Inflorescence stems of the mdr1 mutant display altered gravitropism and phototropism

Environmental and Experimental Botany 70 (2011) 244–250

Contents lists available at ScienceDirect

Environmental and Experimental Botany journal homepage: www.elsevier.com/locate/envexpbot

Inflorescence stems of the mdr1 mutant display altered gravitropism and phototropism Prem Kumar, Katherine D.L. Millar, John Z. Kiss ∗ Department of Botany, Miami University, Oxford, OH 45056, USA

a r t i c l e

i n f o

Article history: Received 21 May 2010 Received in revised form 12 August 2010 Accepted 28 September 2010 Keywords: ABCB19 Auxin Gravitropism MDR Phototropism

a b s t r a c t Auxins are a major group of growth regulators that are involved in all stages of plant growth and development, and these molecules play a key role in the response phase of both phototropism and gravitropism. Polar auxin transport within tissues is mediated by auxin influx and auxin efflux carriers that are asymmetrically distributed across cell membranes. The MDR (multidrug resistance) family of proteins has been identified in plants and is involved in the transport of auxin. In these studies, we examined tropistic responses in the mdr1 (=abcb19) mutant of Arabidopsis thaliana using conventional reorientation studies as well as a computer-based feedback system to investigate the kinetics of root gravitropism and phototropism. Time course of curvature studies show that tropisms were not affected in light-grown seedlings of mdr1 mutants, with the exception of an enhancement of gravitropism in hypocotyls. In mature plants, mdr1 inflorescence stems exhibit attenuated gravitropism but have enhanced phototropic curvature in response to unidirectional blue illumination. Thus, the decreased auxin flow in the mdr1 mutant has a more profound effect in inflorescence stems compared to hypocotyls of seedlings, possibly due to alterations in membrane trafficking pathways in gravity-perceiving endodermal cells of stems. Our data add support to the hypothesis that hypocotyls and stems differ in their cellular mechanisms of gravitropism. © 2010 Elsevier B.V. All rights reserved.

1. Introduction Since plants are sessile in nature, they have developed systems to deal with changing environmental conditions. One general mechanism to deal with environmental inputs is a tropism, directed growth response to external stimuli. Of these tropisms, the two best characterized are phototropism and gravitropism, growth in response to light and gravity, respectively. Light is important for the energy needed for photosynthesis as well as providing directional (i.e., for phototropism) and developmental signals (Whippo and Hangarter, 2006). Throughout their entire life cycle, plants use gravity to orient and coordinate their growth to maximize access to light, water and nutrients (Blancaflor and Masson, 2003). Gravitropism can be divided into three temporal phases: perception, transduction, and response (Kiss, 2000). Gravity sensing/perception occurs in specialized cells in the roots (columella cells) and shoots (endodermal cells) of all flowering plants, and amyloplasts are the organelles that serve as gravity sensors. Signal transduction occurs when dissipation of the potential energy of gravistimulated amyloplasts results in the production of chemi-

∗ Corresponding author. Tel.: +1 513 529 4200; fax: +1 513 529 4243. E-mail address: [email protected] (J.Z. Kiss). 0098-8472/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.envexpbot.2010.09.019

cal signals that ultimately trigger a growth response (Palmieri and Kiss, 2006). The third phase of gravitropism is characterized by differential growth leading to gravitropic curvature (Rashotte et al., 2000; Perrin et al., 2005). The growth response is elicited by concentration gradients of the plant growth regulator auxin that form across reoriented organs such that more of this hormone is present in the lower portion, as compared to the upper portion of the organs (reviewed in Trewavas, 1992). In flowering plants, phototropism has been shown to be induced specifically by blue light (Christie, 2007). Stems and stem-like organs are typically positively phototropic, growing toward the light, and roots are typically negatively phototropic in response to unilateral blue illumination (Sakai et al., 2000; Correll and Kiss, 2002). While phototropism is predominantly a blue-light effect in flowering plants, red light also has been shown to play a role in the mechanisms of phototropic curvature (Hangarter, 1997). Redlight-based phototropism is well known in older plant lineages such as mosses and ferns (Kern and Sack, 1999; Mittmann et al., 2009). However, more recently, we have characterized red-lightbased phototropism in roots (Molas and Kiss, 2008) and hypocotyls (Millar et al., 2010) of Arabidopsis thaliana. The common element in phototropism and gravitropism is an auxin-mediated response phase that results in differential curvature. According to the classical Cholodny–Went model, gravitropic

