During seedling establishment, blue and red light suppress hypocotyl growth through the cryptochrome 1 (cry1) and phytochrome B (phyB) photosensory pathways, respectively. How these photosensory pathways integrate with growth control mechanisms to achieve the appropriate degree of stem elongation was investigated by combining cry1 and phyB photoreceptor mutations with genetic manipulations of a multidrug resistance-like membrane protein known as ABCB19 that influenced auxin distribution within the plant, as evidenced by a combination of reporter gene assays and direct auxin measurements. Auxin signaling and ABCB19 protein levels, hypocotyl growth rates, and apical hook opening were measured in mutant and wild-type seedlings exposed to a range of red and blue light conditions. Ectopic/overexpression of ABCB19 (B19OE) greatly increased auxin in the hypocotyl, which reduced the sensitivity of hypocotyl growth specifically to blue light in long-term assays and red light in high-resolution, short-term assays. Loss of ABCB19 partially suppressed the cry1 hypocotyl growth phenotype in blue light. Hypocotyl growth of B19OE seedlings in red light was very similar to phyB mutants. Altered auxin distribution in B19OE seedlings also affected the opening of the apical hook. The cry1 and phyB photoreceptor mutations both increased ABCB19 protein levels at the plasma membrane, as measured by confocal microscopy. The B19OE plant proved to be a useful tool for determining aspects of the mechanism by which light, acting through cry1 or phyB, influences the auxin transport process to control hypocotyl growth during de-etiolation.
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Of the many environmental cues that influence the early stages of post-germination plant development, light is probably the most influential. When deprived of light in the laboratory or by being buried in soil or leaf litter, dicot seedlings typically develop long juvenile stems, closed apical hooks, and unexpanded, non-green cotyledons. This etiolated growth habit is a strategy for successfully positioning the apex of the seedling into an environment lit well enough to support photosynthesis. Light in the blue and red wavelength bands are potent signals to a seedling that such an environment has been reached. Photomorphogenesis ensues, adapting the plant biochemically, physiologically, and morphologically for photoautotrophy and competition for light.
In many species, including Arabidopsis thaliana, the hypocotyl is the aforementioned juvenile stem structure. Rapid expansion of its cells, on the order of 10% h−1, drives hypocotyl elongation until light suppresses this growth. Blue light can initiate growth suppression in less than a minute (Parks et al., 1998); red light within a few minutes (Parks and Spalding, 1999). After the hypocotyl reaches its new lower growth rate in the light, the tightly curved apical hook subtending the cotyledons begins to open because of a reversal of the growth differential that created and maintained it while pushing through the soil. The hook opens completely within approximately 6 h (Liscum and Hangarter, 1993; Miller et al., 2007; Wang et al., 2009), the two cotyledons begin to separate to unprotect the shoot apical meristem and the first photosynthetically competent organs of the next generation begin to form. In Arabidopsis, in which most of the molecular and genetic work has been performed, the photoreceptor proteins that initiate these critical and profound developmental changes are the phytochromes phyA–phyE, the cryptochromes cry1 and cry2, and the phototropins phot1 and phot2. With respect to hypocotyl growth suppression, which is the focus of the present report, the major contributors are the blue light receptor cry1 and the red light receptor phyB, although transient and even opposing roles for others can be resolved when growth is measured with high resolution (Folta and Spalding, 2001a,b; Folta et al., 2003b). The ‘opposing roles’ refers to a hypothesis that the appropriate rate of stem elongation is the result of light-induced inhibition and counteracting promotion (Parks et al., 2001). Thus, a mutant such as cry1 or phyB has a long hypocotyl in light because the inhibitory pathway is impaired more than the promotive pathway. A mutant with an abnormally short hypocotyl in light such as spa1 may exhibit defective growth promotion (Parks et al., 2001).
