Light carries most of the essential information needed for plant growth and development (Von Arnim and Deng, 1996; Fankhauser and Chory, 1997; Chory, 2010). Photoreceptors such as phytochromes, cryptochromes, and UVR8 perceive light as a developmental signal (Li et al., 2011; Liu et al., 2011; Tilbrook et al., 2013). Vegetation shade reduces plants’ access to sufficient light. Shade-intolerant plants such as Arabidopsis trigger a suite of responses, collectively called shade avoidance syndrome (SAS), including elongation of hypocotyls, stems and petioles, hyponasty (upward bending of leaves) and early flowering (Franklin, 2008; Casal, 2012). SAS is a default developmental program and suppressed under normal light by phyB along with phyD and phyE (Franklin and Quail, 2010; Casal, 2013).

Phytochromes exist in two forms: the red light (666 nm)-absorbing P_r form, and far-red light (730 nm)-absorbing P_fr form (Li et al., 2011). However, monochromatic red (R) or far-red (FR) light usually does not convert 100% of P_r to P_fr or vice versa. Thus, a dynamic equilibrium is established between the two phy forms (P_r and P_fr) depending on the quality of light (Possart et al., 2014). Under white light (high R:FR), the active phyB, P_fr, migrates to the nucleus and interacts with various PIFs and drives their degradation (Lorrain et al., 2008). The onset of low R:FR ratio shifts the steady-state equilibrium toward the inactive Pr form (Franklin and Quail, 2010). As a result, PIFs become stabilized and more abundant (Lorrain et al., 2008). Genome-wide analysis revealed that PIF4 and PIF5 preferentially bind to auxin biosynthetic and signaling gene promoters (Hornitschek et al., 2012; Leivar et al., 2012b) and activate their expression under low R:FR light. PIF7 also functions as a major regulator for shade-induced hypocotyl elongation and is dephosphorylated in response to shade (Li et al., 2012). HFR1 physically interacts with PIF4 and PIF5 and inhibits their binding to the target promoters, thus negatively regulating shade responses (Hornitschek et al., 2009).

CONSTITUTIVE PHOTOMORPHOGENIC 1 (COP1) is a repressor of photomorphogenesis and encodes an RING-finger E3 ubiquitin ligase. Under darkness, COP1 accumulates in the nucleus and targets degradation of positive regulators of photomorphogenesis such as HY5, HYH, LAF1, and HFR1 (Deng et al., 1991; Ballesteros et al., 2001; Lorrain et al., 2006). As well, COP1 is required for activating shade signaling. Mutants of COP1 can barely sense vegetation shade, whereas overexpression of COP1 leads to a constitutive shade avoidance phenotype (Pacín et al., 2013). The molecular mechanisms of COP1-mediated shade responses are emerging. COP1 rapidly accumulates in the nucleus under low R:FR, and this nuclear accumulation is essential for full shade responses (Pacín et al., 2013). Further evidence indicates that COP1 enhances HFR1 degradation under shade, leading to an increase of PIF-mediated gene expression, auxin levels, and thus stem growth (Pacín et al., 2016). COP1 and SPA1 together participate in hypocotyl and leaf petiole elongation in response to low R:FR by targeting the HFR1degradation by 26S proteasome (Rolauffs et al., 2012). The cop1 bbx21 bbx22 triple mutant is responsive to the shade, which suggests that the shade insensitivity phenotype of cop1 is mediated through both BBX21 and BBX22 (Crocco et al., 2010). However, how COP1 regulates SAS at the molecular level needs further investigation.

Phytohormones play important roles in light control of plant development (Lau and Deng, 2010; Kurepin and Pharis, 2014). The interplay of jasmonates (JAs) and light signaling is accepted (Kazan and Manners, 2011; Hsieh and Okamoto, 2014). Although low R:FR light perturbs JA signaling (Moreno et al., 2009; Robson et al., 2010; Cerrudo et al., 2012; De Wit et al., 2013; Chico et al., 2014; Leone et al., 2014), modulation of shade signaling by JA or its signaling components is still under examination. Recent evidence has revealed the interplay between JAs and shade, which is critical for growth-defense balance. This process may involve resource allocation between growth and immunity (Yang et al., 2012; Ballaré, 2014; Mazza and Ballaré, 2015).

The jar1 mutants show reduced sensitivity to exogenous JA and enhanced susceptibility against soil fungus, Pythium irregulare (Staswick et al., 1992, 1998). Later, jar1-1 was mapped to the same locus as fin219-1 (Staswick et al., 2002), a suppressor of cop1 mutant under darkness (Hsieh et al., 2000). FIN219 (GH3.11) belongs to a GH3 family of proteins, and its expression is rapidly induced by auxin (Hsieh et al., 2000). FIN219/JAR1 encodes JA-amino-synthetase, which conjugates isoleucine (Ile) with jasmonic acid (JA) to form the bioactive jasmonoyl-L-isoleucine (JA-Ile) (Staswick and Tiryaki, 2004). JA-Ile is an oxylipin that profoundly affects plant developmental and stress responses (Bari and Jones, 2009; Browse, 2009).