P. Kumar et al. / Environmental and Experimental Botany 70 (2011) 244–250

and phototropic curvature in roots and stems are brought about by a lateral auxin gradient that promotes differential cell elongation (reviewed in Trewavas, 1992; Blancaflor and Masson, 2003; Molas and Kiss, 2009). Polar auxin transport within tissues is mediated by auxin influx (uptake auxin from the cells) and auxin efflux carriers (export auxin) that are asymmetrically distributed across the membranes (Blancaflor and Masson, 2003). The genes encoding auxin influx and efflux carriers have been isolated and characterized (reviewed in Kleine-Vehn and Friml, 2008). Molecular genetic studies have shown that root gravitropism in Arabidopsis requires AUX1, auxin influx carrier. Similarly genetic studies in agravitropic mutants have identified the PIN proteins as auxin efflux carriers along with other proteins. The localization of PIN proteins determines the localization of auxin transport in plants, and the PIN proteins in Arabidopsis are separated temporally and spatially (Teale et al., 2006). The actin cytoskeleton is hypothesized to regulate polar localization of PIN proteins by potentially participating in the vesicular cycling of PIN1 protein (Muday et al., 2003). Consistent with this hypothesis, studies using actin cytoskeleton disrupting drugs have shown that the polar auxin transport and PIN1 cycling between plasma membrane and the endosomal compartments are reduced (Geldner et al., 2001). Pharmacological and biochemical studies suggest that PIN and ARG1 (Boonsirichai et al., 2003), a protein that also plays a role in gravitropism (Kumar et al., 2008), share a common vesicle trafficking pathway between the plasma membrane and the intracellular components. As a result of auxin transport, dissociation of auxin occurs thereby releasing protons (H+ ) into the cytoplasm (Muday, 2000) resulting in alkalinization. Similarly an increase in the cytosolic pH has been observed in cells of plants that were reoriented (Fasano et al., 2001). Such an increase in cytosolic pH is lacking in arg1 mutants (Boonsirichai et al., 2003), indicating that auxin transport mediated by efflux carriers may be blocked due to impaired ARG1 in the mutants. Another group of membrane transport proteins that have been shown to play an important role in auxin transport belong to the MDR (multidrug resistance) family (Noh et al., 2001). The MDR transporters are a sub-group of a broader family of proteins known as the ABC (ATP-binding cassette) transporters (Luschnig, 2002). These proteins have a modular structure with two basic structural elements consisting of a hydrophobic transmembrane domain of six membrane-spanning alpha helices and a cytoplasmic domain involved in ATP-binding (Jasinski et al., 2003). Several studies have demonstrated that mdr1 mutants have enhanced gravitropism and phototropism in seedlings (Noh et al., 2001; Lewis et al., 2007). These alterations in tropisms are due to an improper localization of the PIN1 auxin efflux protein (Noh et al., 2003) which result in impairment of 80% of the auxin flow in the case of roots (Lewis et al., 2007). More specifically, the mdr1 mutant lacks most of the basipetal auxin transport in hypocotyls and inflorescence stems, but it is defective in acropetal (but not basipetal) auxin transport in roots (reviewed in Lewis et al., 2009). While it has been shown that the MDR1 protein (also known as ABCB19 and PGP19; Lewis et al., 2009) is localized to inflorescence stems as well as in seedlings (Noh et al., 2001), there is no information to date on the role of MDR1 on tropisms in these stems. Thus, the goal of this study was to test the hypothesis that MDR1 plays a role in gravitropism and phototropism of inflorescence stems, and, if so, refine the model for the relationship between gravitropism and auxin transport in stems. We also confirmed and extended observations of the role of MDR1 in tropisms in roots and hypocotyls of seedlings, including studies with a high resolution computer-based feedback system to study both red-light and blue-light-based root phototropism.

