Root-localized phytochrome chromophore synthesis is required for photoregulation of root elongation and impacts root sensitivity to jasmonic acid in Arabidopsis.

Plants exhibit organ- and tissue-specific light responses. To explore the molecular basis of spatial-specific phytochrome-regulated responses, a transgenic approach for regulating the synthesis and accumulation of the phytochrome chromophore phytochromobilin (PΦB) was employed. In prior experiments, transgenic expression of the BILIVERDIN REDUCTASE (BVR) gene was used to metabolically inactivate biliverdin IXα, a key precursor in the biosynthesis of PΦB, and thereby render cells accumulating BVR phytochrome deficient. Here, we report analyses of transgenic Arabidopsis (Arabidopsis thaliana) lines with distinct patterns of BVR accumulation dependent upon constitutive or tissue-specific, promoter-driven BVR expression that have resulted in insights on a correlation between root-localized BVR accumulation and photoregulation of root elongation. Plants with BVR accumulation in roots and a PΦB-deficient elongated hypocotyl2 (hy2-1) mutant exhibit roots that are longer than those of wild-type plants under white illumination. Additional analyses of a line with root-specific BVR accumulation generated using a GAL4-dependent bipartite enhancer-trap system confirmed that PΦB or phytochromes localized in roots directly impact light-dependent root elongation under white, blue, and red illumination. Additionally, roots of plants with constitutive plastid-localized or root-specific cytosolic BVR accumulation, as well as phytochrome chromophore-deficient hy1-1 and hy2-1 mutants, exhibit reduced sensitivity to the plant hormone jasmonic acid (JA) in JA-dependent root inhibition assays, similar to the response observed for the JA-insensitive mutants jar1 and myc2. Our analyses of lines with root-localized phytochrome deficiency or root-specific phytochrome depletion have provided novel insights into the roles of root-specific PΦB, or phytochromes themselves, in the photoregulation of root development and root sensitivity to JA.

Light can have opposing effects in different plant tissues: light exposure inhibits hypocotyl elongation, whereas light promotes cotyledon expansion and root development (for review, see Bou-Torrent et al., 2008). The occurrence of distinct organ-or tissue-specific, light-regulated processes in plants is supported by tissue-specific microarray analyses. For example, dis-tinct subsets of light-regulated genes can be identified from cotyledons versus roots in Arabidopsis (Arabidopsis thaliana; Jiao et al., 2005;Ma et al., 2005) or from shoots versus roots in rice (Oryza sativa; Jiao et al., 2005). Red (R) and far-red (FR) light-absorbing phytochromes are expressed in roots of Arabidopsis (Tóth et al., 2001;Salisbury et al., 2007), as are blue (B) light photoreceptors (Tó th et al., 2001;Galen et al., 2007). UV-B is also absorbed by roots, and its absorption impacts root and seedling development in Arabidopsis (Tong et al., 2008;Leasure et al., 2009). The lightdependent growth responses of roots and the localization of photoreceptors in roots themselves implicate root-localized light perception as an important attribute for photomorphogenic adaptation in plants.
Light can penetrate several millimeters into the upper layers of soil, and the depth of light penetration depends on the soil composition (Tester and Morris, 1987;Mandoli et al., 1990). These findings suggest that roots can perceive light directly in natural environments and that root development is likely impacted by light under certain physiological conditions. Indeed, light is expected to function as a useful signal for roots to perceive their depth in the soil stratum. Certainly, phytochromes in seeds perceive light that penetrates the soil, as it is well recognized that seed germination is impacted by light (for review, see Shinomura, 1997;Seo et al., 2009). In addition to localized detection of light, roots may also be impacted by light perceived by aerial plant tissues through internal tissue piping effects (Mandoli and Briggs, 1982).
Recent results indeed have demonstrated that photoreceptors, including phytochromes and B light-absorbing phototropins, affect root growth and development in natural environments. Galen et al. (2007) have shown that phototropin localization in roots is correlated with increased root growth efficiency in the natural environment. In soil-grown plants, phytochrome also has been shown to have a role in root development (i.e. phytochrome B [phyB] regulates the initiation of lateral roots for plants grown on a soil/compost mixture; Salisbury et al., 2007). This finding suggests that roots in a more natural environment (i.e. growing under soil) exhibit phytochrome-dependent regulation of root development.
In addition to a role in root development, the photoreceptor phytochrome also regulates other distinct aspects of photomorphogenesis in plants, including seed germination, cotyledon expansion and leaf development, inhibition of hypocotyl elongation, and the induction of flowering (Chen et al., 2004;Mathews, 2006). In higher plants, photoactive phytochrome consists of an apoprotein covalently attached to the linear tetrapyrrole chromophore, phytochromobilin (PFB; Terry et al., 1993). All higher plant phytochromes, including phyA to phyE in Arabidopsis (Sharrock and Quail, 1989;Clack et al., 1994), use this chromophore for their photoregulatory activities (Terry et al., 1993).
Apart from the above-mentioned role for phyB, the roles of phytochromes in roots have not been studied extensively in soil-grown plants or natural environments. However, roles for phytochromes in the regulation of distinct aspects of root development have been identified definitively in controlled photobiological studies. For example, R light absorbed by rootlocalized phyA and phyB impacts root phototropism (Kiss et al., 2003) and inhibits root elongation rates (Correll and Kiss, 2005) in Arabidopsis. Recent microarray analyses for roots exposed to R light indicate that the expression of many genes, including a number of genes involved in photomorphogenesis and root development, are regulated by light in roots (Molas et al., 2006). FR light also impacts root growth, i.e. stimulating phyA-dependent root elongation in Arabidopsis seedlings (Kurata and Yamamoto, 1997).
Results regarding the impact of global phytochrome deficiency on root elongation in chromophore biosynthetic mutants, which exhibit deficiencies in all members of the phytochrome family, are unresolved. Previously, a elongated hypocotyl1 (hy1) mutant (hy1 [21.84N]) was shown to have longer roots than the wild type control under white (W) light illumination (Muramoto et al., 1999), whereas in separate reports, additional hy1 mutants (hy1-100 and hy1-101) appear to have slightly shorter roots than the ecotype Columbia (Col-0) wild-type parent (Zhai et al., 2007). Notably, HY1, which encodes a heme oxygenase, is expressed in roots (Davis et al., 1999(Davis et al., , 2001Emborg et al., 2006), and expression of HY1 is down-regulated by treatment with the plant hormone jasmonic acid (JA; Zhai et al., 2007). JA is a regulator of plant growth and development, in addition to its recognized role in regulating defense responses (Browse, 2005). JA generally inhibits leaf growth (Zhang and Turner, 2008) and root growth (Staswick et al., 1992) in Arabidopsis. Inhibition of root growth by JA has been widely used to identify mutants impaired in JA biosynthesis and/or signaling based on insensitivity to root growth inhibition upon exogenous application of JA or JA analogs. Notably, phytochrome chromophore deficiency is associated with altered JA-mediated root inhibition (Zhai et al., 2007), suggesting a link between phytochrome chromophore biosynthesis and JA signaling pathways.
An initial report on a molecular link between JA and phytochrome signaling described a mutant displaying a FR-specific long-hypocotyl phenotype, which was found to have a mutation in the FAR-RED-INSENSI-TIVE219 (FIN219) gene (Hsieh et al., 2000). The fin219 mutation was found to be allelic to the jasmonate resistant1 (jar1) mutation (Staswick et al., 2002). JAR1 encodes a JA-amino synthetase, catalyzing the conjugation of JA to Ile, required for optimal signaling in jasmonate responses in Arabidopsis (Staswick and Tiryaki, 2004). Both jar1 and fin219 mutants display a long-hypocotyl phenotype under continuous FR (FRc) compared with the wild type (Chen et al., 2007), suggesting that mutants with defects in JA biosynthesis also are impaired in phytochrome-mediated signaling in FR. Additionally, the PHYTOCHROME AND FLOWERING TIME1 gene, which encodes a component that negatively regulates phytochrome signaling, is required for JA-dependent expression of defense genes in Arabidopsis (Kidd et al., 2009). It appears that phyA is a critical player in the interaction with JA, as a phyA mutant recently has been shown to be hyposensitive in JA root inhibition assays (Robson et al., 2010).
Here, we describe the characterization of light-dependent root development in transgenic Arabidopsis plants exhibiting induced phytochrome chromophore deficiency. Previous studies using tissue-specific promoters to induce localized phytochrome chromophore deficiencies through targeted expression of the BILIVERDIN REDUCTASE (BVR) gene enabled the identification of distinct light-impaired phenotypes (Montgomery, 2009;Warnasooriya and Montgomery, 2009;Warnasooriya et al., 2011). To examine the role of root-localized phytochromes in the photoregulation of root growth and development in Arabidopsis, we analyzed roots of promoter-driven BVR lines in addition to a newly generated GAL4-based enhancer-trap transactivation BVR line exhibiting root-specific phytochrome chromophore deficiency. The analyses of these plants, which all exhibit differential accumulation of a phytochrome chromophore-degrading enzyme in distinct tissues, provide insights into the impact of shoot-versus root-derived phytochrome signals on the photoregulation of root lengths and indicate that root-localized phytochrome chromophore deficiencies are correlated with light-dependent defects in root development and root sensitivity to JA in Arabidopsis.