At the photoreceptor end of seedling photomorphogenesis, many mechanistic details and even molecular structures are known (Chen et al., 2004; Wang, 2005; Bae and Choi, 2008), as are their intracellular sites of action (Kevei et al., 2007; Wu and Spalding, 2007; Fankhauser and Chen, 2008) and means of affecting gene expression (Casal and Yanovsky, 2005; Sellaro et al., 2009). Much less detailed is the current mechanistic description of the ultimate effect on growth. Earlier studies showed that cell wall-yielding properties, rather than hydraulic parameters, were the proximate causes of light-induced growth inhibition (Cosgrove, 1988; Kigel and Cosgrove, 1991). This is also true of the way in which the hormones auxin and gibberellin exert their effects on stem elongation (Cosgrove and Sovonick-Dunford, 1989; Taylor and Cosgrove, 1989; Schopfer, 2006). Thus, it is reasonable to suppose that light acts through hormones to achieve some of its effects on the wall to control stem-cell elongation. The case of phototropism provides the best established supportive example. Laterally redistributed auxin, resulting from the asymmetric activation of phototropin by unilateral blue light, causes the different rates of cell expansion responsible for phototropic curvature (Esmon et al., 2006). In the case of hypocotyl growth inhibition induced by high-irradiance light, genomic and physiological evidence also points to auxin as a mediator. Genetic changes causing elevated levels of free auxin lead to increased hypocotyl elongation in the light (Romano et al., 1995). Mutants that overproduce auxin, such as sur1 (Boerjan et al., 1995), sur2 (Delarue et al., 1998) and yucca (Zhao et al., 2001), develop long hypocotyls in a light-dependent fashion. The hypocotyls of light-grown axr1 mutants, which have impaired auxin responses, are much shorter than those of the wild type (Collett et al., 2000). High-irradiance blue light alters the expression of auxin response factor genes in a cry1-dependent manner (Folta et al., 2003b). But the mechanism is not likely to be as simple as light downregulating auxin sensitivity or auxin levels to suppress elongation, because exogenous auxin does not substantially counteract the inhibitory effects of light (Tian and Reed, 2001).
Auxin transport through tissues and organs is integral to its action (Teale et al., 2006). Therefore, the photocontrol of growth could depend on light-dependent changes in the auxin transport mechanism. This is not a new idea (Jensen et al., 1998), but recent research has opened up new ways to investigate it. The present work is based on the previous finding that a member of the large superfamily of ATP-binding cassette (ABC) transporters, originally called MDR1, then PGP19 and now ABCB19 (Verrier et al., 2008), is required for the normal transport of auxin from the shoot apex to the cotyledons (Lewis et al., 2009), down the hypocotyl (Noh et al., 2001; Murphy et al., 2002) and through the root, towards the tip (Lewis et al., 2007). Mutations in the ABCB19 gene blocked 50–80% of these auxin streams, so abcb19 mutants (hereafter b19) could be used to investigate the role of auxin transport in the photocontrol of hypocotyl elongation during de-etiolation. Also, a related gene now known as ABCB1 was previously shown to lengthen hypocotyls in light when overexpressed, and to shorten them when expressed in the antisense orientation (Sidler et al., 1998). With these observations as a general background, the current study compared the effects of b19 loss-of-function mutations with the effects of B19 overexpression in different light conditions and photoreceptor mutant backgrounds to investigate the intersection of auxin transport/distribution and growth control during de-etiolation.
ABCB19 overexpression and its effect on auxin distribution
The natural level and locale of B19 expression was determined before it was manipulated to alter the auxin distribution in seedling hypocotyls. Figure 1a shows that a GFP-tagged ABCB19 protein (hereafter GFP-B19) under the control of the native promoter, previously shown to be functional (Wu et al., 2007), was observed at the plasma membrane of cortical cells in both etiolated and light-grown hypocotyls (Figure 1a,c). It was difficult to obtain clear images of cells at the center of the hypocotyl because of the depth of the organ, but the GFP signal appeared to emanate from the vascular cylinder. Thus, B19 may be expressed in all cells of the hypocotyl, except the epidermis (Figure 1a). The same GFP-B19 fusion protein was expressed under the control of the constitutive 35S promoter. Confocal microscopy investigation showed Pro35S:GFP-B19 to generate greater signal than ProB19:GFP-B19, and to express in epidermis, where the native promoter was not effective. This was true in etiolated (Figure 1b) and light-grown (Figure 1d) seedlings. Thus, the Pro35S:GFP-B19 line (hereafter B19OE) can be considered an ectopic/overexpressing line.