245

2. Materials and methods 2.1. Plant material and culture conditions The wild-type (WT) plants used in our studies were of the Wassilewskija (WS) strain of Arabidopsis thaliana. The mdr1 (multidrug resistance) mutant was isolated using T-DNA insertional mutagenesis as described by Noh et al. (2001). These seeds were provided courtesy of Dr. Edgar P. Spalding of the University of Wisconsin, Madison, WI, USA. The MDR1 protein also is known as ABCB19 and PGP19 (Lewis et al., 2009). For studies with seedlings, seeds were surface sterilized in 70% (v/v) ethanol with 0.002% (v/v) Triton X-100 for 5 min, rinsed two times for 1 min in 95% (v/v) ethanol, and washed several times in sterile double distilled water (Kumar and Kiss, 2007). Seeds were then placed onto sterile cellulose film placed on top of 1.2% (w/v) agar containing one-half-strength Murashige and Skoog salts with 1% (w/v) sucrose and 1 mM MES at pH 5.5 in square (100 × 15mm) Petri plates. The Petri plates were sealed with parafilm and placed on their edge so that the surface of agar was vertical and the seedlings were grown for 4 d in white fluorescent light (70 ␮mol m−2 s−1 ) from above at 23 ◦ C. For studies with inflorescence stems of mature plants, seeds were surface sterilized as described above and then sown in 10 × 10 × 8.5 cm plastic pots according to Kumar and Kiss (2006). The pots were placed in continuous white light from 34 W fluorescent bulbs at fluence of 70 ␮mol m−2 s−1 . When the inflorescence stems were 6–12 cm long (20–40 d after sowing), plants were used for experiments. 2.2. Tropism experiments and analyses For the gravitropism studies, plates or pots with light-grown plants were reoriented at 90◦ from the vertical in darkness at 22 ◦ C. Photographs were taken with a digital camera using dim green light (fluence rate < 0.8 ␮mol m−2 s−1 ) at defined intervals, and previous studies have demonstrated that this dim green safe light had no measurable effect on the tropistic responses of Arabidopsis seedlings (Fitzelle and Kiss, 2001). In the phototropism experiments, plates or pots with lightgrown plants were placed vertically and exposed to continuous, unidirectional blue light (90◦ from the vertical) with a fluence rate of 15–20 ␮mol m−2 s−1 through a blue Plexiglas filter (Rohm and Haas No. 2424; transmission maximum 490 nm). Photographs were taken with a digital camera from the ambient light from the experiment. Growth and curvature of hypocotyls and roots were measured by using the program Image-Pro Plus (version 5.01, Media Cybernetics, Silver Spring, MD, USA). Tropistic curvature was measured as the change in angle from the starting point. Gravitropic curvature was categorized as positive (toward the gravity vector) or negative (away from the gravity vector). In phototropism, plant organs responding toward the light source were assigned a positive value, and those responding away from illumination were assigned a negative value. Statistical differences were determined by using a Student’s t-test with the program Sigma Stat (ver. 2, Systat, Chicago, IL, USA). 2.3. Computer-based feedback system to study root gravitropism and phototropism Seedlings were grown in the light as described above, except that round (60 × 15 mm) Petri dishes were used. In gravitropism experiments, a computer-controlled feedback system developed and described by Mullen et al. (2000) was used to maintain the root tip at 90◦ relative to vertical. In phototropism experiments,

P. Kumar et al. / Environmental and Experimental Botany 70 (2011) 244–250

the feedback system was used to maintain the root angle to the vertical (parallel to the root axis) during continuous, unidirectional red light (660 nm from an LED) stimulation of 10–20 ␮mol m−2 s−1 at 90◦ relative to the root axis as described by Kiss et al. (2003) and Molas and Kiss (2008). In these gravtropism and phototropism experiments, a root of a seedling was positioned in the center of a rotatable vertical stage with individual steps of the motor corresponding to 0.17◦ (Kiss et al., 2003). Roots were imaged every 45 s using infrared illumination from a 940 nm LED (Radio Shack, Fort Worth, TX, USA) and a CCD camera. The software analyzed the images, and if the root tip deviated from 0◦ , the software activated the stepper motor to the rotating stage to constrain the tip segment of the root to remain at 0◦ . The recorded value of the rotation of the stage, which corresponds to tropistic curvature, is indicated in the figures.

80

A. Root--Gravitropism

mdr WT

70

Curvature (degrees)

246

60 50 40 30 20 10 0

*

0

4

8

12

16

20

24

Time (h) B. Root--Phototropism 0

Since previous studies suggested a difference in tropisms between WT and the mdr1 mutant (Noh et al., 2003), we investigated both gravitropism and phototropism in light-grown seedlings and inflorescence stems of Arabidopsis thaliana. In roots of seedlings, both gravitropic (Fig. 1(A)) and negative blue-lightinduced phototropic curvature (Fig. 1(B)) were not significantly different (p > 0.05) comparing the mdr1 mutant to the WT throughout the time course. In order to confirm these results, we also used a high resolution feedback system (Mullen et al., 2000) to study the detailed kinetics of gravitropism and phototropic curvature in roots of the mutant compared to the WT (Fig. 2). Both qualitative (Fig. 2) and quantitative (Fig. 3(A)) observations show that the kinetics of gravitropism is not significantly different (p > 0.05) in mdr1 relative to the WT. In addition, this feedback system is needed to measure and effectively study positive red-light-based phototropism in roots (Kiss et al., 2003). Results from these experiments showed that this redlight phototropism was not significantly different (p > 0.05) in the mdr1 mutant compared to the WT (Fig. 3(B)). In terms of the hypocotyls of seedlings, gravitropism in the mdr1 mutant was significantly greater (p < 0.05) compared to the WT at the end of the time course experiments (Fig. 4(A)). However, there were no significant differences in blue-light-based positive phototropism between the mdr1 mutant and the WT (Fig. 4(B)). While tropisms have been studied in mdr1 seedlings, to date, there have been no published reports on inflorescence stems of