Phytochrome or PFB Impacts Root Elongation in Arabidopsis
We compared light-dependent root lengths in lines exhibiting phytochrome chromophore deficiencies using either constitutive (Montgomery et al., 1999) or tissuespecific, promoter-driven (Warnasooriya and Montgomery, 2009) BVR lines. We confirmed the accumulation of BVR protein in the roots of these lines using anti-BVR immunohistochemical analysis (Fig. 1). We detected BVR accumulation in the roots of only the 35S::pBVR3 (plastid-localized BVR) and 35S::cBVR1 (cytosolic BVR) lines ( Fig. 1, B and C), as determined previously using immunoblot analyses (Warnasooriya and Montgomery, 2009;S.N. Warnasooriya and B.L. Montgomery, unpublished data). We determined that only those lines that have BVR-induced phytochrome chromophore deficiencies in roots themselves (i.e. 35S::pBVR3 or 35S::cBVR1) had noticeably longer roots relative to the Nossen (No-0) wild type when grown under continuous white (Wc) illumination ( Fig. 2A). When we quantified root lengths for No-0 wild-type and BVR transgenic lines, we determined that 35S::pBVR3 and 35S::cBVR1 lines have significantly longer roots (P , 0.0001) than the No-0 wild-type parent (Fig. 2B). The roots of 35S::pBVR3 and 35S::cBVR1 seedlings are approximately 45% and approximately 90% longer than the roots of No-0 wild-type seedlings, respectively. Notably, the roots of CAB3::pBVR2 seedlings, which exhibit mesophyll-specific phytochrome deficiency (Warnasooriya and Montgomery, 2009), also were marginally longer by approximately 21% than those of the No-0 wild type (P = 0.0165; Fig. 2B), although this difference was not to the same degree as observed for 35S::pBVR3 and 35S::cBVR1. By contrast, the root lengths of MERI5::pBVR1 seedlings, which exhibit shoot-apex-specific BVR accumulation (Warnasooriya and Montgomery, 2009), were not significantly different from those of the No-0 wild type (P = 0.324; Fig. 2B).
As the differences observed for promoter-driven BVR lines indicated that highly significant differences observed in root lengths were for those lines in which the phytochrome chromophore was depleted in roots themselves, we generated a line exhibiting root-specific phytochrome chromophore deficiency using a GAL4based bipartite enhancer-trap transactivation system that has been successfully employed in Arabidopsis (Laplaze et al., 2005) as well as in rice (Johnson et al., 2005). M0062 exhibits root-specific GFP expression and, thus, GAL4 accumulation (http://www.plantsci.cam.ac. uk/Haseloff/assembly/page167/index.html; Haseloff, 1999). After a cross of the M0062 enhancer-trap line with a GAL4-responsive UASBVR line, we isolated a transgenic BVR enhancer-trap line, M0062/UASBVR, that exhibits root-localized BVR protein accumulation (Fig. 3G). In this line, which has root-localized PFB deficiency and thus root-specific holophytochrome deficiencies, we observed that roots of Wc-grown seedlings were longer than the C24 ecotype background ( Fig. 2A), similar to the response observed for the 35S promoterdriven BVR lines. When quantified, the roots of M0062/ Figure 1. Whole-mount immunolocalization of BVR protein accumulation in roots of wild-type and promoter-driven BVR seedlings. No-0 wild-type (A and F), 35S::pBVR3 (B and G), 35S::cBVR1 (C and H) CAB3::pBVR2 (D and I), and MERI5::pBVR1 (E and J) seedlings were grown on Phytablend medium containing 1% (w/v) Suc for approximately 4 d at 22°C under Wc illumination of 100 mmol m 22 s 21 . Seedlings were incubated with anti-BVR primary antibody at a 1:4,000 dilution, except for 1:3,000 dilution for 35S::cBVR1. The top row shows fluorescence images and the bottom row shows DIC images for each seedling. Each image is a representative slice from a Z-series with 0.5-mm interval size and was captured using 543-nm laser excitation with a 203 lens objective. Fluorescence images were collected using a 560-to 615-nm band-pass filter. Bars = 50 mm.
In comparative studies, we examined root lengths of two phytochrome chromophore-deficient mutants, hy1-1 and hy2-1 ( Fig. 2A). Whereas roots of the hy1-1 mutant were on average shorter than the C20 wildtype parent by approximately 15%, roots of the hy2-1 mutant were on average approximately 23% longer than the roots of the C20 wild type (Fig. 2B). Neither of these observed differences in root length was found to be statistically significant (P = 0.1524 and P = 0.0859, respectively), as compared with the lengths of roots of the C20 wild-type parent. Ecotypic differences in root lengths were observed for wild-type lines, with the Col-0 wild type having longer roots than other ecotypes (Supplemental Fig. S1), as de- Figure 2. Photomorphogenesis and root elongation responses of the wild type, phytochrome chromophore-deficient, and JA-insensitive mutants grown under Wc illumination. No-0 wildtype (WT), 35S::pBVR3, 35S::cBVR1, CAB3:: pBVR2 (CAB-2), MERI5::pBVR1 (MERI-1), C24 wild-type, F3 seedlings of a M0062 3 UASBVR cross (M0062/UASBVR), C20 wild-type, hy1-1, hy2-1, Col-0 wild-type, jar1, and myc2 lines were grown at 22°C on Phytablend medium containing 1% (w/v) Suc with no added MeJA for 10 d at 22°C under Wc illumination of 100 mmol m 22 s 21 . A, Images of seedlings. Bars = 1 cm. B, Bars represent means 6 SD of root lengths in mm (n $ 10 for each of six independent experiments). For statistical significance tests, comparisons were made relative to cognate wild-type lines: a P , 0.0001, b P , 0.01, c P , 0.05. For information on the range of seedling lengths observed for each line, see summarized frequency distribution data (Supplemental Table S1). Figure 3. Whole-mount immunolocalization of BVR protein accumulation in roots of wild-type and enhancer-trap BVR seedlings. C24 wild-type seedlings (WT; A-D) and F3 seedlings of a M0062 3 UASBVR cross (M0062/UASBVR; E-H) were grown on Phytablend medium containing 1% (w/v) Suc for approximately 4 d at 22°C under Wc illumination of 100 mmol m 22 s 21 . Fluorescence images of seedlings incubated without (A and E) or with (C and G) anti-BVR primary antibody (Ab) at a 1:2,000 dilution are shown. DIC images (B, D, F, and H) are shown for each seedling. Each image is a representative slice from a Z-series with 0.5-mm interval size, captured using 543-nm laser excitation with a 203 lens objective. Fluorescence images were collected using a 560-to 615-nm band-pass filter. Bars = 50 mm.

