation zone of the root after gravistimulation (Young, Evans & Hertel 1990; Larkin et al. 1996; Rashotte, DeLong & Muday 2001).
Although auxin is clearly involved in the gravitropic response of inflorescence stems (Fukaki et al. 1996), the process of establishing the gradient and the role of basipetal auxin transport are not well characterized in this plant organ. An auxin gradient across the elongation zone does occur in association with shoot and coleoptile gravitropism (Parker & Briggs 1990; Li, Hagen & Guilfoyle 1991; Kaufman et al. 1995; Philippar et al. 1999; Long et al. 2002), but a unilateral redirection of basipetal auxin transport does not provide a mechanism as it does in the root, rather auxin is likely to be redistributed at multiple positions along the shoot axis. Redistribution of PIN3, an auxin efflux facilitator protein, may be specialized for lateral transport in the stem and thereby facilitate lateral auxin transport (Friml et al. 2002).
To identify components of the gravity signal transduction pathway that link amyloplast sedimentation and auxin transport, the gravity persistent signal (gps) mutants were isolated using a cold effect to separate the perception events from the response (Wyatt et al. 2002). Plants perceive the gravity stimulus at 4 °C, but do not respond; however, a response to that stimulus occurs when plants are returned to a vertical orientation at room temperature (Fukaki et al. 1996). In contrast, after a cold gravistimulation and a return to vertical at room temperature [the gravity persistent signal (GPS) treatment], the gps1 mutant failed to bend, gps2 bent in the wrong direction and gps3 continued to bend past the expected curvature (Wyatt et al. 2002). Wyatt et al. (2002) showed that amyloplast sedimentation was normal in all three gps mutants after GPS treatment, indicating that the perception was not affected by the mutation. Basipetal auxin transport in wild-type Arabidopsis was abolished at 4 °C; but, auxin transport was restored when plants were returned to room temperature (Wyatt et al. 2002). These data suggest that the GPS response was caused by an event or events that link gravity perception to auxin transport.
Because the mutations in all the gps mutants affected the events prior to auxin transport, basipetal auxin transport studies were performed to assess the affect of the gps mutations on auxin transport. In addition, we report the effect of the gps mutations on the asymmetric expression of an auxin-responsive promoter fused to a β-glucuronidase (GUS) reporter gene across the Arabidopsis inflorescence stems. Both measurement of basipetal auxin transport and assessment of lateral gradients of auxin-induced gene expression have provided insights into the effects of the gps mutations on the establishment of the auxin gradient after gravistimulation in inflorescence stems.
MATERIALS AND METHODS
Seed from wild-type ecotype Wassilewskija (WS), Arabidopsis and each gps mutant were soaked in 70% ethanol for 2 min and then surface-sterilized with 30% bleach for 30 min. The seeds were washed five times with distilled water and re-suspended in 0.1% type M agar. Seeds were sown into wet perennial mixture (Scotts-Sierra Horticultural Products Company, Marysville, OH, USA) using a pipette and the flats were covered with plastic wrap until the seeds germinated. The plants were grown under long-day conditions (16 h of light and 8 h of darkness), 60 µmol s−1 m−2 light.
Gravistimulation of plants
Arabidopsis plants were grown in soil until the inflorescence stems reached 8–10 cm. Plants were subjected to one of the following three treatments, after which, tritiated IAA transport or PIAA2::GUS gene expression were assayed: (1) gravistimulated at 4 °C for 1 h, followed by room-temperature analysis (GPS treatment). (2) gravistimulated at 4 °C for 1 h, followed by 4 °C analysis; or (3) subjected to constant gravistimulation at room temperature with room-temperature analysis.
