Introduction
============

Because plants are dependent on light for photosynthesis they have developed a complex system of photoreceptors and downstream responses enabling them to optimize growth to their light environment ([@ref-22]). One critical aspect of plant light responses is neighbor detection and shade avoidance ([@ref-7]; [@ref-17]). Plants detect the presence of neighbors by changes in the light quality: since photosynthetic tissue absorbs more red light (R) than far-red light (FR), foliar shade uniquely lowers the R:FR ratio. Changes in the R:FR ratio are detected by phytochrome photoreceptors that exist in two photoconvertible forms, the red light absorbing form, Pr, and the far-red light absorbing form, Pfr. In high R:FR conditions, such as direct sunlight, type II phytochromes are converted from Pr to Pfr and translocated from the cytoplasm to the nucleus ([@ref-51]). Once in the nucleus phytochrome binds to and triggers the degradation of a family of bHLH transcription factors known as PHYTOCHROME INTERACTING FACTORS (PIFs), thereby inhibiting elongation and other phenotypes associated with foliar shade or darkness ([@ref-33]; [@ref-37]).

The PIF proteins were originally identified as phytochrome binding factors but are now known to be regulated not only by light but also to integrate signals from the circadian clock, high temperature, and hormone signaling ([@ref-27]). They have partially overlapping roles in regulating multiple aspects of development, including promotion of cell elongation and inhibition of both seed germination and chloroplast maturation.

Auxin has long been thought to play a role in shade avoidance ([@ref-31]; [@ref-44]). As predicted by Morelli and Ruberti, phytochromes were shown to regulate auxin transport through the shoot ([@ref-41]) and shade treatment was demonstrated to alter localization of the PIN3 auxin transporter ([@ref-23]). Shade also increases endogenous auxin levels ([@ref-25]; [@ref-45]) and auxin signaling ([@ref-3]; [@ref-5]; [@ref-20]). Disruption of auxin synthesis by mutation of the *TRYPTOPHAN AMINOTRANSFERASE OF ARABIDOPSIS1 (TAA1)* gene reduced both shade-induced increases in auxin and shade avoidance elongation responses ([@ref-45]; [@ref-50]). Treatment of leaves with an end-of-day far-red pulse (EOD-FR) will convert type II phytochromes from Pfr to Pr and has been found to increase stem elongation ([@ref-18]), similar to low R:FR. Also similar to low R:FR, EOD-FR induces many auxin-responsive genes, while disruption of auxin signaling via the *big/doc1* mutant prevents EOD-FR promotion of petiole elongation ([@ref-24]). These studies strongly implicate auxin in growth responses to shade and EOD-FR.

PIF proteins were first suggested to promote increases in auxin production and sensitivity based on microarray and dose--response studies of plants with perturbed *PIF4* and *PIF5* expression ([@ref-34]). More conclusive evidence came when it was shown that PIF4 regulates auxin biosynthesis in response to high temperature by promoting transcription of auxin biosynthesis genes ([@ref-15]). More recently it has been demonstrated that PIF4, 5, and 7 are required for normal shade avoidance and function by promoting transcription of the *YUCCA* family of auxin biosynthesis genes and potentiating auxin responsiveness ([@ref-20]; [@ref-21]; [@ref-28]; [@ref-14]).

The *YUCCA* family consists of eleven genes encoding flavin monooxygenases that function in tryptophan-dependent auxin biosynthesis ([@ref-9]; [@ref-30]; [@ref-50]; [@ref-52]). They are expressed in developmentally interesting spatiotemporal patterns ([@ref-9]; [@ref-10]). These genes are partially redundant: single knockouts often have no obvious phenotypes but double and higher-order combinations have defects in many aspects of development ([@ref-9]; [@ref-10]).

Although the phytochrome/PIF/YUCCA/auxin connection seems clear, most *yucca* mutant combinations that have been examined to date (*yucca1, 4* or *yucca3, 5, 7, 8, 9*) only show minimal to moderate shade avoidance phenotypes ([@ref-28]; [@ref-45]; [@ref-50]). More recently, as part of a large phenotypic profiling experiment we reported that the *yucca2, 5, 8, 9,* quadruple mutant has a strong shade avoidance phenotype ([@ref-35]). Because of the centrality of *YUCCA* genes to the current shade avoidance model, here we analyze that mutant strain in more detail, beginning with why we decided to make the *yucca2, 5, 8, 9* quadruple in the first place.