Phytochrome-Dependent Regulation of Root Elongation under Specific Wavelengths of Light
To determine the effect of phytochrome depletion in roots on the elongation of roots under distinct wavelengths of light, we grew seedlings under continuous blue (Bc), red (Rc), and far-red (FRc) illumination and assessed the lengths of roots relative to cognate wild-type seedlings. We observed roots that were significantly longer in lines with root-localized BVR accumulation (i.e. 35S::pBVR3, 35S::cBVR1, and M0062/UASBVR) relative to cognate wild-type parents under Bc illumination (Fig. 4A). Significantly longer roots also were observed for a phyA mutant relative to the Col-0 wild type (Fig. 4A). A role for phytochromes in the regulation of root length under B light has been postulated previously (Canamero et al., 2006).
Under Rc illumination, the M0062/UASBVR line exhibited significantly longer roots than its parent C24 wild type (Fig. 4B). The phyB mutant had significantly shorter roots than the wild type under these conditions, a phenotype previously observed in R (Shin et al., 2010). Notably, under FRc, smaller and largely nonsignificant differences in root lengths were observed for BVR lines (Fig. 4C), suggesting that globally, phytochromes localized in roots responded more to R and B illumination. However, 35S::cBVR1 seedlings and phyA seedlings were significantly shorter than wild-type seedlings under FRc (Fig. 4C), suggesting a role for shoot-derived phyA on the regulation of root length. The shorter roots observed for a phyA mutant have been reported previously (Kuratu and Yamamota, 1997;Shin et al., 2010).

Root-Specific BVR Phytochrome Depletion Does Not Impact Light-Dependent Changes in Hypocotyl Elongation
The observation that constitutive BVR-expressing seedlings and CAB3::pBVR seedlings exhibit elongated hypocotyls in Rc, FRc, and Bc light indicates a lack of phytochromes within the shoots of these seedlings (Lagarias et al., 1997;Montgomery et al., 1999;Montgomery, 2009;Warnasooriya and Montgomery, 2009). To confirm the root-specific inactivation of phytochromes in the M0062/UASBVR line and rule out residual activity of the BVR enzyme in the shoots of these seedlings, we assessed whether light-dependent hypocotyl inhibition was impacted in this line under Bc, Rc, or FRc light. The positive control 35S::cBVR1 line exhibited a significantly longer hypocotyl compared with the No-0 wild-type parent under Bc, Rc, and FRc (P . 0.0001 for all conditions; Fig. 5), as reported previously (Montgomery et al., 1999). By comparison, the hypocotyl lengths of the M0062/ UASBVR line were not significantly different from the C24 wild type under Bc (P = 0.7146), Rc (P = 1.0), or FRc (P = 1.0; Fig. 5), confirming root-specific impacts of BVR-mediated phytochrome deficiencies in the M0062/UASBVR line.