Basipetal auxin transport
Basipetal auxin transport was measured by pulse chase as described (Rashotte et al. 2003) with slight modifications. These assays were performed with 3-[5(n)-3H] indolylacetic acid, which contains the tritiated IAA label on the indole ring (Amersham Pharmacia Biotech, Piscataway, NJ, USA). Nine plants were used for each treatment per experiment, with the experiment repeated three times. After gravistimulation at either 4 °C or room temperature, a 2.5 cm segment was cut 5 cm from the base of the inflorescence stem. This segment represents the elongation zone of the stem. The stem segments were inverted and placed in an Eppendorf tube (Fisher Scientific, Hampton, NJ, USA) with 15 µL 3H-IAA solution (400 nm3H-IAA in 1.05 µm cold IAA with 5 mm 2[N-morpholino] ethanesutortic acid (MES), 1% sucrose) for 10 min with the top end submerged in the solution. The segments were removed from the 3H-IAA and the tip was washed with buffer (5 mm MES, 1% sucrose) to remove any residual 3H-IAA. The segments were transferred to 15 µL of cold IAA solution (1.45 µm cold IAA in 5 mm MES, 1% sucrose) for 50 min. The segments were then cut into 0.5 cm sections. These were placed in 2.5 mL scintillation vials containing scintillation fluid (Universal Liquid Scintillation Counting Cocktail, Fisher Scientific, Hampton, NJ, USA), and the radioactivity in each of the sections was measured using the scintillation counter (LS 6500, Beckman Coulter, Fullerton, CA, USA).
For the 4 °C treatment, the plants were gravistimulated and cut in a cold room and the assay was performed at 4 °C using pre-chilled 3H-IAA (4 °C). For basipetal auxin transport at room temperature, plants were gravistimulated at room temperature for 10 min and auxin transport was measured. The amount of auxin transported after gravistimulation at room temperature was judged to be equivalent to the amount transported after return to room temperature in the GPS treatment (data not shown).
The statistical significance of these values was determined using a Student's t-test. The 3H-IAA transported in each segment was compared between wild type and each mutant.
Auxin-responsive GUS expression in the inflorescence stems
To identify an appropriate Arabidopsis transgenic line containing an auxin-responsive promoter: GUS fusion functional in the inflorescence stem, we obtained seeds of several fusion lines (DR5::GUS, SAUR::GUS, PIAA3::GUS and PIAA2::GUS) (Tian, Uhlir & Reed 2002). Seeds from the fusion lines was planted as described previously and grown until the inflorescence stems reached 8–10 cm. The elongation zone of the inflorescence stem, 3–5 cm from shoot apex, was cut and washed in 0.1 m phosphate buffer, pH 7.0 (Sigma, Saint Louis, MO, USA). Immediately, the tissue was covered with a minimal volume of 2 mm X-Gluc [5-Bromo-4-chloro-3-indolyl-beta-D-glucuronic acid (Bio-world, Dublin, OH, USA)] dissolved in a 100 mm phosphate buffer containing 0.5% Triton X-100 (MP Biomedicals, Inc, Irvine, CA, USA), 0.5 mm potassium ferrocyanide, 0.5 mm potassium ferricyanide (Fisher Scientific), and the solution was vacuum-infiltrated into the tissue for 20 min at 68 Kpa. The tissue was then incubated in the dark at 37 °C and observed hourly until the GUS product first appeared in the vascular and epidermal tissue of the stem.
The PIAA2::GUS (Luschnig et al. 1998) was selected for expression of the GUS gene in the inflorescence stem of Arabidopsis, and the transgenic line was crossed into all three gps mutant backgrounds. The F2 plants were selected for the GUS expression in the respective gps mutant background. The progeny was allowed to self-pollinate and the F3 plants with 8–10 cm inflorescence stems were used to select for plants showing expression of the GUS transgene and the mutant phenotype. Assessment of auxin-induced gene expression was conducted on the F3-generation plants.
As described previously, seeds from the wild type or gps1, gps2 or gps3 containing the PIAA2::GUS lines were planted and plants were grown until the inflorescence stems reached 8–10 cm; stems were subjected to the three gravistimulation regimes; and after gravistimulation, the region of inflorescence stem showing gravitropic bending was cut and subjected to GUS staining. After about 3–4 h of incubation, the desired intensity of blue was obtained and samples were placed in 95% EtOH overnight to clear. Although incubation time varied slightly from experiment to experiment, the sensitivity of the wild type and mutant plants were similar within each experiment. The incubation time for each experiment was similar for all stems within an experiment. The orientation of the samples with respect to gravity was maintained throughout the process. All Arabidopsis stems were hand-sectioned at the area of curvature, which was ≈0.5–1 cm long. Stems were cut with a surgical scalpel using a Nikon SMZ 1500 dissection scope (Nikon USA, Melville, NY, USA). Samples were viewed under a Nikon SMZ1500 dissection scope and images were taken using a Nikon CoolPix5400 digital camera (Nikon USA).