Curvature (degrees)

3. Results

-5 -10 -15

mdr

-20

WT

-25 -30

0

4

8

12

16

20

24

Time (h) Fig. 1. Time course of gravitropism (A) and phototropism (B) studies of roots of light-grown, 4-day old seedlings of WT and the mutant mdr1. Phototropic curvature is in response to blue light. Mean curvature at each data point was calculated for 50 seedlings (A) and 54 seedlings (B). Error bars represent SE. There is no statistically significant difference (p > 0.05) in curvature between the two genotypes.

mature plants. We did observe significant differences in both gravitropism and phototropism in stems of the mdr1 mutant compared to the WT (Figs. 5 and 6). In contrast to our observations with hypocotyls, we found gravitropic curvature in mdr1 stems to be significantly reduced (p < 0.05) compared to curvature in WT stems throughout the time course experiments (Fig. 6(A)). However, in terms of blue-light-induced positive phototropism in stems, the

Fig. 2. Representative images of root gravitropism as assayed with the computer-based feedback system in seedlings of WT and mdr1. The time indicates intervals following reorientation, and robust curvature was observed in both genotypes.

300 280 260 240 220 200 180 160 140 120 100 80 60 40 20 0 -20

A. Root--Gravitropism--Rotato

WT

-10 -20

-50 -1

0

1

2

3

4

5

6

7

8

9

0

4

8

12

16

20

24

Time (h) 80

B. Hypocotyl--Phototropism--Blue

70

B. Root--Phototropism--Red--Rotato Curvature (degrees)

Rotation (degrees)

mdr

10

WT mdr

mdr WT

60 50 40 30 20 10 0

0

4

8

12

16

20

24

Time (h) -1

0

1

2

3

4

5

6

7

8

9

10

Fig. 3. Time course of curvature studies of roots of light-grown WT and mdr1 seedlings during gravitropism (A) and red-light-based phototropism (B) as assayed with the feedback system. For both tropisms, there was no significant difference (p > 0.05) in curvature between the two genotypes. Each data point represents a mean of 14–19 root tips.

mdr1 mutant exhibited a significantly greater curvature (p < 0.05) compared to that of the WT. In general, there were few differences in growth rate by organ of the light-grown mdr1 mutant compared to the WT (Table 1). However, in the case of the hypocotyl and inflorescence stem during phototropism studies, the growth rate of the mdr1 mutant was attenuated (p < 0.05) relative to the WT. Interestingly, in mdr1 inflorescence stems, while there was a greater magnitude of phototropic curvature (Fig. 6(B)), growth of the mutant was significantly less (p < 0.05) compared to the WT (Table 1). Thus, the difference in phototropism cannot be attributed to differences in growth rate between the mutant and WT stems. Table 2 summarizes gravit-

Table 1 Summary of the growth rates (␮m h−1 ) of the mdr1 mutant compared to WT in lightgrown seedlings and inflorescence stems of mature plants during gravitropism and blue-light-based phototropism experiments. Values are mean ± SE, and a significant difference in growth is indicated by asterisks. Number of plants measured is indicated in (). Organ

Tropism

WT

Root

Gravitropism Phototropism Gravitropism Phototropism Gravitropism Phototropism

268.9 251.8 59.5 134.9 188.3 510.0

Stem

WT

-40

Time (h)

Hypocotyl

*

-30

Time (h) 300 280 260 240 220 200 180 160 140 120 100 80 60 40 20 0 -20

247

A. Hypocotyl--Gravitropism

0

mdr

Curvature (degrees)

Rotation (degrees)

P. Kumar et al. / Environmental and Experimental Botany 70 (2011) 244–250

mdr1 ± ± ± ± ± ±

21.1 (45) 12.8 (34) 6.2 (53) 3.9 (65) 15.7 (61) 12.7 (66)