Root-Localized PFB or Phytochromes Impact the Arabidopsis Root Elongation Response to Jasmonates
Previous results indicated that phytochrome chromophore-deficient plants exhibit altered JA accumulation . Light-dependent root elongation responses of the wild type, phytochrome chromophore-deficient, and phytochrome apoprotein mutants. No-0 wild-type (WT), 35S::pBVR3, 35S::cBVR1, C24 wildtype, F3 seedlings of a M0062 3 UASBVR cross (M0062/UASBVR), C20 wild-type, hy1-1, hy2-1, Col-0 wild-type, phyA, and phyB lines were grown on Phytablend medium containing 1% (w/v) Suc for 10 d at 22°C under Bc light of 30 mmol m 22 s 21 , Rc light of 50 mmol m 22 s 21 , and FRc light of 10 mmol m 22 s 21 . Bars represent means 6 SD of root lengths in mm (n $ 10 for each of three independent experiments). For statistical significance tests, comparisons were made relative to cognate wild-type lines: a P , 0.001, b P , 0.01, c P , 0.05. that is correlated with changes in root elongation (Zhai et al., 2007). Inhibition of root elongation is promoted by JA and bioactive derivatives of JA (Staswick et al., 1992). We used our collection of BVR lines that exhibit phytochrome chromophore deficiency in various tissues to determine whether phytochrome chromophore deficiency in the root itself is correlated with the observed link between changes in JA levels and associated root elongation responses. Specifically, we conducted experiments to investigate the impact of root-localized phytochrome chromophore depletion on the elongation response of roots in JA-dependent root inhibition assays. We grew No-0 wild-type, 35S::pBVR3, 35S::cBVR1, CAB3::pBVR2, and MERI5::pBVR1 seedlings in the presence and absence of 20 mM methyl jasmonate (MeJA) and measured the lengths of roots. In our analyses, plants with root-localized phytochrome chromophore deficiencies were hyposensitive to JA (Fig. 6A). Seedlings of the lines 35S::pBVR3, 35S::cBVR1, M0062/UASBVR, hy1-1, and hy2-1 had significantly longer roots than their cognate wild-type parent when grown in the presence of 20 mM MeJA (Fig. 6B). 35S::pBVR3, 35S::cBVR1, hy1-1, and hy2-1 plants were approximately 73% to 100% longer than the wild-type parent grown under identical conditions, whereas M0062/UASBVR seedlings with root-specific phytochrome deficiency were approximately 21% longer than roots of C24 (Fig. 6B). The response for seedlings with root-localized phytochrome deficiencies was similar to that observed for characterized JA-insensitive mutants, jar1 and myc2, which were approximately 100% and 61% longer than the Col-0 wild-type parent, respectively (Fig. 6B). This observation agrees with prior results for jar1 (Staswick et al., 1992(Staswick et al., , 2002Lorenzo et al., 2004) and myc2 (Lorenzo et al., 2004;Yadav et al., 2005;Gangappa et al., 2010).
As the root lengths of lines with root-localized BVR expression were already longer than the wild-type parents in the absence of JA (Fig. 2), we measured the relative JA sensitivity of these lines by calculating the ratio of the root length for a particular line in the absence of exogenous MeJA treatment to root length in the presence of JA. We determined that a line exhibiting rootlocalized, plastid-targeted BVR accumulation had a lower relative JA sensitivity than the wild type: 3.45fold longer roots for 35S::pBVR3 in the absence of JA as compared with 4.75-fold longer roots for the No-0 wildtype parent (Table I). This difference represents a reduction of approximately 27% in sensitivity to JA treatment in the 35S::pBVR3 line. The roots of the M0062/UASBVR line were 4.46-fold longer in the absence of added MeJA as compared with the lengths of seedlings grown in the presence of MeJA. This result represents a reduction of approximately 7% in sensitivity to exogenous JA relative to the C24 wild type. By comparison, the hy1-1 mutant exhibited an approximately 50% reduction in sensitivity to JA, whereas JA sensitivity was reduced by approximately 37% for the hy2-1 mutant relative to the C20 wild type. The results for lines with phytochrome deficiencies in roots are less than the approximately 67% and 49% reductions in JA sensitivity calculated for the jar1 and myc2 mutants relative to the Col-0 wild type, respectively. Notably, the 35S::cBVR1 line, which exhibits cytosolic BVR expression, was at least as sensitive to JA-mediated inhibition of root elongation as the No-0 wild type: the 35S::cBVR1 line exhibited 4.95-fold longer roots in the absence of MeJA than in the presence of 20 mM MeJA (Table I), although the roots were significantly longer than the No-0 wild type both with and without MeJA treatment. The CAB3::pBVR lines, which exhibited marginally Phytochrome-Mediated Root Responses longer roots than the wild type, showed no reduction in JA sensitivity (Table I). The observance of longer roots for 35S promoter-driven BVR lines is apparent not only at 20 mM MeJA but throughout a range of MeJA concentrations (Supplemental Fig. S2). Notably, the No-0, Col-0, and C24 ecotypes appear to be less sensitive to JA than the C20 ecotype: No-0, Col-0, and C24 roots are approximately 4.8 times longer in the absence of JA than when grown in the presence of 20 mM JA, whereas C20 roots are 5.76 times longer in the absence of JA (Table I).