Basipetal IAA transport
Basipetal auxin transport in the inflorescence stems of wild type and the gps mutants was measured during three treatments: at 4 °C after gravistimulation at 4 °C; at room temperature after 4 °C gravistimulation (GPS treatment); and at room temperature, after gravistimulation at room temperature. Inflorescence stem sections of 2.5 cm in the elongation zone were cut and decapitated. These decapitated stem sections were incubated with the apical end in tritiated IAA for 10 min, followed by a 50 min chase with unlabeled IAA. The radioactivity was then measured in individual 0.5 cm segments of the stem. The rate at which auxin was transported was indicated by how far the radioactive auxin front travelled in the inflorescence stem segments.
At 4 °C, auxin transport was abolished in all mutants and wild type (Fig. 1a), which is consistent with the studies conducted by Wyatt et al. (2002). However, when returned to room temperature after a 4 °C gravistimulation (GPS treatment), auxin transport was evident in all the mutants (Fig. 1b). Accumulation of 3H-IAA in each segment of the inflorescence stems was compared directly between each mutant and wild type using a Student's t-test. In gps1, transport was elevated as compared to wild-type, as indicated by an increase in 3H-IAA accumulation in the upper 3–5 cm of the inflorescence stem (Fig. 1b). This was somewhat surprising. We had hypothesized, based on the no-response phenotype, that auxin transport would not be restored after return to cold. Clearly, IAA transport was not abolished in this mutant under conditions that blocked gravitropic bending. Furthermore, the results were inconsistent with changes in auxin metabolism or retention that would indirectly reduce auxin transport and, thus, the gravitropic response. In gps2 and gps3, there were no significant differences in the auxin transport during GPS treatment when compared to wild type (Fig. 1b).
The amount of auxin transported in inflorescences that were gravistimulated at room temperature in gps1 and gps2 was not significantly different from that of wild type (Fig. 1c), indicating that auxin transport was unaffected by these mutations under constant gravistimulation at room temperature. These results were consistent with the room-temperature phenotype of these mutants. However, a significant increase in the basipetal IAA transport in gps3 mutants was seen during constant gravistimulation at room temperature (Fig. 1c) when compared to the wild type. This increase in the basipetal auxin transport of the gps3 during room-temperature gravistimulation was not anticipated. The gps3 showed no gravitropic phenotype at room temperature.
Gravity-induced lateral redistribution of auxin-induced gene expression
Four lines of Arabidopsis (DR5::GUS, SAUR::GUS, PIAA3::GUS and PIAA2::GUS), each containing an auxin-responsive promoter fused to the GUS reporter gene, were screened to identify a line that had detectable GUS expression in Arabidopsis inflorescence stems. GUS expression was observed in the inflorescence stems of the PIAA2::GUS line (Fig. 2a–d), but not in the DR5::GUS, SAUR::GUS or PIAA3::GUS lines (data not shown). PIAA2::GUS also showed excellent GUS expression in the root, cotyledons and leaves, but showed little to no GUS expression in the hypocotyls of the plants (data not shown). Because we were most interested in expression in the inflorescence stems, we chose PIAA2::GUS to assess the lateral redistribution of auxin after gravistimulation.
After gravistimulation, inflorescence stems were cut into 1.5 cm segments, fixed and treated for GUS expression. Both immediately prior to and after rotation (the 0 time point after gravistimulation), GUS staining was observed uniformly throughout the elongation zone of the inflorescence stem (Figs 2a & 3). An increase in GUS expression in the vascular tissue on the lower side of the inflorescence stems was first observed ≈20 min after gravistimulation and was concurrent with initial curvature. GUS expression in the vascular tissue increased along the length of the elongation zone on the lower side from 20 min to 60 min after gravistimulation (Fig. 3). After 60 min of gravistimulation, the reorientation of the inflorescence stem had reached ≈ 45° and was associated with increased PIAA2::GUS expression in the vascular tissue, cortex and epidermis on the lower side of the elongation zone (Fig. 3), identical to that seen after the GPS treatment (Fig. 2b–d). These results indicated a gradual increase of the PIAA2::GUS expression on the lower side of the gravistimulated inflorescence stems, first in the vascular tissue and later extending to the cortex and epidermis, that occurs in association with the gravity-induced curvature.