To better understand the role of the *YUCCA* genes in shade avoidance and EOD-FR response we used live imaging of an auxin reporter (eDR5*::Luciferase*) and found a rapid increase in auxin response following an end-of-day far-red (EOD-FR) pulse. We found that the kinetics of the eDR5 reporter response to EOD-FR were similar to the kinetics of *YUCCA2, 5, 8,* and *9* upregulation, suggesting that these genes are the critical *YUCCAs* for response to EOD-FR. We tested this idea by generating a *yucca2, 5, 8, 9* quadruple mutant and found that these genes are essential both for upregulation of the auxin reporter and for both EOD-FR and low R-FR shade-induced increases in hypocotyl and petiole elongation. These results conclusively show that the *YUCCA* genes are required for a normal EOD-FR and shade avoidance response.

Materials & Methods
===================

Plasmids
--------

eDR5*::LUC+* is described in [@ref-12]. The pZP-eDR5::*LUC2* plasmid was constructed in two steps. First, the *luciferase+* gene in the eDR5::*LUC* plasmid ([@ref-12]) was replaced with the *luciferase2* (*luc2*) gene (from pGL4.10, Promega, Madison, WI) using the HindIII and XbaI sites in the two plasmids. Second, the *e*DR5*::LUC2* cassette was removed from the resulting plasmid using the BamHI and PstI sites and cloned into the BamHI and PstI sites of pPZPXomegaLUC+ (a derivative of pPZP221 ([@ref-19]) that contains the RbcS E9 polyadenylation region). The resulting plasmid confers resistance to spectinomycin in bacteria and gentamycin in plants.

Plant materials and growth conditions
-------------------------------------

Plant transformations were performed by floral dip as previously described ([@ref-11]). eDR5*::LUC2* transformants were selected on gentamycin-containing growth media. The T-DNA and transposon insertion lines were obtained from the Arabidopsis Biological Resource Center (ABRC), the Cold Spring Harbor Lab (CSHL) or GABI-Kat. Mutant *yucca* lines and plants carrying *YUCCA* promoter-*GUS* constructs were obtained from Yunde Zhao and have been previously described ([@ref-8]; [@ref-9]). Multiple mutant combinations were obtained by repeated crossing and PCR genotyping using described primers ([@ref-8]; [@ref-9]). Homozygous *athb-2* mutants were obtained from SALK line_106790 ([@ref-1]; [@ref-36]). Homozygotes were identified by PCR genotyping using standard techniques and the primers listed in [Table 1](#table-1){ref-type="table"}. A reverse-transcription PCR assay was used to confirm that no wild-type message was made.

10.7717/peerj.2574/table-1

###### PCR primers.

![](peerj-04-2574-g007)

  Gene       Primer type   Sequence                      Final concentration
  ---------- ------------- ----------------------------- ---------------------
  *AtHB-2*   LBb1          GCGTGGACCGCTTGCTGCAACT        500 nM
  *AtHB-2*   LP            TTGGTTGAAATAAAACGAAAAGTG      500 nM
  *AtHB-2*   RP            CGTCACTGATTCCTCTTGAGC         500 nM
  *AtHB-2*   qPCR          ACATGAGCCCACCCACTAC           200 nM
  *AtHB-2*   qPCR          GAAGAGCGTCAAAAGTCAAGC         200 nM
  *PP2a*     qPCR          TAACGTGGCCAAAATGATGC          200 nM
  *PP2a*     qPCR          GTTCTCCACAACCGCTTGGT          200 nM
  *YUC2*     qPCR          ACCCATGTGGCTAAAGGGAGTGA       900 nM
  *YUC2*     qPCR          AATCCAAGCTTTGTGAAACCGACTG     300 nM
  *YUC3*     qPCR          CGTCCCTTCATGGCTTAAGGACAAC     900 nM
  *YUC3*     qPCR          GACGCACCAAACAATCCTTTTCTCG     50 nM
  *YUC5*     qPCR          ATGATGTTGATGAAGTGGTTTCCTCTG   300 nM
  *YUC5*     qPCR          ATCAGCCATGCAAGAATCAGTAGAATC   300 nM
  *YUC6*     qPCR          GAGACGCTGTGCACGTCCTA          300 nM
  *YUC6*     qPCR          AGTATCCCCGAGGATGAACC          300 nM
  *YUC8*     qPCR          ATCAACCCTAAGTTCAACGAGTG       50 nM
  *YUC8*     qPCR          CTCCCGTAGCCACCACAAG           300 nM
  *YUC9*     qPCR          TCTCTTGATCTTGCTAACCACAATGC    300 nM
  *YUC9*     qPCR          CCACTTCATCATCATCACTGAGATTCC   50 nM