To detect any auxin homeostasis change resulting from altered B19 levels or distributions, the ProDR5:GUS auxin reporter was introduced into each genotype mentioned above. Histochemical analysis showed profoundly higher GUS activity in B19OE seedling hypocotyls than in the wild type or the b19 mutant when the seedlings were grown in darkness (Figure 2a), after transfer to blue light (Figure 2b) or after transfer to red light (Figure 2c). The auxin transport inhibitor naphthylphthalamic acid (NPA) suppressed the high ProDR5:GUS signal in B19OE seedlings to wild-type levels (Figure S1), indicating that altered auxin transport was the basis of the high reporter activity. Similar results were obtained in light-grown seedlings, especially in the upper hypocotyl (Figure 2b). However, direct measurements of free indole-3-acetic acid (IAA) content in entire seedlings indicated similar levels in B19OE and in the wild type (19.4 ± 1.2 versus 19.5 ± 1.3 pg mg−1 FW, n = 4). The apparent paradox of normal auxin levels in seedlings showing such high ProDR5:GUS activity in the shoot may be explained by much less auxin in the apex of B19OE roots (Figure 2b). This high shoot, low root interpretation suggested by the histochemical staining is valid only if the sensitivity of the signaling mechanism linking auxin to the DR5 promoter is similar in both the wild type and the overexpressor. Figure 2c shows that ProDR5:GUS was similarly induced by a wide range of exogenous IAA concentrations in B19OE and in the wild type: the reporter construct was equally sensitive to auxin in both genotypes. Thus, ectopic overexpression of B19 resulted in altered auxin distribution within seedlings, with no change in the overall auxin content. The higher auxin level in the hypocotyl was used to investigate the role of auxin in light-induced hypocotyl growth and development.
Effects of altered auxin distribution on hypocotyl growth in light
Figure 3a shows the effect of increasing fluence rate of blue or red light on hypocotyl length. Figure 3 shows the effect of increasing fluence rate of blue or red light on hypocotyl length. Figure 3a shows that growth suppression by blue light was particularly affected in B190E. Figure 3a shows that growth suppression by blue light was particularly affected in B19OE. At 50 μmol m−2 sec−1, B19OE seedlings were approximately 80% taller than wild-type or b19 seedlings. Red light is considerably less inhibitory than blue light in general, acting through phytochromes instead of cryptochromes. The effect of B19 manipulation, and by inference auxin distribution, was observed in red light, but to a lesser extent than in blue light (Figure 3b). The results in Figure 3 are generally like those obtained with ABCB1 except growth in red light was slightly more affected than growth in blue light (Sidler et al., 1998). The results in Figure 3 give evidence that seedling photomorphogenesis and auxin action are interrelated by a mechanism that involves B19. These 7-day experiments capture what can be considered to be the near-final state of hypocotyl growth because little elongation occurs beyond this time. To characterize the genotypes at a time when the hypocotyls are still growing, a modified 4-day protocol was employed (2 days in the dark before transfer to the indicated light treatment for 2 days). The hypocotyl end-point lengths were measured at the end of these 4 days, and are co-plotted with the 7-day results. Together, the plots enable a visualization of the changes that occur in a fluence-rate-dependent manner between 4 and 7 days in the different genotypes. Hypocotyls after 4 days were shorter than after 7 days, as expected. The effect of increasing fluence rate and the effect of the genetic manipulation were similar at the 4- and 7-day points. One interesting inference that can be drawn from the time-point comparisons is that growth promotion as a result of B19 overexpression continued to accrue between 4 and 7 days in 50 μmol m−2 sec−1 blue light.
An examination of response time courses was conducted using a new method for measuring hypocotyl elongation and shape changes (Wang et al., 2009), to learn when the growth-rate differences between the genotypes differing in auxin distribution began to manifest themselves. Figure 4a shows the well-established rapid inhibition of hypocotyl elongation caused by the onset of irradiation with high fluence-rate blue light (50 μmol m−2 sec−1) in the wild type, and the subsequent sustained phase of low growth rate. B19OE responded essentially like its Ws wild type throughout the 12-h experiment, but b19-1, a T-DNA insertion allele in the Ws background, showed an even stronger inhibition for approximately 6 h, beginning approximately 1 h after the onset of irradiation. Essentially the same result was obtained with b19-3 and its corresponding Col-0 wild type (Figure 4b). Wild-type seedlings treated with NPA before the onset of irradiation responded very much like the (phenocopied) b19-1 mutant. The data in Figure 4a show that loss or gain of B19, and by inference auxin, had a modest effect on the initial 12 h of hypocotyl growth inhibition induced by blue light. A previous study also found that exogenous auxin by itself had only a modest effect on growth inhibition, but that when combined with active GA4, auxin was much more effective (Folta et al., 2003b) at counteracting blue light. Thus, Figure 3 indicates auxin and B19 have a role to play in hypocotyl elongation in blue light, but Figure 4 indicates the role is not strong in the early part of the response, consistent with the mechanism being dependent on other factors as well.