When the effect of the GPS treatment on expression of the PIAA2::GUS was examined, the results in wild type were quite similar to those during constant gravistimulation at room temperature. Asymmetries in GUS expression became detectable after return to room temperature with similar kinetics as under constant gravistimulation and were localized on the lower side of the inflorescence relative to the gravity vector (Fig. 2a–d). The elevated expression on the lower side of the inflorescence was clear in both longitudinal (Fig. 2c) and cross-sectional (Fig. 2d) views. The elevated expression of this construct in response to gravity was visible in the vascular tissue and outer layers of inflorescence tissue.
Asymmetric expression of IAA2::GUS in the gps mutants
Auxin-induced gene expression after gravistimulation was assessed using the PIAA2::GUS transgenic line in the gps mutant background under three experimental conditions: (1) during gravistimulation at 4 °C; (2) during gravistimulation at room temperature; and (3) at room temperature following 4 °C gravistimulation (GPS treatment). The PIAA2::GUS line served as a control. GUS expression in all mutants during gravistimulation at room temperature and at 4 °C was similar to that of the control, PIAA2::GUS line (data not shown).
However, after GPS treatment, the gps1 mutant containing a PIAA2::GUS construct showed no lateral redistribution of GUS staining in the inflorescence stems (Fig. 2e–h), in contrast to the wild-type PIAA2::GUS line (Fig. 2a–d), which has a clear lateral distribution of auxin-induced gene expression. Cross sections of the stems showed equal distribution of GUS staining throughout the circumference of the stem (Fig. 2h) indicating no asymmetry in auxin distribution in gps1 after the GPS treatment. These results were consistent with the gps1 no-response phenotype.
GUS-expression studies on the inflorescence stems of the gps2 mutant containing a PIAA2::GUS construct indicated improper distribution of auxin, as compared to the control. GUS expression was increased on the upper side of the gps2 inflorescence stems after GPS treatment (Fig. 2i–l). The inverted GUS expression in the inflorescence stem of gps2 was consistent with its wrong-way phenotype after GPS treatment.
Inflorescence stems of the gps3 mutant containing a PIAA2::GUS construct showed an extended region of GUS expression in the inflorescence stem (Figs 2m–p & 4a–d) as compared to the control (Fig. 2a–d). This indicated an increased region of auxin redistribution after GPS treatment in the gps3 inflorescence stems that coincides with an extended region of gravitropic curvature. This result was consistent with an elongated auxin gradient that would result in the over-response phenotype.
Gravity is a fundamental stimulus governing plant growth and development. Although research into the mechanisms responsible for gravity response has been ongoing for almost 150 years, very little is known about the components of the signal transduction pathway that link perception of the gravity stimulus to the redistribution of the plant growth regulator auxin, which results in differential cell elongation and curvature. To specifically identify components of the signal transduction pathway, the gps mutants were isolated using a cold effect to separate the perception events from the response (Wyatt et al. 2002). Although plants perceive the gravity stimulus at 4 °C, they do not respond; however, when returned to vertical at room temperature, they bend in response to the previous cold gravistimulation (Fukaki et al. 1996). Amyloplast sedimentation occurs at 4 °C, but basipetal auxin transport is abolished. However, when plants are returned to room temperature, auxin transport is restored, indicating that the cold treatment affects an event or events that link gravity perception to auxin transport (Wyatt et al. 2002). Taking advantage of this cold effect on gravity signal transduction, three mutants were identified: gps1 failed to bend in response to the cold gravistimulation; gps2 bent in the wrong direction; and gps3 over-responded, bending past the expected orientation. Because the mutations in the gps mutants were expected to affect the events prior to auxin redistribution, both basipetal auxin transport and lateral auxin redistribution was examined in each of the gps mutants.