For seedling stage EOD-FR analysis, seeds were surface sterilized with 70% ethanol, 0.1% TritonX-100 for 5 min, stratified for four days at 4 °C, then sown on medium containing 1/2X MS with minimal organics (Sigma M6899) and 0.7% agar (Sigma A1296). Seeds were grown in custom chambers outfitted with Quantum Devices Snaplite LEDs under short-day (8 hour day/16 hour night) conditions with 35 μmol m^−2^ s^−1^ "red" (peak wavelength 670 nm, half power spectral bandwidth 655--685 nm) and 5 μmol m^−2^ s^−1^ "blue" (peak wavelength 470 nm, half power spectral bandwidth 455--485 nm). EOD-FR treatment consisted of a 30 min, 14 μmol m^−2^ s^−1^ "far-red" (peak wavelength 730 nm, half power spectral bandwidth 715--745 nm) pulse given nightly for 1 or 4 nights before measurement. LED chamber temperature was 21 °C.

For seedling stage high and low R:FR analysis, seedlings were grown in the same custom chambers as described above for seedling EOD-FR analysis. Light conditions were continuous illumination with 35 μmol m^−2^ s^−1^ "red" (peak wavelength 670 nm, half power spectral bandwidth 655--685 nm) and 5 μmol m^−2^ s^−1\ "^ blue" (peak wavelength 470 nm, half power spectral bandwidth 455--485 nm). After 24 hours, "far-red" (peak wavelength 730 nm, half power spectral bandwidth 715--745 nm) illumination was added to bring the red-to-far-red ratio (R:FR) to 2. After an additional 48 hours the R:FR ratio in one chamber was lowered to 0.5 and plants were grown for an additional 4 days. The chambers assigned to high and low R:FR were swapped for each trial.

For analysis of juvenile plants under EOD-FR seeds were sown as above but plants were grown under 12/12 or short day (8 hr light:16 hr dark) conditions at 22 °C in a Conviron E7 chamber for approximately 18 days with cool white and incandescent lights (75 μmol m^−2^ s^−1^ PAR, R:FR 1.4). Two days prior to the EOD-FR pulse, plants were transferred to the LED chambers using the same light and temperature conditions as for seedlings (short day 35 μmol m^−2^ s^−1^ red, 5 μmol m^−2^ s^−1^ blue light; 21 °C.) and then pulsed as above.

For analysis of juvenile plants under high and low R:FR, stratified seeds were sown on soil and grown under long days in a Conviron walk-in chamber with cool white bulbs and far-red LEDs (Orbitec) (16 h light/8 h night; 100 μmol m^−2^ s^−1^ PAR, R:FR 1.8, 22 °C). Two week old plants were transferred to shelves in the same chamber with increased FR (100 μmol m^−2^ s^−1^ PAR, R:FR 0.5) to stimulate the shade avoidance response or kept under high R:FR for ten days. Leaves were scanned and petiole length measured as described ([@ref-29]). Plants for were grown under these same high R:FR conditions but were not transferred to low R:FR.

For *N* −1-naphthylphthalamic acid (NPA; Chem Service, PS-343, <http://www.chemservice.com>) treatment of eDR5*::LUC* juvenile plants, seeds were sown and grown as above. 24 hours and 1 hour prior to EOD-FR treatment each plate of plants was sprayed with 1.5ml of DMSO containing 100 μM NPA or an equivalent volume of DMSO alone. Powdered NPA was dissolved in DMSO and stored at −20 °C.

Quantitative RT-PCR
-------------------

Columbia and *athb-2* seedlings were grown as described above for seedling EOD-FR except that they had 30 min EOD-FR pulses on days 3 through 7 and were harvested on day 7, one hour after the end of the final EOD-FR pulse. RNA was prepared with Plant RNeasy (Qiagen) and cDNA prepared with Superscript II (Invitrogen). Real-time qRT-PCR was performed using an iCycler IQ™ 5 (Bio-Rad) in self-made buffer (final concentration: 40 mM Tris-HCL, pH 8.4, 100 mM KCl, 6 mM MgCl2, 8% glycerol, 20 nM fluorescein, 0.4× SYBR Green I (Molecular Probes), 1× bovine serum albumin (New England Biolabs), and 1.6 mM dNTPs) using primers described in [Table 1](#table-1){ref-type="table"}, 10 ng of RNA-equivalent cDNA and Taq polymerase. Each of five to six independent cDNA preparations was assayed two times for each transcript analyzed. Data presented are normalized to the expression level of the control gene *PP2a* (At1g13320; [@ref-13]). Transcript abundance was calculated using the relative expression software tool (REST-MCS; ([@ref-38])).