Expression of JA-Inducible Genes Is Impaired in Lines with Root-Localized Phytochrome Deficiencies
To determine whether the JA-hyposensitive phenotype of lines with BVR-induced phytochrome deficiency in roots is correlated with a molecular phenotype in these lines, we investigated the expression of JA-inducible marker genes OPDA-REDUCTASE3 (OPR3) and VEGE-TATIVE STORAGE PROTEIN1 (VSP1) using quantitative reverse transcription PCR (qPCR) analyses. OPR3 encodes a JA biosynthetic enzyme, whereas VSP1 is a JAresponsive marker gene. In qPCR analyses using RNA extracted from whole seedlings, we determined that all promoter-driven BVR lines exhibited reduced expression of OPR3 independent of externally applied MeJA, with the 35S::cBVR1 line exhibiting the greatest reduction relative to No-0 wild-type levels (Fig. 7). However, the gene expression in these lines was still highly sensitive to applied MeJA (i.e. all lines showed 6-to 7-fold greater expression of OPR3 when 20 mM MeJA was supplied; Fig.  7). The root-specific M0062/UASBVR line exhibited only minor differences in OPR3 expression relative to C24 (Fig. 7). Notably, chromophore-deficient hy1-1 and hy2-1 mutants did not show as severe defects in OPR3 expression (i.e. only hy1-1 exhibited a 12% reduction relative to the C20 wild type in the presence of JA; Fig.  7). The myc2 mutant exhibited slight reductions in OPR3 expression independent of MeJA treatment (Fig. 7). The jar1 mutant showed a minor reduction in OPR3 expression in the presence of MeJA, similar to prior observations obtained using northern-blot analyses (Chung et al., 2008;Koo et al., 2009).
When assessing the expression of VSP1, we observed that all lines exhibited extremely low levels of VSP1 expression in the absence of MeJA (Fig. 7). In the presence of 20 mM MeJA, all BVR lines, with the exception of the root-specific M0062/UASBVR line, exhibited reduced VSP1 expression relative to the cognate wild type (Fig. 7). The root-specific M0062/UASBVR line exhibited approximately 50% greater expression of VSP1 under these conditions (Fig. 7). hy1-1 and hy2-1 mutants exhibited increased VSP1 expression independent of MeJA treatment, as observed previously (Zhai et al., 2007). The jar1 and myc2 mutants showed reduced expression of VSP1 independent of MeJA treatment (Fig. 7), which has been noted previously for jar1 (Tuominen et al., 2004;Gangappa et al., 2010).
agar media, which allows direct exposure of the entire body of plants to light, has been debated. However, the growth habits of Arabidopsis in shallow soils and highly disturbed environments and the recognition that light penetrates the upper layers of soil increase the likelihood that Arabidopsis roots are exposed to light and exhibit light-dependent responses in natural environments. Very early studies indicate a role for phytochrome in specific aspects of root development in pea (Pisum sativum; Furuya and Torrey, 1964), bean (Phaseolus vulgaris; Jaffe, 1970), and Convolvulus arvensis (Tepfer and Bonnett, 1972). Furthermore, recent results demonstrate that phyB mutant Arabidopsis plants grown on soil showed a similar defect in the initiation of lateral roots, as when the phyB mutant seedlings were grown on agar plates (Salisbury et al., 2007). Therefore, the impact of light perception and subsequent signaling by phytochromes can occur in Table I. Fold difference values for root lengths determined for seedlings with respect to cognate wild types grown in the absence versus presence of MeJA No-0 wild-type, 35S::pBVR3, 35S::cBVR1, CAB3::pBVR2, MERI5::pBVR1, C24 wild-type, M0062/ UASBVR, C20 wild-type, hy1-1, hy2-1, Col-0 wild-type, jar1, and myc2 seedlings were grown with or without added MeJA. Fold difference values for average root lengths for seedlings grown on 0 mM JA relative to 20 mM JA are indicated (n $ 10 for each of six independent experiments). The percentage reduction in fold difference of root lengths relative to the cognate wild type was calculated.  qPCR was conducted using RNA from No-0 wildtype (WT), 35S::pBVR3, 35S::cBVR1, CAB3:: pBVR2, MERI5::pBVR1, C24 wild-type, M0062/ UASBVR, C20 wild-type, hy1-1, hy2-1, Col-0 wild-type, jar1, and myc2 lines. Expression of UBC21 (At5g25760), which is a control gene encoding a ubiquitin-conjugating enzyme, was analyzed as a reference. Black bars, 2JA; white bars, +JA (20 mM). Quantification by qPCR was performed with three independent experiments. Fold difference (Fold diff.) for levels of transcript accumulated in a test line relative to the cognate wild-type line is shown below each graph.
Phytochrome-Mediated Root Responses more natural growth conditions, just as when seedlings are grown on plates.