The main photoreceptor responsible for growth inhibition in blue light is cry1, as evidenced by its fourfold longer hypocotyl compared with the wild type after long-term blue light (Ahmad and Cashmore, 1993). Figure 4b shows the well-established cry1 hypocotyl response to blue light, namely an initial inhibition, mediated by phot1 (Folta and Spalding, 2001b; Folta et al., 2003a), followed by an escape from inhibition after 30 min and resumption of fast growth. Previous work indicated that this fast growth phase in cry1 resulted from a combination of gibberellin and auxin action (Folta et al., 2003b). Loss of B19 partially suppressed the fast growth of cry1, indicating that auxin transport plays a role in the escape from inhibition that produces the cry1 long hypocotyl phenotype.
In red light, a controlling role for B19 in growth suppression was even more evident in the time-course results. Over/ectopic expression of B19 counteracted red light-induced growth suppression, whereas the b19-1 mutation enhanced it (Figure 5a). In addition, over/ectopic B19 expression delayed the onset of red light-induced growth inhibition mediated by phyA (Parks and Spalding, 1999), by approximately 15–30 min. Long-term inhibition of hypocotyl growth by red light is mediated by phyB in Arabidopsis. This can be seen in the fast growth of phyB seedlings that followed the initial phyA-mediated phase of inhibition (Parks and Spalding, 1999). The sustained escape from inhibition displayed by phyB seedlings required B19, as evidenced by the significant decline in the growth rate of a b19 phyB double mutant (Figure 5b). Thus, for much of the 10-h light period assayed with high resolution, B19-dependent auxin transport can be thought of as part of the growth-promoting activity that counteracts photoreceptor-mediated suppression.
Effects of blue or red light on ABCB19 and auxin levels
The ProB19:GFP-B19 fusion protein was used as a tool to investigate the possible regulation of its expression by photoreceptors that could help explain the growth results in Figures 3–5. Quantitative confocal microscopy performed on the most rapidly elongating region of the hypocotyl (Figure S2) showed that blue light suppressed levels of B19 protein in the wild type (Figure 6a), consistent with the notion that blue light reduces auxin transport to the growing cells to slow elongation. Inexplicably, the cry1 mutant showed fourfold more B19 protein than the wild type, even in seedlings that had never been exposed to any light, except the actinic laser of the confocal microscope (Figure 6a). Apparently, cry1 somehow prevents B19 from accumulating, even in the absence of blue light. Perhaps cry1 seedlings are predisposed to grow faster in blue light because they lack cryptochrome-mediated growth suppression, and have elevated the B19-dependent growth promotion capacity. Unlike blue light, 5 h of red light did not reduce B19 levels in the wild type (Figures 6b and S2). But loss of phyB caused a similarly large increase in B19 protein, even in etiolated seedlings never exposed to red light (Figure 6b), which could be expected to promote growth based on the results presented in Figures 3b and 5a. Perhaps phyB seedlings grow faster in red light because of a predisposition for auxin-mediated growth promotion, an influence established before the onset of irradiation. Not all photoreceptor mutations raise B19 levels. In phot1 mutants, B19 levels were lower than in the wild type (Figure 6c). Phototropin1 initiates blue-light-induced growth inhibition, but the inhibition is transient, lasting, at most, 0.5 h (Folta and Spalding, 2001b; Folta et al., 2003a).
One conclusion to be drawn from the results in Figure 6 is that higher levels of B19 promote faster hypocotyl growth by increasing auxin in the growing cells. To test this idea, the auxin-responsive ProDR5 element was used to drive GFP expression in wild-type and mutant seedlings. Confocal microscopy was used to quantify the reporter signal strength in apical regions of the hypocotyl, where cells were elongating most rapidly. Figure 7a and Figure S3 show that ProDR5:GFP expression was lower in b19 hypocotyls and higher in cry1 hypocotyls compared with the wild type, regardless of the light conditions. The expression of ProDR5:GFP in the b19 cry1 double mutant was intermediate between the two single-mutant values. These auxin signaling assays performed 5 h after the onset of blue light roughly correlated with hypocotyl growth rates after equivalent lengths of irradiation (Figure 4b). However, the correlation between ProDR5 activity and growth rate has exceptions. ProDR5 activity is very high in B19OE hypocotyls (Figure 2a,b), but evidence of a faster growth rate was found only in the end-point analyses (Figure 3a), not in the time-course results (Figure 4a).