The fin219/jar1 mutant exhibits an insensitive long-hypocotyl phenotype under continuous far-red (cFR) light, which indicates its role in phyA-mediated signaling (Hsieh et al., 2000; Chen et al., 2007). Moreover, FIN219/JAR1 physically interacts with COP1 under darkness and cFR light and negatively regulates COP1 under FR light. Artificial induction of FIN219 protein accumulation via overexpression inhibited COP1 nuclear localization and hence stabilized HY5 protein (Wang et al., 2011). In addition, FIN219 might contribute to signaling of other hormones such as auxin, ethylene, gibberellin and abscisic acid by regulating a large number of bHLH transcription factors (TFs) (Chen et al., 2015).

Under low R:FR light, both phyA and fin219 mutant seedlings show enhanced hypocotyl elongation as compared with the wild type (Johnson et al., 1994; Yanovsky et al., 1995; Robson et al., 2010). Although the phyA phenotype is attributed to a FR light-mediated high irradiance response, the molecular mechanism underlying fin219-mediated shade avoidance phenotype is still under debate.

In the present study, we examined the functional significance of FIN219 under simulated shade in Arabidopsis seedlings. With a combination of genetic and biochemical experiments, we demonstrate that FIN219 negatively regulates shade avoidance responses by modulating key shade signaling components. Moreover, FIN219-mediated shade avoidance responses were independent of phyA-mediated high irradiance responses.

Hypocotyl elongation is a key adaptation of Arabidopsis seedlings to avoid shade light. Here, we report that FIN219/JAR1, a JA-conjugating enzyme, plays a negative role in regulating shade responses such as hypocotyl elongation and expression of shade components. The double mutant phyA 211fin219-2 had a synergistic effect in response to simulated shade, which suggests that phyA and FIN219/JAR1 work in parallel pathways to regulate shade signaling, which is consistent with the additive levels of TFs PIL1 and ATHB2 and auxin-response genes IAA29 and SAUR68 under the same condition. Moreover, FIN219/JAR1 and PHYA levels were down- and upregulated by shade, respectively. Intriguingly, PHYA and COP1 levels were affected in the fin219 mutants under simulated shade. In particular, FIN219/JAR1 overexpression under shade could change COP1 subcellular localization from the nucleus to the cytoplasm. Further Co-IP studies under shade revealed that FIN219/JAR1, phyA and COP1 interacted with each other. These data indicate that FIN219/JAR1 plays a vital role in regulating shade responses likely by modulating the expression and subcellular location of shade components.

FIN219/JAR1 is a JA-conjugating enzyme, responsible for the formation of JA-Ile and involved in the regulation of plant development and defense responses likely via crosstalk with different hormones and TF-mediated signaling pathways (Chen et al., 2015). Mutation in the FIN219/JAR1 locus resulted in enhanced shade-induced hypocotyl elongation with different degrees (Figures 1A,B) (Robson et al., 2010), whereas its overexpression led reduced elongation responses (Figures 1A,B), which suggests a negative role of FIN219 in shade signaling. Since FIN219 is an auxin and JA inducible gene and its function in JA signaling is well known, how it modulates other hormone signaling pathways is largely unknown. Our previous studies also indicated that FIN219 could crosstalk with other different hormones (Chen et al., 2015). The fin219-2 is a T-DNA insertion knockout mutant (Wang et al., 2011), whereas jar1-1 is an EMS mutant (Staswick et al., 2002) and fin219-1 is a mis-regulated mutant with changes of methylation status in the promoter of its gene (Hsieh et al., 2000). Thus, jar1-1 and fin219-1 mutants contain reduced levels of proteins compared to wild type. It could be possible that in these mutant lines (jar1-1 and fin219-1), FIN219 perturbs other hormone signaling pathways and makes plants more sensitive to shade. This is our hypothesis that needs further investigation to clarify. In addition, COP1 is necessary for hypocotyl and petiole elongation under shade light (Rolauffs et al., 2012). Furthermore, B-box-containing 21 (BBX21) acts downstream of COP1 to negatively regulate the shade response (Crocco et al., 2010). FIN219/JAR1 appears to affect COP1 levels under shade (Figures 2B,C). Intriguingly, BBX21 negatively regulated FIN219 transcript levels under canopy shade (Crocco et al., 2010), so FIN219-COP1-BBX21 may form a regulatory feedback loop in response to shade environments. Recent studies revealed that phyB and PIFs form a mutually negative feedback loop under continuous red but not shade light (Leivar et al., 2012a). Thus, a local loop regulation initiated by FIN219-COP1-BBX21, leading to reduced levels of active JA-Ile, may play a vital role in fine-tuning shade light signaling.