Root-Localized Phytochromes Contribute to Tissue-Specific Control of Root Development
Phytochromes impact root development in Arabidopsis. We initiated in planta experiments to determine at the molecular level whether roots are the site of light perception for this response. GAL4 enhancertrap lines are T-DNA insertion lines with diverse expression patterns of the yeast transcription factor, GAL4, whose expression depends on the presence of native genomic enhancer sequences. The GAL4responsive mGFP5 gene marks the expression pattern mediated by genomic enhancers in green fluorescence (Haseloff, 1999;Laplaze et al., 2005). The BVR gene under control of the upstream activation sequence (UAS) element is silently maintained in the absence of GAL4 in the UASBVR parent. Progeny from genetic crosses between a UASBVR transgenic line and a root-specific GAL4 enhancer-trap line exhibited rootspecific accumulation of the BVR protein (Fig. 3G). These lines did not display any light-dependent changes in hypocotyl elongation, indicating that phytochrome function in shoots is not impacted in the M0062/UASBVR line (Fig. 5). However, we observed distinct growth phenotypes in lines that exhibit rootlocalized BVR accumulation (i.e. 35S::pBVR, 35S:: cBVR1, and M0062/UASBVR lines) as compared with lines that lack root-localized BVR expression (i. e. No-0 wild-type, CAB3::pBVR, and MERI5::pBVR lines; Fig. 2). Roots for lines with root-localized, BVRinduced phytochrome deficiencies are highly significantly different with regard to length than roots of seedlings of wild-type or parental lines under Wc illumination (Fig. 2).
Notably, roots in lines with root-localized BVR accumulation also are longer under Bc and Rc illumination (Fig. 4, A and B). These findings suggest that R and B light are perceived directly by roots and subsequently effect root elongation. A tissue-specific role for the cry1 and cry2 proteins in roots was not supported in a prior study (Canamero et al., 2006); thus, our data indicate a role for phyA in roots in the tissue-specific regulation of root elongation under Bc illumination. On the contrary, root-specific phytochrome depletion had little effect on root elongation under FRc (Fig. 4C). However, phyA stimulates root elongation, as apparent from seedlings lacking phyA throughout the whole organism ( Fig. 4C; Kurata and Yamamoto, 1997;Shin et al., 2010). Thus, phyA may impact root elongation largely from shoot-derived signals. In support of this suggestion, FR light is the wavelength most effectively conducted axially from shoots to roots , and a prior study, although limited to arrays with 7,000 elements, identified no FR-regulated genes locally in dark-adapted roots exposed to FR (Sato-Nara et al., 2004). Considering the results for Bc, Rc, and FRc, we have demonstrated a root-specific role for phytochromes in the photoregulation of root elongation under distinct light conditions in the absence of altered phytochrome responsiveness in shoots in the M0062/UASBVR line.
As the 35S::pBVR3 line exhibits plastid-localized BVR expression, whereas the 35S::cBVR1 and M0062/ UASBVR lines exhibit cytosolic BVR expression, the observed phenotypes are likely associated with PFB or phytochrome deficiency, as opposed to changes in plastid metabolism that could be correlated with plastid-localized BVR expression in 35S::pBVR lines (Franklin et al., 2003). Thus, the elongated roots of BVR lines exhibiting root-localized phytochrome deficiencies under Wc, Bc, and Rc likely represent the disruption of a phytochrome-dependent phenotype (e.g. the noted R-dependent inhibition of root elongation; Correll and Kiss, 2005) or disruptions in the coaction of phytochromes and cryptochromes (Ahmad and Cashmore, 1997;Guo et al., 2001;Usami et al., 2004). Furthermore, the occurrence of marginally longer roots in CAB3::pBVR2 relative to the No-0 wild type under Wc (Fig. 2B), and comparative analyses of root-specific versus constitutive phytochrome deficiencies under distinct wavelengths of light (Fig. 4), indicate that shoot-localized phytochromes exhibit some long-distance control over root elongation. Previous results have demonstrated that shoot-derived signals impact root development in Arabidopsis (Salisbury et al., 2007). Ultimately, the reduced response observed for lines with either root-specific or mesophyll-specific BVR accumulation relative to constitutive BVR accumulators suggests that mesophyll-localized, or shootderived, phytochromes contribute to long-distance regulation of root elongation, particularly under FR light, as the root-specific BVR line did not differ significantly from the wild type under these conditions (Fig. 4C). However, these results provide strong evidence that root-localized phytochromes contribute locally to the photoregulation of root development in Arabidopsis.

Alterations in JA Sensitivity Are Correlated with Phytochrome-Chromophore Deficiency in Roots
JA is known to inhibit germination and root elongation in Arabidopsis (Staswick et al., 1992). Different Arabidopsis ecotypes exhibit distinct sensitivities to treatment with JA (Table I; Rao et al., 2000;Matthes et al., 2008). Of the ecotypes tested in our study, the C20 ecotype appears to be more sensitive to treatment with MeJA than any other ecotype (Table I). 35S:: pBVR3 and M0062/UASBVR lines with induced phytochrome-chromophore deficiency in roots exhibit reduced JA sensitivity in root inhibition assays, whereas the roots of plants lacking root-localized phytochrome chromophore depletion largely exhibit wild-type phenotypes in these assays (Table I). The fact that only specific lines exhibiting BVR accumulation in roots (i.e. either constitutive 35S::pBVR3 or root-specific, enhancertrap BVR expression) exhibit significant reduction in sensitivity to JA treatment relative to the wild type provides evidence that PFB or phytochrome deficiency itself in the root contributes significantly to light-dependent, JA-responsive elongation phenotypes. Although shoot-specific phytochromes can impact root elongation through interorgan signaling, as observed for the CAB3:: pBVR2 line (Fig. 2), which exhibits shoot-specific phytochrome deficiency (Warnasooriya and Montgomery, 2009), root-localized phytochromes are most important for JA sensitivity, as the CAB3::pBVR2 line has no alternation in JA sensitivity (Table I).
Chromophore-deficient mutants hy1-1 and hy2-1 exhibited disparate responses in the absence of JA, with hy1-1 being shorter and hy2-1 being longer than the identically treated wild-type parent (Fig. 2). In the presence of JA, both lines were longer than the wild type, exhibiting reduced sensitivity to JA ( Fig. 6; Table  I). This result observed for hy1-1 is distinct from what was previously reported for hy1-100 (Zhai et al., 2007). The hy1-100 allele exhibited shorter roots than its wildtype parent, although the responsiveness to JA was similar to that of the wild type (Zhai et al., 2007). Our distinct observation may result from ecotypic differences or differences in growth conditions. In this regard, there are known ecotypic differences in the response to JA responsiveness (Table I; Rao et al., 2000;Matthes et al., 2008) and root elongation responses in light-grown seedlings (Supplemental Fig. S1; Beemster et al., 2002;Passardi et al., 2007). In our studies, the hy1-1 mutant response resembles the JA-insensitive mutants jar1 and myc2 at the phenotypic level: shorter roots than the wild type in the absence of MeJA and longer roots in the presence of exogenously applied MeJA. By contrast, the hy2-1 mutant is more similar to other lines with rootlocalized phytochrome chromophore deficiency. These lines all have longer roots than the wild type independent of JA treatment and have reduced sensitivity to JA. Notably, 35S::cBVR1 is unique among the tested lines with root-depleted PFB, as it does not exhibit a significant difference in its sensitivity to JA (Table I). This line has significantly longer roots than the wild type in the absence or presence of JA (Figs. 2 and 5), but it shows a relative response to JA that is at least equal to that observed for the wild-type parent (Table I).
Root-specific phytochrome deficiency alone was correlated with an increased level of expression of the JAresponsive gene VSP1 in the presence of exogenously applied MeJA among BVR lines (Fig. 7). It has been shown that a repressor of JA signaling is regulated by phyA in shoots but not in roots of Arabidopsis (Robson et al., 2010). Thus, phyA can regulate distinct components that impact JA signaling in a tissue-specific manner. In comparison with root-specific BVR expression, 35S promoter-driven BVR lines exhibited reduced levels of expression of VSP1 and OPR3 genes in response to MeJA treatment (Fig. 6). The lower levels in BVR lines, other than the root-specific line, suggest that BVR lines have lower JA biosynthetic capacity. These results are somewhat complicated, however, by the fact that light and MeJA synergistically impact VSP1 expression (Berger et al., 1995). Recently, it has been determined that phyA is required for VSP1 induction in the presence of JA (Robson et al., 2010). In BVR lines, both light signaling and JA responsiveness are impacted; thus, the combined impact of PFB or phytochrome deficiency and JA treatment on VSP1 expression in these lines is likely complex.
The disparate results observed with regard to the regulation of JA-associated genes in the root-specific BVR line relative to other BVR lines may be associated with a strictly localized degradation of the phytochrome chromophore or a lack of phytochrome in the M0062/UASBVR line. This localized disruption of PFB accumulation does not appear to result in a major impact on JA biosynthesis in the whole seedlings, which were used for RNA isolation for the gene expression studies. Notably, however, the result observed for the M0062/UASBVR line strongly suggests a need for phytochromes or the phytochrome chromophore specifically in roots for a wild-type, JAmediated root inhibition response. Taken together, these data support a molecular link between phytochrome and JA signaling and further demonstrate that these pathways interact in roots themselves. These results correspond strongly with recent results showing that JA perception by the root itself is required for the inhibition of root growth in Arabidopsis, as application of the hormone to roots, but not leaf application, led to decreased root growth (Schmidt et al., 2010). Furthermore, these results provide additional support for a tissue-specific role of phyA in regulating JA responses. Prior work demonstrated that JA-mediated responses in the shoot and in response to wounding are not impacted in a phyA mutant, whereas functional phyA and root growth inhibition in response to JA are definitively linked (Robson et al., 2010). Our analyses further implicate root-localized phytochromes as the pool important for this link.
In summary, Arabidopsis lines that exhibit rootlocalized BVR accumulation have defects in the photoregulation of root elongation. Specific lines with root-localized BVR expression, including 35S::pBVR3 and M0062/UASBVR, also exhibit reduced sensitivity to the plant hormone JA. These results provide evidence that root-specific phytochrome chromophore, or root-localized phytochromes themselves, are critical for apposite photoregulation of root elongation and impact JA sensitivity.