In red light, the correlation was strong. ProDR5 activity was high in phyB (Figure 7b) and B19OE hypocotyls (Figure 2a,c) after 5 h of red light, and so was the growth rate (Figures 3b and 5). A lower growth rate in red light correlated with lower ProDR5 activity in b19 and b19 phyB mutants (Figures 3b, 5 and 7b). In both red and blue light, b19 mutations suppressed the fast growth of the photoreceptor mutants. The promotive influence on hypocotyl growth in phyB and cry1 mutants depends to a significant extent on B19, apparently through its influence on auxin levels in the growing portion of the hypocotyl.
ABCB19 and apical hook opening in blue light
The apical hook is created by a growth rate differential across the hypocotyl at the apex. Light reverses the growth differential as cells flow through the hook region to open it (Silk, 1980). Previous high-resolution studies have shown that the process begins approximately 1 h after the onset of blue light, or approximately 30 min after growth reached its initial minimum value (Miller et al., 2007; Wang et al., 2009). The cry1 mutation did not affect hook opening (Wang et al., 2009).
B19 protein distribution in the hook region indicated a potential role in this important aspect of photomorphogenesis. Usually, the protein was localized primarily to the plasma membrane of epidermal cells only on the concave flank of the tightly folded hook, which is the slower growing side (Figure 8a). Occasionally (in approximately 10% of seedlings), a non-membrane-localized distribution was observed (Figure 8b). This special concentration of B19 on the concave, slower growing side of the hook overlapped with the region of high ProDR5 signal strength (Figure 9). Thus, again, there is a strong correlation between the expression level of B19 and the auxin level, which may reflect B19 function in auxin transport or the fact that its expression is auxin dependent (Noh et al., 2001). An important difference between the hook and hypocotyl elongation results is that the high-B19, high-auxin area is the slowest growing part of the hook, whereas the previous figures showed a correlation with faster growth. One possible explanation is that the high auxin content on the concave side of the hook is surpraoptimal, present at an inhibitory concentration.
Expressing B19 under the control of the 35S promoter produced an expression pattern that was often like that of the ProB19:GFP-B19 plants (Figure 8c), but in approximately 10% of seedlings the GFP-B19 signal was seen more uniformly across the hook, and sometimes non-plasma-membrane signal was observed (Figure 8d). The non-plasma-membrane signal may be evidence that B19 occasionally does not efficiently reach or is more readily retrieved from the plasma membrane in the slower growing, high-auxin cells on the concave side. Delocalization from the plasma membrane may be part of a mechanism for slowing the expansion of these cells to maintain the hook.
Figure 9 shows that the auxin asymmetry across the wild-type hook, which may be caused by asymmetric B19 localization, dissipated as the hook opened. The b19 mutant was not discernibly different from the wild type, but B19OE was dramatically different. The strong ProDR5:GUS staining in the B19OE hook formed a gradient that persisted through the 4-h point when the wild-type gradient had disappeared. Seedlings of all three genotypes maintained in darkness but treated with NPA showed open hooks and no auxin gradient (Figure 9), further evidence that auxin transport is the key process in determining hook shape.
The HYPOTrace image-processing technology used to measure hypocotyl growth with high resolution automatically quantifies the hook angle (Wang et al., 2009). Figure 10a shows that the rate of hook opening in response to blue light in B19OE seedlings was substantially slower than in the wild type. This is consistent with the more persistent auxin gradient across the hook, which presumably results from higher and/or ectopic expression of B19. The b19 mutation did not significantly affect the rate of hook opening, consistent with its apparent lack of effect on the trans-hook auxin gradient (Figure 9). Because B19 functions together with its closest relative ABCB1 (formerly known as PGP1), the b1 b19 double mutant was examined to see if its apical hook auxin gradient and opening response were more severely affected than the b19 single mutant. The double mutant hook was not as tightly closed in darkness as the other genotypes, and it opened more slowly in response to blue light (Figure 10a). ProDR5:GUS created no staining in the double mutant hook region. Thus, B1 and B19 together form the auxin gradient that light diminishes to open the apical hook. In continuous darkness, the apical hook remained fully closed over the 12-h assay period (Figure 10b). Application of NPA (Figure 10b) to block polar auxin transport initiated hook opening, as well as dissipating the trans-hook auxin gradient (Figure 9). This indicates that auxin transport generates the trans-hook auxin gradient, and that its dissipation is responsible for hook opening. Manipulation of the trans-hook auxin gradient by over/ectopic expression of B19 indicates that this ABC transporter plays a role in establishing the gradient, and therefore in hook morphology. The lack of an effect in the b19 mutant may indicate that similar functions are performed by other family members, such as ABCB1.