FIN219/JAR1 acts as a major enzyme for the formation of physiologically active JA-Ile to regulate plant growth and defense responses (Staswick et al., 2002; Wang et al., 2011). Its protein levels should be strictly modulated in response to developmental and environmental cues. So far, multiple photoreceptors, including phyA, and different hormones such as auxin and JAs can regulate FIN219 levels. Current work indicated that pGR219 with FIN219 overexpression has always severely stunted phenotype (Figure 1). The expression levels of IAA29 and SAUR68 in pGR219 are always similar with Col-0 under white light (Figures 5B,D). Moreover, PIF5 and PAR1 have opposing effects on shade responses, which suggests that FIN219 may trigger major effects on shade-responsive genes mainly under shade conditions. Besides, PIF5 positively regulates and PAR1 negatively affects shade responses. In pGR219, the PIF5 expression is less and PAR1 expression is greater than wild type (Figures 5A,C). However, these gene expressions do not alter with response to shade in pGR219. In addition, our previous studies indicated that FIN219 overexpression could exclude COP1 from the nucleus to the cytoplasm even in the dark without greatly altering COP1 level (Wang et al., 2011). Shade also resulted in COP1 accumulation in the nucleus (Figures 7D,E). In contrast, pGR219 under shade showed more COP1 accumulation in the cytoplasm and substantial reduction of COP1 in the nucleus compared to wild-type Col-0 under the same condition, which suggests that the accuracy of COP1 subcellular location is critical for shade responses in addition to expression of shade components. Thus, the PGR219 with pronounced short-hypocotyl phenotype under white light and shade conditions may have substantial effects on plant growth and development in addition to light signaling.

Shade light illumination affects a number of genes, including TFs (Sessa et al., 2005; Roig-Villanova et al., 2006). PIF-related TFs are essential for shade-induced responses (Lorrain et al., 2008; Casal, 2013). As expected, wild-type PIF5 transcript levels did not change greatly under shade light. However, FIN219 negatively regulated PIL1 and PIF5 gene expression under low R:FR light (Figures 1C, 5A). As well, the expression of several TFs such as HFR1, ATHB2, PIL1, and PAR1/2 were rapidly induced under shade light (Carabelli et al., 1996; Salter et al., 2003; Sessa et al., 2005; Roig-Villanova et al., 2006). HFR1 and PAR1/2 negatively regulate shade responses (Roig-Villanova et al., 2007; Hornitschek et al., 2009), whereas ATHB2 and PIL1 are positive (Steindler et al., 1999; Salter et al., 2003) and negative regulators, respectively (Li et al., 2014). PAR1 expression was positively regulated by FIN219 under shade (Figure 5C). Therefore, FIN219 may regulate the expression of early shade-induced genes positively and negatively. In addition, PIF5 and PAR1 modulate auxin biosynthesis and signaling (Roig-Villanova et al., 2007; Hornitschek et al., 2012). In agreement, the expression of auxin-response genes IAA29 and PAR1 target SAUR68 is associated with PIF5 and PAR1 expression, respectively. As well, the PIF5 and PAR1 expression was higher in the double mutant phyA211 fin219-2 than in each single mutant under shade (Figures 6A,C), so FIN219 may act independently of phyA-mediated signaling to modulate auxin signaling in response to shade.

In addition, PAR1 transcripts are repressed by PHYA and PHYB overexpression under simulated shade (Roig-Villanova et al., 2006). Its transcripts are also suppressed by phyA and phyB under FR and R light, respectively. The PHYA protein level in fin219 mutants was similar to that in wild type (Figure 2B) and it was greater in PGR219 under white light (Figure 2C), which suggests that PAR1 transcripts would be less than wild type under white light. However, in Figure 5C, PAR1 expression was even greater in both fin219 mutants and PGR219 than in wild type under white light, which implies that PAR1 expression patterns in fin219 mutants and PGR219 may involve hormone effects such as JAs and auxin. The detailed mechanisms remain elusive.