Plasmid Construction
We isolated the pUAS1380-BVR (hereafter UASBVR) construct using the following method. The full-length BVR coding region was cloned using primers UASBVR_S (5#-CGTCTAGAATGGATGCCGAGCCAAAG-3#) and UASBVR_AS (5#-CGAGATCTTTACTTCTTCTGGTGGCAAAG-3#) with introduced XbaI and BglII restriction sites (underlined), respectively. The BVR coding region was PCR amplified using pASK-FLBVR (B.L. Montgomery and J.C. Lagarias, unpublished data) as a template, and the resulting PCR product was restricted with XbaI and BglII enzymes (New England Biolabs). The digested PCR product was ligated to the similarly digested pUAS1380 plant transformation vector using the TaKaRa DNA Ligation Kit version 2.1 (Takara Bio U.S.A.). The integrity of the plasmid was confirmed using restriction digestion and DNA sequencing analyses.

Plant Growth Conditions
Seeds were surface sterilized with 35% (v/v) commercial bleach and 0.025% (v/v) SDS solution and then rinsed with ultrapure water (Milli-Q; Millipore) as described previously (Warnasooriya and Montgomery, 2009). Seeds were planted in 100-3 100-3 15-mm square petri dishes on medium containing 13 MS salts, 0.8% (w/v) Phytablend, 0.05% (w/v) MES (Sigma), and 1% (w/v) Suc, adjusted to pH 5.7 with KOH, with or without 20 mM MeJA (Sigma). Imbibing seeds were cold stratified at 4°C for 3 d in darkness. Plates with stratified seeds were kept vertically in a humidity-controlled chamber under defined light conditions for 10 d at 22°C.

Light Sources
For Wc growth, seeds on Phytablend medium were grown in a Percival CU-36L5 Tissue Culture Chamber under cool-white fluorescent illumination of 100 mmol m 22 s 21 . Rc, Bc, and FRc sources were those described previously (Warnasooriya and Montgomery, 2009). We measured Wc, Rc, and Bc fluence rates using a LI-250A Light Meter (LI-COR) equipped with a quantum sensor (LI-COR), and FRc was measured using a StellarNet EPP2000 spectroradiometer (Apogee Instruments).