276.8 245.5 43.3 93.4 178.4 310.2

± ± ± ± ± ±

17.2 (77) 19.7 (77) 3.9 (57) 3.9 (57)** 18.1 (57) 20.3 (57)**

Fig. 4. Time course of gravitropism (A) and phototropism (B) of hypocotyls of lightgrown seedlings of WT and the mutant mdr1. Phototropic curvature is in response to blue light. Mean curvature at each data point was calculated for 37–41 seedlings (A) and 45 seedlings (B). Error bars represent SE. A statistically significant difference (p > 0.05) of curvature at each time interval is indicated by asterisks.

ropism and phototropism in the various organs of the mdr1 mutant compared to the WT. 4. Discussion 4.1. Inflorescence stems of mdr1 mutants have an attenuated gravitropism and enhanced phototropism Since auxin is a key modulator of tropisms and MDR1 is involved in auxin transport, several reports have considered the role of this protein in tropisms in plant seedlings (Noh et al., 2003; Lewis et al., 2007). However, the present study is the first to assay gravitropism and phototropism in inflorescence stems of mature plants. Previous reports have demonstrated that the MDR1 protein is expressed in inflorescence stems of mature plants as well as in seedlings (Noh et al., 2001). Table 2 Summary of the tropistic responses in light-grown seedlings and inflorescence stems of mature plants in the mdr1 mutant relative to the WT. + indicates a promotion; − indicates an inhibition; and O indicates no effect. Organ

Tropism

mdr1

Root

Positive gravitropism Negative phototropism – blue Positive phototropism – red Negative gravitropism Positive phototropism – blue Negative gravitropism Positive phototropism – blue

O O O + O − +

Hypocotyl Stem

248

P. Kumar et al. / Environmental and Experimental Botany 70 (2011) 244–250

Fig. 5. Representative images of inflorescence stems of WT and mdr1 light-grown plants during gravitropism and phototropism studies. The time indicates intervals following reorientation or the beginning of unidirectional blue illumination. Gravitropic curvature is attenuated in the mdr1 mutant relative to the WT, and, conversely, phototropic curvature is enhanced in the mutant relative to the WT.

Interestingly, while stems of mdr1 exhibit attenuated gravitropism, they have enhanced phototropic curvature in response to unidirectional blue illumination (Table 2). The enhanced phototropism may be a direct effect of auxin transport caused by the mdr1 mutation or it may be an indirect effect of the attenuated gravitropism found in this mutant. The latter scenario would be similar to the increased phototropic curvature that is found in roots of gravitropically-impaired starchless mutants of Arabidopsis (Vitha et al., 2000; Ruppel et al., 2001; Kiss et al., 2002). 4.2. Tropisms in mdr1 seedlings and comparisons to previous studies Time course of curvature studies show that tropisms in roots and hypocotyls of seedlings were not affected in mdr1 mutants, with the exception of an enhancement of gravitropism in hypocotyls (Fig. 4(B)). Thus, our observations confirm the results of Lewis et al. (2007) who showed that there is no alteration in the time course of gravitropic curvature in roots of light-grown seedlings of mdr1. However, along with this group, we noted that during vertical orientation of the Petri dishes, the roots of mdr1 exhibited greater undulation (not shown), which they termed “spurious curvature” (Lewis et al., 2007). Thus, there is an overall effect of MDR1 on gravitropic orientation in roots without a direct effect on the kinetics of gravitropic curvature following reorientation. This observation may be related to the fact that the mdr1 mutant is defective in acropetal (but not basipetal) auxin transport in roots, which in turn, could cause the greater undulation that both groups observed in mdr1 roots.

In addition, the previous studies using dark-grown seedlings showed that gravitropism is enhanced in hypocotyls of the mdr1 mutant (Noh et al., 2003), and we now show a similar enhancement in light-grown seedlings of mdr1. In contrast, while Noh et al. (2003) demonstrated enhanced blue-light-based phototropism in dark-grown hypocotyls, we found a similar magnitude of phototropic curvature in hypocotyls of light-grown seedlings of the mdr1 mutant. The differences between results of the two studies may be due to the fact dark-grown seedlings (Noh et al., 2003) exhibit a greater magnitude and sensitivity to light and gravity compared to light-grown material (our study). Thus, at least in the case of seedlings, it would be easier to detect differences between the mutant and WT in dark-grown plants. 4.3. Red-light-based phototropism in roots A phytochrome-mediated positive phototropism in response to unidirectional red light has been reported in seedlings of Arabidopsis thaliana (Kiss et al., 2003; Molas and Kiss, 2008). This tropism is relatively weak and generally requires special instrumentation to measure the resulting curvature (i.e., a computer-based, feedback system; Kiss et al., 2003). Red-light-based root phototropism, similar to the other time course studies in roots, is not significantly different (p > 0.05) in the mdr1 mutant compared to the WT (Fig. 3). Interestingly, the magnitude of red-light root phototropism in these studies using the Wassilewskija ecotype (approximately 70◦ ) is much greater than what we had observed in other studies with the Columbia (5–10◦ ; Kumar and Kiss, 2007; Molas and Kiss, 2008) or Landsberg ecotypes (Kiss et al., 2003). Thus, these data