GUS staining
------------

Columbia, YUCCA5::*GUS*, YUCCA8::*GUS* and YUCCA9::*GUS* seeds were grown as described for juvenile plants above. On day 2 in the LED chamber half of the plants were treated with an EOD-FR pulse. Two hours after the pulse plants were taken for GUS analysis. Plants were harvested in 80% acetone on ice and kept in acetone for 30 min. They were then washed twice with pre-staining solution (100 mM NaPO~4~, pH 7.0, 0.1% (v/v) Triton X-100, 2 mM potassium ferrocyanide, 2 mM potassium ferricyanide, 1 mM EDTA), after which they were vacuum-infiltrated for 10 min with GUS-infiltration buffer (pre-staining solution + 1 mM X-gluc). Images were taken with a Zeiss Discovery-V12 stereo microscope and AxioCam MRC (Zeiss).

Imaging and analysis
--------------------

For hypocotyl length measurements, whole seedlings were placed on transparency film and scanned with a flatbed scanner (Microtek ScanMaker 8700, <http://www.microtek.com>). For luminescence measurements, 24 hours prior to luciferase imaging each plant plate was sprayed with 1.5 ml of 3 mM D-luciferin (Biosynth AG) in 0.1% Triton X-100. Bioluminescence was captured with an XR/Mega-10Z ICCD camera (Stanford Photonics) and Piper Imaging software (Stanford Photonics) or an iKon M-934 CCD camera (Andor) controlled by LabView software (National Instruments). Photo analysis software ImageJ ([@ref-40]) was used to measure both hypocotyl lengths and bioluminescence. Subsequent data analysis was performed in R ([@ref-39]) using base packages and the add-on packages ggplot2 ([@ref-49]), reshape2 ([@ref-48]), lme4 ([@ref-2]), lmerTest ([@ref-26]), and arm ([@ref-16]).

![EOD-FR induction of eDR5::*LUC* luminescence.\
(A--C) Mean luminescence of 5-day-old seedlings (A), 3-week-old juveniles (B), or 3-week-old juveniles in the presence of NPA (C) moved to darkness (solid black line) or treated with a 30 minute EOD-FR pulse prior to transfer to darkness (dashed red line). Dotted lines indicate SEM. Time 0 indicates the beginning of the EOD-FR treatment. *n* = 4 − 11 plants for each treatment. Representative plots for one of three independent experiments are shown. (D--F) False-color images of eDR5::LUC plants. Representative DMSO treated plant 40 (D) or 240 (E) minutes after EOD-FR pulse showing increase in petiole luminescence after treatment. (F) NPA treated plants 240 minutes after EOD-FR do not have observable petiole luminescence but show increased luminescence in the leaves and apices. (G) Mean luminescence of 3-week-old juveniles treated with DMSO (compare with (C)). For growth conditions see "seedling stage EOD-FR analysis" (A) and "analysis of juvenile plants under EOD-FR" (B-G) in Materials and Methods.](peerj-04-2574-g001){#fig-1}

Data and scripts
----------------

The raw data and scripts to recreate plots are available on GitHub: <https://github.com/MaloofLab/Mueller-Moule-PeerJ-2016>.

Results and Discussion
======================

End-of-day far-red treatment rapidly increases auxin responses
--------------------------------------------------------------

It is clear that changes in auxin biosynthesis and sensitivity are critical to shade avoidance responses ([@ref-3]; [@ref-21]; [@ref-28]; [@ref-14]). To examine phytochrome/auxin pathway interactions in real-time we used an enhanced version of the synthetic auxin responsive promoter DR5 ([@ref-46]) to drive the expression of firefly luciferase (*LUC*; [@ref-47]), eDR5*::LUC* ([@ref-12]). We initially used an end-of-day far-red (EOD-FR) pulse that, like low R:FR, will reduce the amount of active type II phytochromes, increases expression of auxin responsive genes ([@ref-24]), and increases stem elongation ([@ref-18]). Plants treated with EOD-FR displayed a strong increase in eDR5*::LUC* bioluminescence peaking two to three hours after the treatment, consistent with prior reports on eDR5*::GUS* ([@ref-5]). This response is found in both seedling stage ([Fig. 1A](#fig-1){ref-type="fig"}) and juvenile ([Fig. 1B](#fig-1){ref-type="fig"}) plants and occurred in cotyledons, hypocotyls, petioles, the shoot apex, and developing leaves ([Figs. 1D](#fig-1){ref-type="fig"} and [1E](#fig-1){ref-type="fig"}).