B19OE as a tool for studying auxin-mediated development
A key attribute of B19OE that made it useful for exploring the relationships between ABC transporter control of auxin distribution and seedling photomorphogenesis was its substantial effect on auxin distribution (Figure 2) without excessive pleiotropy. In some cases B19 over/ectopic expression perturbed the system in more useful ways than b19 mutations. Hook opening is a good example, as b19 had little effect on the auxin gradient or the response. B19OE, on the other hand, provided good evidence that hook behavior is controlled by auxin (Figures 9 and 10). Genetically removing another ABCB family member was necessary before loss of function could affect hook auxin and opening. But then the hook was already partly opened in darkness, probably because of a major change in auxin distribution. Despite pleiotropic complications such as irregularly curved hypocotyls, slow growth and dwarfism at maturity (Noh et al., 2001, 2003; Geisler et al., 2003; Blakeslee et al., 2007; Nagashima et al., 2008), the b1 b19 double mutant here supported a significant conclusion. An auxin gradient across the hook could not be detected, yet an angle of 120° was nonetheless formed in the double mutant. That a hook can form in this situation argues against auxin alone being the determinant of the growth differential responsible for apical hook maintenance. That the hook is not as tightly closed as in the wild type indicates that the B1 and B19 proteins have a role in hook maintenance.
Auxin may rarely or never act alone, but instead its effects are often found to be interrelated with other hormones. Figures 9 and 10 show a controlling role for auxin in hook maintenance and light-induced opening, but ethylene and gibberellin are also known to play roles (Raz and Ecker, 1999; Vriezen et al., 2004). With the present work as background and the HYPOTrace tool for response quantification, the Arabidopsis apical hook may serve as a useful system for studying the integration of the multiple hormone actions that are believed to be important in seedling photomorphogenesis (Symons and Reid, 2003; Alabadí and Blázquez, 2009).
Promotion of growth by auxin in light not dark
Loss of B19 was previously shown to reduce polar auxin transport down the hypocotyl of etiolated seedlings by 80% (Noh et al., 2001), and B19OE was here shown to increase auxin signaling dramatically in the etiolated hypocotyl (Figure 2a), yet the large effects on auxin transport and signaling caused by B19 manipulations or NPA treatment had no distinguishable effect on hypocotyl growth rate before exposure to light (Figures 4 and 5). Only after some time in red or blue light did the persistent perturbations of auxin transport and/or distribution (Figure 2b,c) begin to affect the growth rate (Figures 4 and 5). One explanation that fits previous observations of Arabidopsis hypocotyls is that growth control in darkness is mechanistically distinct from growth control in light, and that only the latter depends on auxin transport (Jensen et al., 1998). Transition from one to the other occurs during photomorphogenesis. One tentative conclusion is that B19 counteracts light-induced growth inhibition to help achieve a hypocotyl elongation rate appropriate for the prevailing conditions, by a mechanism that involves auxin transport. Overexpression of B19 strengthens this counteraction, whereas mutation weakens it, allowing photoreceptor-dependent inhibition to be more effective (Figures 3–5). This interpretation fits with the transcriptome analysis of Folta et al. (2003b), which showed auxin-regulated gene expression changes at the onset of cry1 fast hypocotyl growth, and with several other studies that have found auxin to promote elongation in light-grown seedlings (Boerjan et al., 1995; Romano et al., 1995; Delarue et al., 1998; Zhao et al., 2001). This postulated light-dependent, B19-mediated, auxin-regulated growth may share a pathway or operate entirely independently of the light-induced inhibition it counteracts. The fact that the phyB and b19 phyB responses to red light begin to diverge after 4 h of illumination may be evidence of B19 action that is independent of phyB. The ‘push’ on growth by 4 h is independent of the phyB-mediated inhibition it is counteracting.