Phytochromes were shown to play vital roles in shade signaling (Devlin et al., 1999; Franklin and Whitelam, 2005; Casal, 2012). phyB is stable in light and functions as a major photoreceptor in suppression of shade-mediated hypocotyl elongation. Under a high R:FR ratio, the phenotype of the phyB mutant was similar to the wild type under shade. Furthermore, phyD and phyE were redundant to phyB in repressing shade responses, whereas phyC did not play a role in shade responses (Franklin and Quail, 2010). Functional roles of phyA involved in the repression of shade responses remain largely unknown. Here, we found that PHYA levels were increased by simulated shade (low R:FR ∼ 0.05) (Figures 2, 4), which agrees with the report by Martínez-García et al. (2014). An increase in PHYA level on exposure to shade even for 5 h (Figure 4) is likely related to the phyA function in fine-tuning the chlorophyll biosynthesis in response to partial shading (Brouwer et al., 2014). Moreover, phyA appears to positively regulate FIN219 levels in short-term exposure to shade (Figure 4A), rather than long-term exposure to shade (Supplementary Figure S2B). In turn, increased FIN219 levels leading to enhanced JA-Ile levels resulted in reduced the chlorophyll content under shade. This speculation is consistent with JAs being able to decrease chlorophyll content and reduce photosynthesis in light (He et al., 2002; Zhai et al., 2007). In contrast, with longer exposure to shade, FIN219 level was reduced in the wild type (Figure 2 and Supplementary Figure S2B) and positively regulated PHYA level (Figure 2B). Thus, reduced FIN219 level under shade may involve other mechanisms, and both FIN219 and phyA may regulate each other in response to low R:FR light.

CONSTITUTIVE PHOTOMORPHOGENIC 1 is a negative regulator of photomorphogenesis and encodes an E3 ubiquitin ligase. Under the dark, COP1 accumulates in the nucleus and results in the degradation of positive regulators such as HY5 of photomorphogenesis. Upon light exposure, COP1 is inactivated by migrating to the cytoplasm and other unknown mechanisms. Shade light can trigger COP1 accumulation rapidly in the nucleus, and it positively modulates the shade responses (Pacín et al., 2013). Moreover, COP1 physically interacts with FIN219 under the dark and continuous FR light and restricts its nuclear accumulation on overexpression of FIN219 (Wang et al., 2011). We found COP1 mainly accumulated in the cytoplasm and unable to migrate to the nucleus in pGR219 under the conditions examined, including shade for 1 h (Figures 7B–E), which significantly affects hypocotyl elongation (Figures 1A,B). Thus, abundant FIN219 protein affects the total content of active COP1 protein in the nucleus. Ethylene promotes hypocotyl elongation in the light by triggering COP1 nuclear accumulation, which enhances HY5 degradation (Yu et al., 2013). FIN219 levels responsible for the formation of JA-Ile, an active form of JA, are likely tightly regulated in response to low R:FR to modulate the subcellular localization of COP1.

FIN219/JAR1 is a cytoplasmic protein in FR light and remains in the same location even under shade (Hsieh et al., 2000; Figure 7). Co-IP studies indicated that FIN219/JAR1 dose-dependently interacted with phyA and COP1 proteins under shade (Figure 8). FIN219 overexpression excluded COP1 from the nucleus to the cytoplasm under FR light (Wang et al., 2011). As well, ectopic expression of FIN219 under low R:FR light changed the COP1 subcellular location from the nucleus to the cytoplasm because COP1 was localized in the nucleus under shade light. However, FIN219 level in the wild type was reduced by shade light (Figure 2) and was increased by 5-h shade as compared with white light (Figure 4). FIN219 and COP1 interaction likely occurred in the cytoplasm at the early stage of shade light exposure. This speculation is consistent with transient expression at 1-h shade that resulted in more accumulation of GFP-COP1 in the cytoplasm by ectopic expression of FIN219 (pGR219) (Figure 7).

In addition, the levels of PHYA were increased and stabilized by continuous and transient shade light (Figures 2, 4), which leads to suppression of hypocotyl elongation, with an antagonistic effect on phyB deactivation by low R:FR light. PhyA and phyB likely function dynamically to modulate hypocotyl elongation in response to changes in R:FR ratios in natural environments. In addition, FIN219 and phyA positively regulated each other under shade (Figures 2, 4, 8). phyA is activated by FR light and migrates to the nucleus. Whether FIN219 affects phyA subcellular localization, leading to their interaction in the cytoplasm upon exposure to shade light, remains to be elucidated.

FIN219/JAR1 is a JA-conjugating enzyme and interacts with multiple partners, including COP1 (Chen et al., 2007; Wang et al., 2011; Figure 8). These data suggest that FIN219 may have a dual function with an enzymatic and protein–protein interaction activities. Collectively, our data show that FIN219/JAR1, a JA-conjugating enzyme, functions as a negative regulator in shade signaling and may work with phyA and COP1 in response to shade. Alternatively, low R:FR ratio may inactivate phyB by reducing its abundance in the nucleus and decrease FIN219/JAR1 levels, thereby leading to increased PIF5 level and reduced PAR1 level and increased COP1 accumulation in the nucleus. These events activate downstream genes such as IAA29 and SAUR68 and shade responses, including hypocotyl elongation (Figure 9).

SS and H-LH designed the experiments; SS and H-WJ performed experiments; SS and H-LH did data analyses; SS and H-LH wrote the manuscript.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.