Initially, we hypothesized that a lack of gravitropic bending in gps1 after the GPS treatment was caused by an inability of the plants to restore basipetal auxin transport after the cold treatment. However, our data clearly indicated that auxin transport was not only restored but increased under GPS treatment (Fig. 1b). Thus a reduction or lack of basipetal auxin transport was not the cause for the gps1 no-response phenotype. Although the increase in auxin transport in gps1 is puzzling, it is not without precedent. Buer & Muday (2004) have shown that flavonoid-deficient transparent testa 4 (tt4) mutants, which have a delayed root gravitropic response, have elevated basipetal auxin transport in the root. Conversely, Noh et al. (2003) showed that a group of hyper-gravitropic mutants, the AtMDR mutants, had reduced auxin transport. In gps2, which bends the wrong direction, and gps3, the over-achiever, basipetal auxin transport was not significantly different than in wild type, and these experiments did not provide much insight into the causes of the mutant phenotypes (Fig. 1a).
We also documented gravitropic bending as it related to the auxin-induced gene expression using an auxin-responsive promoter reporter gene fusion (PIAA2::GUS). In PIAA2::GUS inflorescence stems, GUS expression began to localize to the lower side of the stems within 20 min after gravistimulation, but asymmetric expression was restricted to the vascular tissue (Fig. 3). Within 60 min, auxin-induced GUS expression extended into the cortical and epidermal cells in the lower half of the gravistimulated inflorescence stem (Fig. 3). Although the auxin gradient in the cortical and epidermal cells does not seem to precede bending, the inability to observe asymmetries in auxin using the GUS reporter constructs, preceding gravitropic bending, is not uncommon (Rashotte et al. 2001) and may reflect the limited sensitivity of the assay.
Assessment of auxin-induced gene expression in the gps mutants provided insight into the causes of the mutant phenotypes. In PIAA2::GUS (representing wild type), an increase in auxin on the lower side of the gravistimulated stem tissue was associated with increased elongation and bending. In gps1, the auxin-induced gene expression analyses indicated a lack of differential redistribution of auxin after GPS treatment (Fig. 2e–h). Auxin was uniformly distributed around the stem, which could result in the lack of gravitropic bending in gps1. Improper redistribution of auxin to the upper side of the inflorescence stem in gps2 after GPS treatment (Fig. 2i–l) was consistent with the wrong-way phenotype of the mutant. This unusual phenotype was analogous to the growth of peanut gynophores toward the ground after fertilization to allow fruit development to occur below the surface of the soil. Moctezuma & Feldmann (1999) showed that auxin accumulated on the upper side of the peanut gynophores, facilitating redirection of the gynophores by asymmetric cell elongation. The auxin-induced GUS expression in gps2 indicated that the phenotype of gps2 was caused by improper auxin redistribution (i.e. increased auxin on the upper side of the inflorescence stem during the GPS treatments), which resulted in bending, thus directing the stem toward the gravity vector. A likely mechanism for this auxin gradient is redistribution of auxin efflux carriers or facilitators to the opposite side of the membrane. The PIAA2::GUS studies with gps3 indicated an increased region of lateral auxin redistribution on the lower side of the gravistimulated inflorescence stem (Figs 2m–p & 4a–d). This extended region of lateral auxin redistribution could result in asymmetric growth over a greater region of the inflorescence stem resulting in the over response phenotype seen in gps3 under the GPS condition.
In conclusion, the gravitropic phenotype of the gps1, 2 and 3 mutants may result from the improper distribution of auxin. The gps1 (no-response) phenotype was most likely the result of a lack of auxin redistribution after GPS treatment, even though basipetal auxin transport was increased. The gps2 (wrong-way) phenotype was the result of an inversion of lateral auxin redistribution after the GPS treatment. The gps3 (over-response) phenotype was the result of auxin redistribution, causing an extended elongation region of the inflorescence stem. The gps mutants represent potentially three independent aspects of signal transduction in the gravitropic response: the (gps1) lack of auxin redistribution; determination of the polarity of the response (gps2); and definition of the region of bending (gps3). Cloning and characterization of the genes involved should provide exciting new insights into the molecular components that establish auxin redistribution in gravitropism.
The assistance of Shari Brady in the auxin transport experiments is greatly appreciated. This work was partially supported by the United States Department of Agriculture (grant no. 2002-35304-1233 to S.E.W.), the American Society of Gravitational Space Biology (to S.E.W.) and the National Aeronautics and Space Administration (NASA) (grant no. NAGW-4984 awarded to the NASA Specialized Center of Research and Training in Gravitational Biology, to G.K.M.).