Photoreceptor control of ABCB19, polar auxin transport and auxin levels
The integration of light and auxin effects on growth may be achieved at the level of auxin transport from the site of biosynthesis, and redistribution within target organs (Tian and Reed, 2001). Recent work suggests that phyA and phyB control the distribution of auxin between the shoot and root (Salisbury et al., 2007). Previously, it has been demonstrated that phytochrome causes a reduction in the IAA content of epidermal but not cortical cells of pea stems, which correlated with red light-induced growth inhibition (Behringer and Davies, 1992). Such changes in transport and distribution by light are probably achieved by altering the subcellular distribution of auxin efflux carriers such as PIN2 in the root (Laxmi et al., 2008) or, as was recently shown for phototropism, by delocalizing PIN1 from the plasma membrane on the lit side of the hypocotyl (Blakeslee et al., 2004).
Directly relevant to the present study is the recent demonstration that red light acting through phytochromes reduces polar auxin transport in Arabidopsis hypocotyls, probably by reducing B19 protein levels (Nagashima et al., 2008). This previous observation along with the finding that red light-induced growth inhibition was stronger in b19 mutants and weaker in B19OE (Figures 3b and 5) leads to a model in which phytochrome inhibits hypocotyl elongation through reduced B19-dependent auxin transport. The effects of overexpression and antisense manipulations of the related B1 gene performed by Sidler et al. (1998) fit a version of this model in which B1 and B19 function together, which is believed to be the case for reasons discussed above. Time-course data become especially useful at this point, because they allow this working model to be refined. Growth rate was reduced by approximately 50% (Figure 5) before any reduction in B19 protein could be detected with western blots (Nagashima et al., 2008). Instead of initiating hypocotyl inhibition, the B19 reduction detected by Nagashima et al. (2008) between 2 and 4 h of red light coincided with the transition from phyA-mediated to phyB-mediated hypocotyl growth inhibition (Parks and Spalding, 1999; Figure 5c). Therefore, the better model may be that red light inhibits hypocotyl elongation first through phyA (Parks and Spalding, 1999) and then through a phyB effect on B19-mediated auxin transport. One observation that does not neatly fit is the lack of effect of red light on B19 levels in the elongating zone of the hypocotyl (Figure 6b). One possibility is that a reduction occurred shortly after this 5-h measurement point. Another possibility is that the key reduction occurs in a region above the elongating zone, thereby affecting the delivery of auxin to the expanding cells. Even more worthy of clarification is the strong effect of photoreceptor mutation on B19 levels in seedlings grown in the absence of light. The effect, quantified in Figure 6b and obvious in the results of Nagashima et al. (2008), is of unknown significance.
A theme of the present work is that hypocotyl growth control mechanisms, whether mediated by phytochromes or cryptochromes, must ultimately converge on the process of cell expansion, and that the hormone auxin could be an agent of integration in this respect. The effects of red and blue light on B19 levels (Figure 6; Nagashima et al., 2008) indicate B19-mediated auxin transport may be a point of convergence. In red light, the overall correlations between B19 protein, auxin and growth rate were more consistent than in blue light. The discrepancies in blue light, such as reduced B19 protein levels (Figure 6a; Nagashima et al., 2008) without significantly reduced auxin levels in the elongation zone (Figure 7a), despite strong growth inhibition (Figure 4), may be evidence of a more complicated web of responses operating in this condition. It is already known that nuclear cry1 acts through plasma membrane anion channels to inhibit growth after the phot1 phase is complete (Cho and Spalding, 1996; Folta and Spalding, 2001b; Wu and Spalding, 2007), but that the anion channel-dependent phase cannot account for the full cry1 phenotype (Parks et al., 1998). So, although there may be a general correlation between the levels of B19 protein, auxin and growth rate – the faster growing genotypes had more, and the slower growing genotypes had less – there is a need for more studies employing genetic manipulation of photoreceptors and auxin transport proteins, live cell imaging, and high-resolution methods for measuring responses, to connect the various components into pathways that positively and negatively affect growth rate to achieve the rate of hypocotyl growth appropriate for the prevailing conditions.