P. Kumar et al. / Environmental and Experimental Botany 70 (2011) 244–250

Curvature (degrees)

0

* A. Stem--Gravitropism

-10 -20 -30

*

-40

*

-50

*

*

-60

mdr WT

*

-70 -80 -90 -100

0

4

8

12

16

20

24

Time (h) 35

*

B. Stem--Phototropism--Blue Curvature (degrees)

30

*

25

mdr

* WT

20 15 10 5 0

* 0

4

8

12

16

20

24

Time (h) Fig. 6. Time course of gravitropism (A) and phototropism (B) of inflorescence stems of WT and the mutant mdr1. Phototropic curvature is in response to blue light. Mean curvature at each data point was calculated for 74–79 plants (A) and 68–85 plants (B). Error bars represent SE. A statistically significant difference (p < 0.05) of curvature at each time interval is indicated by asterisks.

support previously published works which showed variation in phytochrome-mediated responses among ecotypes of Arabidopsis thaliana (Alconada-Magliano et al., 2005). 4.4. The role of auxin transport during tropistic curvature Auxins are a key group of endogenous plant growth regulators that are involved in all stages of plant growth and development throughout the life cycle of the plant. This molecule has been shown to play a key role in the response phase of both gravitropism and phototropism by the “fountain” model of flow in the root and shoot in which auxin induces differential growth leading to tropistic curvature (Blancaflor and Masson, 2003; Molas and Kiss, 2009). A surprising result of the study by Lewis et al. (2007) is that acropetal transport of auxin in roots of the mdr1 mutant can be impaired by 80% without affecting the time course of gravitropic curvature following reorientation. The data in our present study along with the data from the Lewis et al. (2007) group support the refinement of the standard auxin flow model in which there are reflux loops in the stele rather than a simple acropetal streaming of auxin, as outlined in their 2007 paper. This addition to the model would account for the fact that gravitropic curvature following reorientation was not altered in the mdr1 mutant while roots from these seedlings exhibited a high degree of undulation in vertically-held dishes. In terms of the mechanisms of hypocotyl gravitropism, our results are in agreement with Noh et al. (2003) who showed enhanced gravitropism and suggested that MDR1 functions in the response phase rather than the stimulus sensing phase of tropisms.

249

Interestingly, compared to hypocotyls, gravitropism is diminished in inflorescence stems of mdr1 mutants, which suggests that the decreased auxin flow has a more profound effect in inflorescence stems compared to hypocotyls of seedlings. Thus, our data also add support to the hypothesis that hypocotyls and stems differ in their cellular mechanisms of gravitropism (Weise et al., 2000). The gravitopic response in inflorescence stems can be genetically separable from the response in hypocotyls in Arabidopsis. For instance, the srg3 mutant has an altered response in inflorescence stems but not in hypocotyls (Fukaki et al., 1996). Conversely, the rgh mutant has an alteration in hypocotyl gravitropism but is normal in the inflorescence stems (Fukaki et al., 1997). These differences may be related to the developmental origin of these organs in that the inflorescence stem develops from the shoot apical meristem originating from the apical domain of the early embryo whereas the hypocotyl is an embryonic organ that differentiates from the central domain of the early embryo (Fukaki et al., 1996). One idea to explain the differences in effects of MDR1 on gravitropism in hypocotyls versus inflorescence stems is to also consider the dynamics in gravity-perceiving endodermal cells in the latter organ. A great deal of research suggests that the vesicle trafficking in endodermal cells of inflorescence stems is important in gravity perception (Hashiguchi et al., 2010), but there is no information available on trafficking in these cells in hypocotyls. Thus, there may be significant alterations in membrane trafficking pathways in endodermal cells of inflorescence stems in mdr1 mutants that ultimately affect the auxin transport proteins in these plants, thereby, causing the observed decrease in gravitropic curvature in stems. However, further research is needed to clarify how MDR1 regulates auxin flow in inflorescence stems to modulate gravitropism as well as phototropism in this organ.