Whole-Mount Immunohistochemical Analysis
Seedlings (3.5 d old) grown on vertically placed plates as described above were subjected to whole-mount in situ protein localization to visualize proteins in root tips, lateral roots, and embryos as described previously with limited modifications (Sauer et al., 2006). Seedlings were treated with the paraformaldehyde-based fixative solution for 30 min followed by washing with 13 phosphate-buffered saline (PBS) twice for 10 min and with sterile water twice for 5 min. Fixed seedlings were mounted on Poly-Prep slides (Sigma) in a droplet of water and were air dried for 2.5 h at room temperature. Cell walls were digested with 2% (w/v) Driselase (Sigma) in 13 PBS for 30 min at 37°C. Slides were washed with 13 PBS three times for 10 min. Tissues were permeabilized with 3% (v/v) IGEPAL CA-630 containing 10% (v/v) dimethyl sufoxide for 1 h at room temperature, followed by washing with 13 PBS four times for 10 min. Blocking with 3% (v/v) bovine serum albumin fraction V (Roche) was carried out for 1 h at room temperature. Fixed and permeated seedlings were incubated with rabbit anti-BVR antibody (QED Bioscience) at 1:2,000, 1:3,000, or 1:4,000 dilution as indicated in 13 PBS or with 13 PBS alone for control samples overnight at 4°C. Excess primary antibody was removed by washing slides with 13 PBS three times for 10 min. Following incubation with the primary antibody and washing, seedlings were incubated with goat anti-rabbit IgG (H+L) conjugated to HiLyte Plus 555 (AnaSpec) at 0.005 mg mL 21 dilution in 13 PBS for 6 h at 37°C. To remove excess secondary antibody, slides were washed with 13 PBS four times for 10 min. Drops of antifade mountant medium (Citifluor) were placed on treated seedlings and covered with coverslips. Slides were stored overnight at 4°C before imaging. Root tips of seedlings were imaged on an inverted Axiovert 200 Zeiss LSM 510 Meta confocal laser scanning microscope (Carl Zeiss MicroImaging) using differential interference contrast (DIC) optics and fluorescence excitation/emission filters. A 203 0.75 Plan Apochromat objective lens was used for imaging. DIC imaging was performed using the 543-nm laser. Fluorescence from the secondary antibody was collected using a 543-nm laser for excitation and a 560-to 615-nm band-pass filter for emission. Images were acquired using the LSM FCS Zeiss 510 Meta AIM imaging software.

Root Length Measurements
Prior to conducting detailed root length analyses at 10 d, we conducted preliminary observations daily between 3 and 11 d after transferring seeds to experimental growth chambers and observed the same relative differences in root length throughout the observation period (for representative images of wild-type lines at 3, 5, 8, and 10 d, see Supplemental Fig. S1). As the roots of seedlings grown in the presence of JA are shorter, we used a longer time point of 10 d for replicative root length assays. Plates containing 10-d-old seedlings were scanned, and plant images were used to quantify root lengths in endpoint length measurement assays using ImageJ software (National Institutes of Health). Root length assays were repeated at least three times. Two-tailed, unpaired Student's t tests (for normally distributed data) or Mann-Whitney U tests (for nonnormal data distributions) were performed to compare the means of root lengths.

Hypocotyl Inhibition Assays
Sterilized seeds of the No-0 wild type, 35S::cBVR1, the C24 wild type, and M0062/UASBVR were cold stratified at 4°C for 3 d in darkness. Plates were kept in a humidity-controlled chamber with Bc of 30 mmol m 22 s 21 , Rc illumination of 50 mmol m 22 s 21 , FRc illumination of 10 mmol m 22 s 21 , or in darkness for 7 d at 22°C. Seedlings were scanned, and plant images were used to quantify hypocotyl lengths using ImageJ software (National Institutes of Health). The hypocotyl inhibition assay was repeated three times. Percentage dark length and SD of percentage dark length are reported. A two-tailed, unpaired Student's t test was performed to compare the percentage dark length of hypocotyls of transgenic lines relative to cognate wild-type seedlings, except for the C24 wild type and M0062/UASBVR grown in Rc and FRc, where a two-tailed, unpaired Mann-Whitney U test was performed to compare the percentage dark length of hypocotyls of M0062/UASBVR with that of the C24 wild type.
qPCR Analyses qPCR was performed to quantify the levels of transcripts of a JA biosynthetic gene, OPR3 (At2g06050), and a JA-inducible marker gene, VSP1 (At5g24780). Seeds of the No-0 wild type, 35S::pBVR3, 35S::cBVR1, CAB3:: pBVR2, MERI5::pBVR1, the Col-0 wild type, jar1, myc02-05, the C24 wild type, M0062/UASBVR, the C20 wild type, hy1-1, and hy2-1 were planted in 245-3 245-3 18-mm square petri dishes on MS medium with or without 20 mM MeJA (Sigma). Imbibing seeds were cold stratified at 4°C for 3 d in darkness. Plates were kept vertically in a humidity-controlled chamber with Wc illumination of 100 mmol m 22 s 21 for 10 d at 22°C. Ten-day-old whole seedlings were quickly (less than 1 min) harvested and immediately frozen in liquid nitrogen. Using the RNeasy Plant Minikit (Qiagen) including on-column DNase treatment (Qiagen), total RNA was isolated according to the manufacturer's instructions. The quantity of the RNA was analyzed by spectrometry (NanoDrop1000; Thermo Scientific). First-strand cDNA synthesis was performed using the Reverse Transcription System (Promega) with random primers according to the manufacturer's instructions using a 20-mL reaction volume. The incubation times of first-strand cDNA synthesis with total RNA of 0.2 mg were (1) 10 min at room temperature, (2) 1 h (instead of 15 min) at 42°C, (3) 5 min at 95°C, and (4) 5 min at 4°C. The cDNA reaction mixture was diluted 40-fold with nuclease-free water, and 4 mL of the diluted cDNA product was used as template in a 10-mL qPCR using the Applied Biosystems FAST 7500 real-time PCR system in FAST mode with Fast SYBR Green Master Mix (Applied Biosystems), according to the manufacturer's instructions. For transcript analysis, annealing/extension temperature was 60°C for both the OPR3 and VSP1 primer sets. Reactions were performed in triplicate, and products were checked by melting curve analysis. The abundance of transcripts was analyzed using the delta delta Ct (ddCt) method based on relative quantification, with normalizing to the reference transcript UBC21 (At5g25760). All qPCR experiments were repeated with three independent biological replicates. All qPCR procedures and analyses conform to Minimum Information for Publication of Quantitative Real-Time PCR Experiments guidelines (Bustin et al., 2009).

Supplemental Data
The following materials are available in the online version of this article.
Supplemental Figure S1. Representative images showing root lengths of wild-type Arabidopsis ecotypes over time.
Supplemental Figure S2. MeJA response curve for JA-mediated root growth inhibition in wild-type and BVR-expressing lines.
Supplemental Table S1. Frequency distribution analyses for root lengths of the wild type, phytochrome-chromophore deficient, and jasmonic acid-insensitive mutants under Wc illumination.