Arabidopsis thaliana ecotypes Wassilewskija (Ws) and Columbia (Col-0) were the wild types used here. The b19-1 mutant in the Ws background is the line formerly called mdr1-1 (Noh et al., 2001), and b19-3 in the Col-0 background is the same as mdr1-3, which has also been described previously (Lewis et al., 2007). Construction of the plant expressing the ProB19:GFP-B19 molecule in the b19-1 background was described previously (Wu et al., 2007). The cry1-304 mutation used here was previously described by Mockler et al. (1999), and the phyB (SALK_022035) allele was previously described by Seo et al. (2006). The ProDR5:GUS plants used here were in the Ws genetic background, and the ProDR5:GFP plants were in the Col-0 background.
To generate the GFP-B19 fusion construct, the complete B19 cDNA coding sequence was amplified by PCR, and fused in-frame to the C terminus of eGFP in the pEGAD vector, as described previously (Wu et al., 2007). The CaMV 35S promoter was inserted upstream of the GFP-B19 fusion. The resulting Pro35S:GFP-B19 vector was introduced into Agrobacterium tumefaciens GV3101, and the resulting cells were used to transform b19-1 mutant plants using the floral-dip method (Clough and Bent, 1998). Basta resistance was used to identify transformed plants. Lines homozygous for the Pro35S:GFP-B19 transgene were obtained in the T3 generation.
IAA sensitivity analysis based on the MUG assay
Seedlings grown in white light for 3 days were sprayed with an IAA solution of the indicated concentration, using an aerosol sprayer. Three hours after IAA application, 10 seedlings per trial were harvested and subjected to a MUG assay of GUS activity, as described previously (Lewis et al., 2007).
Wild-type and B19OE seedlings were grown for 3 days in white light on half-strength MS medium (0.8% agar, pH 5.7) before entire seedlings were harvested, extracted and subjected to IAA quantification, as described previously (Ljung et al., 2005).
Hypocotyl length measurements
Arabidopsis seeds of the indicated genotype were surface sterilized, plated on solid medium (half-strength MS medium, 0.8% agar, pH 5.7), and stratified at 4°C for 2 days to break dormancy and synchronize germination. After cold treatment, the plates were placed horizontally under continuous light of the indicated wavelength and photon fluence rates, at a temperature of 22°C. On day 7, the seedlings were removed from the plate and imaged with a flatbed scanner. ImageTool v3.0 (UT Health Science Center, http://ddsdx.uthsca.edu/imagetool.asp) was used to quantify hypocotyl length. Each data point represents the mean of 15–20 seedlings in each of three trials.
Time course of hypocotyl growth inhibition and hook opening
Seeds were sown on 1% agar plates containing 1 mM KCl and 1 mm CaCl2 (pH 5.7), and then stratified as described above before being subjected to 30 min of white light to stimulate germination. After 2 days of growth in total darkness, the seedlings were transferred to a new agar plate to be mounted perpendicular to the optical axis of the image-acquisition apparatus described previously (Miller et al., 2007; Wang et al., 2009). Digital images of the seedlings growing in complete darkness and then responding to light were automatically acquired every 10 min, and were then subjected to morphometric analysis to quantify the hypocotyl growth rate and apical hook angle as a function of time using the HYPOTrace method (Wang et al., 2009).
Confocal fluorescence microscopy analysis
Confocal microscopy was performed on a Zeiss LSM 510 laser scanning confocal microscope, equipped with a Meta detector and a C-Apochromat 40× water immersion lens (Zeiss, http://www.zeiss.com). The sample was excited with the 488-nm laser line from a 30-mW argon gas laser. The fluorescence was captured in 10-nm bandwidths, and then linear unmixing was performed to isolate the GFP signal from the background chlorophyll and cell wall. Pixels that were determined to be GFP and chlorophyll were false-colored green and red, respectively. To measure the GFP intensities, high-resolution images of the stem elongation zone (1.2–1.5 mm from the shoot meristem) were obtained. The mean fluorescence intensity was calculated within manually selected regions of interest with Zeiss LSM 510 software. To measure GFP-B19 intensity in enlarged images of the plasma membrane, a thin rectangle area covering the membrane of a cortical cell was selected. To measure the overall GFP intensity of ProDR5-driven GFP, representative hypocotyl regions of approximately 0.1 mm2 were selected for fluorescence quantification.
Funding was provided by NSF Grant DBI-0421266 to EPS.
Accession numbers: The Arabidopsis Genome Initiative locus identifier for the ABCB19 gene (formerly MDR1) is At3g28860; CRY1 is At4g08920; and PHYB is At2g18790.