Acknowledgments We thank Dr. Edgar P. Spalding of the University of Wisconsin for providing seeds of the mdr1 mutant and NASA for financial support through grant NNX09AF11G.

References Alconada-Magliano, T.M., Botto, J.F., Godoy, A.V., Symonds, V.V., Lloyd, A.M., Casal, J.J., 2005. New Arabidopsis recombinant imbred lines (Landsberg erecta × Nossen) reveal natural variation in phytochrome-mediated responses. Plant Physiol. 138, 1126–1135. Blancaflor, E.B., Masson, P.H., 2003. Plant gravitropism. Unraveling the ups and downs of a complex process. Plant Physiol. 133, 1677–1690. Boonsirichai, K., Sedbrook, J.C., Chen, R., Gilroy, S., Masson, P.H., 2003. ARG1 is a peripheral membrane protein that modulates gravity-induced cytoplasmic alkalinization and lateral auxin transport in plant statocytes. Plant Cell 15, 2612–2625. Christie, J.M., 2007. Phototropin blue-bight receptors. Ann. Rev. Plant Biol. 58, 21–45. Correll, M.J., Kiss, J.Z., 2002. Interactions between gravitropism and phototropism in plants. J. Plant Growth Regul. 21, 89–101. Fasano, J.M., Swanson, S.J., Blancaflor, E.B., Dowd, P.E., Kao, T.H., Gilroy, S., 2001. Changes in root cap pH are required for the gravity response of the Arabidopsis root. Plant Cell 13, 907–921. Fitzelle, K.J., Kiss, J.Z., 2001. Restoration of gravitropic sensitivity in starch-deficient mutants of Arabidopsis by hypergravity. J. Exp. Bot. 52, 265–275. Fukaki, H., Fujisawa, H., Tasaka, M., 1996. How do plant shoots bend up? – the initial step to elucidate the molecular mechanisms of shoot gravitropism using Arabidopsis thaliana. J. Plant Res. 109, 137–145. Fukaki, H., Fujisawa, H., Tasaka, M., 1997. The RHG gene is involved in root and hypocotyl gravitropism in Arabidopsis thaliana. Plant Cell Physiol. 38, 804–810. Geldner, N., Friml, J., Stierhof, Y.D., Jurgens, G., Palme, K., 2001. Auxin transport inhibitors block PIN1 cycling and vesicle trafficking. Nature 413, 425–428. Hangarter, R.P., 1997. Gravity, light and plant form. Plant Cell Environ. 20, 796– 800. Hashiguchi, Y., Niihama, M., Takahashi, T., Saito, C., Nakano, A., Tasaka, M., Morita, M.T., 2010. Loss-of-function mutations of retromer large subunit genes suppress the phenotype of an Arabidopsis zig Mutant that lacks Qb-SNARE VTI11. Plant Cell 22, 159–172.

250

P. Kumar et al. / Environmental and Experimental Botany 70 (2011) 244–250

Jasinski, M., Ducos, E., Martinoia, E., Boutry, M., 2003. The ATP-binding cassette transporters: structure, function, and gene family comparison between rice and Arabidopsis. Plant Physiol. 131, 1169–1177. Kern, V.D., Sack, F.D., 1999. Irradiance dependent regulation of gravitropism by red light in protonemata of the moss Ceratodon purpureus. Planta 209, 299–307. Kiss, J.Z., 2000. Mechanisms of the early phases of plant gravitropism. Crit. Rev. Plant Sci. 19, 551–573. Kiss, J.Z., Miller, K.M., Ogden, L.A., Roth, K.K., 2002. Phototropism and gravitropism in lateral roots of Arabidopsis. Plant Cell Physiol. 43, 35–43. Kiss, J.Z., Mullen, J.L., Correll, M.J., Hangarter, R.P., 2003. Phytochromes A and B mediate red-light-induced phototropism in roots. Plant Physiol. 131, 1–7. Kleine-Vehn, J., Friml, J., 2008. Polar targeting and endocytic recycling in auxindependent plant development. Annu. Rev. Cell Dev. Biol. 24, 447–473. Kumar, P., Kiss, J.Z., 2006. Modulation of phototropism by phytochrome E and attenuation of gravitropism by phytochromes B and E in inflorescence stems. Physiol. Plant. 127, 304–311. Kumar, P., Kiss, J.Z., 2007. The SHL1 and SHL5 genes influence both red- and bluelight-based phototropism in Arabidopsis thaliana. Environ. Exp. Bot. 60, 284–289. Kumar, N.S., Stevens, M.H.H., Kiss, J.Z., 2008. Plastid movement in statocytes of the arg1 (altered response to gravity) mutant. Am. J. Bot. 95, 177–184. Lewis, D.R., Miller, N.D., Splitt, B.L., Wu, G., Spalding, E.P., 2007. Separating the roles of acropetal and basipetal auxin transport on gravitropism with mutations in two Arabidopsis multidrug resistance-like ABC transporter genes. Plant Cell 19, 1838–1850. Lewis, D.R., Wu, G.S., Ljung, K., Spalding, E.P., 2009. Auxin transport into cotyledons and cotyledon growth depend similarly on the ABCB19 multidrug resistance-like transporter. Plant J. 60, 91–101. Luschnig, C., 2002. Auxin transport: ABC proteins join the club. Trends Plant Sci. 7, 329–332. Millar, K.D.L., Kumar, P., Correll, M.J., Mullen, J.L., Hangarter, R.P., Edelmann, R.E., Kiss, J.Z., 2010. A novel phototropic response to red light is revealed in microgravity. New Phytol. 186, 648–656. Mittmann, F., Dienstbach, S., Weisert, A., Forreiter, C., 2009. Analysis of the phytochrome gene family in Ceratodon purpureus by gene targeting reveals the primary phytochrome responsible for photo- and polarotropism. Planta 230, 27–37. Molas, M.L., Kiss, J.Z., 2008. PKS1 plays a role in red-light-based positive phototropism in roots. Plant Cell Environ. 31, 842–849. Molas, M.L., Kiss, J.Z., 2009. Phototropism and gravitropism in plants. Adv. Bot. Res. 49, 1–34.

Muday, G.K., 2000. Maintenance of asymmetric cellular localization an auxin transport protein through interaction with the actin cytoskeleton. J. Plant Growth Regul. 19, 385–396. Muday, G.K., Peer, W.S., Murphy, A.S., 2003. Vesicular cycling mechanisms that control auxin transport polarity. Trends Plant Sci. 8, 301–304. Mullen, J.L., Wolverton, C., Ishikawa, H., Evans, M.L., 2000. Kinetics of constant gravitropic stimulus responses in Arabidopsis roots using a feedback system. Plant Physiol. 123, 665–670. Noh, B., Murphy, A.S., Spalding, E.P., 2001. Multidrug resistance-like genes of Arabidopsis required for auxin transport and auxin-mediated development. Plant Cell 13, 2441–2454. Noh, B., Bandyopadhyay, A., Peer, W.A., Spalding, E.P., Murphy, A.S., 2003. Enhanced gravi- and phototropism in plant mdr mutants mislocalizing the auxin efflux protein PIN1. Nature 424, 999–1002. Palmieri, M., Kiss, J.Z., 2006. The role of plastids in gravitropism. In: Wise, R.R., Hoober, J.K. (Eds.), The Structure and Function of Plastids. Springer, pp. 507– 525. Perrin, R.M., Young, L.S., Murthy, U.M.N., Harrison, B.R., Wang, Y., Will, J.L., Masson, P.H., 2005. Gravity signal transduction in primary roots. Ann. Bot. (Lond) 96, 737–743. Rashotte, A.M., Brady, S.R., Reed, R.C., Ante, S.J., Muday, G.K., 2000. Basipetal auxin transport is required for gravitropism in roots of Arabidopsis. Plant Physiol. 122, 481–490. Ruppel, N.J., Hangarter, R.P., Kiss, J.Z., 2001. Red-light-induced positive phototropism in Arabidopsis roots. Planta 212, 424–430. Sakai, T., Wada, T., Ishiguro, S., Okada, K., 2000. RPT2: a signal transducer of the phototropic response in Arabidopsis. Plant Cell 12, 225–236. Teale, W.D., Papanov, I.A., Palme, K., 2006. Auxin in action: signaling, transport and the control of plant growth and development. Nature Rev. Mol. Cell Biol. 7, 847–859. Trewavas, A.J., 1992. Forum: what remains of the Cholodny–Went theory? Plant Cell Environ. 15, 759–794. Vitha, S., Zhao, L., Sack, F.D., 2000. Interaction of root gravitropism and phototropism in Arabidopsis wild-type and starchless mutants. Plant Physiol. 122, 453– 461. Weise, S.E., Kuznetsov, O.A., Hasenstein, K.H., Kiss, J.Z., 2000. Curvature in Arabidopsis inflorescence stems is limited to the region of amyloplast displacement. Plant Cell Physiol. 41, 702–709. Whippo, C.W., Hangarter, R.P., 2006. Phototropism: bending towards enlightenment. Plant Cell 18, 1110–1119.