To investigate the importance of auxin transport in eDR5::*LUC* activation we examined the effect of the auxin transport inhibitor *N*-1-naphthylphthalamic acid (NPA) on eDR5*::LUC* expression. Plants grown on NPA still responded with a peak of luminescence following an EOD-FR treatment ([Fig. 1C](#fig-1){ref-type="fig"}), but in this case the increased bioluminescence was limited to the apex and young leaves ([Fig. 1F](#fig-1){ref-type="fig"}). The magnitude of induction was somewhat lower on NPA because of higher basal luminescence, however the peak strongly resembles the response of the control plants without NPA ([Fig. 1G](#fig-1){ref-type="fig"}) and occurs within a similar time-frame. These results suggest that auxin transport is not required to generate the peak of auxin reporter expression following EOD-FR treatment but that transport is required for increased auxin signaling in the petiole. Alternatively, it is possible that the lack of signal in the EOD-FR, NPA treated petioles is due to increased IAA conjugation that can occur in the presence of NPA.

![Shade and EOD-FR induction of *YUCCA* genes.\
(A) Expression levels of *YUCCA* genes from a published shade-induction microarray experiment ([@ref-42]). (B) mRNA levels in EOD-FR treated wild-type plants. (C) mRNA levels in EOD-FR treated *athb-2* mutant plants. For (B and C) plants were treated for five days with EOD-FR, and samples were taken 1 hour after the last EOD-FR treatment. mRNA levels shown are normalized to untreated plants. Results shown are averages of *n* = 5 − 6 ± SEM. Asterisks mark statistical significance of induction (\* *p*-value ≤ 0.05, \*\* *p*-value ≤ 0.005) calculated by the REST-program ([@ref-38]).](peerj-04-2574-g002){#fig-2}

![Histochemical localization of GUS in transgenic Arabidopsis thaliana plants containing the *YUCCA2::GUS,YUCCA5::GUS*, *YUCCA8::GUS* or *YUCCA9::GUS* constructs.\
(A--D) Whole plants. (E--H) Hypocotyls and shoot-apical meristems. (I--L) Leaves. (M--P) Roots.](peerj-04-2574-g003){#fig-3}

Shade treatment induces expression of four *YUCCA* auxin biosynthetic genes
---------------------------------------------------------------------------

Shade treatment is known to lead to increased expression of some *YUCCA* auxin biosynthetic genes ([@ref-21]; [@ref-28]; [@ref-45]), so it seemed possible that the induction of eDR5 could be due to increased *YUCCA* expression. However, most studies of *yucca* mutants have not found strong shade avoidance phenotypes. One explanation for the observed weak shade phenotypes might be redundancy within the *YUCCA* gene family. To determine if this could be the case we asked which *YUCCA* genes were induced by EOD-FR or shade treatments. We first analyzed a published microarray data set ([@ref-42]) and found that three members of this family, *YUCCA5, 8*, and *9*, were all significantly and rapidly induced by low R:FR (*P* \< 0.002; [Fig. 2A](#fig-2){ref-type="fig"}), suggesting that they would be interesting targets for further analyses. A fourth member, *YUCCA2,* was more modestly induced (*P* \< 0.03). All *YUCCA* genes returned to pre-induction levels after four days, indicating that they are involved in early response to shade conditions. We used quantitative real-time reverse transcription PCR (qRT-PCR) to confirm that *YUCCA2, 5, 8*, and *9* are induced after a series of EOD-FR treatments. One hour after the last EOD-FR treatment all four genes were significantly induced with mRNA levels up to 10 times higher than in control plants ([Fig. 2B](#fig-2){ref-type="fig"}), consistent with previous microarray studies ([@ref-28]; [@ref-45]).

*YUCCA* genes 2, 5, 8, and 9 are expressed in organs responsive to shade-treatment
----------------------------------------------------------------------------------

To determine whether these genes were expressed in tissues relevant to shade avoidance, we examined staining in *YUCCA2, 5, 8,* or *9* promoter::*GUS* fusions ([Fig. 3](#fig-3){ref-type="fig"}). All four genes were expressed in the hypocotyls and leaf veins ([Figs. 3E](#fig-3){ref-type="fig"}--[3L](#fig-3){ref-type="fig"}). *YUCCA2* was also expressed strongly in the primary root, whereas the other three expressed more weakly in primary roots ([Figs. 3M](#fig-3){ref-type="fig"}--[3P](#fig-3){ref-type="fig"}). The *YUCCA2* and *5* genes were expressed in the shoot apical meristem ([Figs. 3E](#fig-3){ref-type="fig"} and [3F](#fig-3){ref-type="fig"}) and in very defined locations in the leaf. In the leaf they were highly expressed in the veins, petioles, and hydathodes ([Fig. 3G](#fig-3){ref-type="fig"}). In the roots *YUCCA5* was highly expressed at the branching points between primary and secondary roots ([Fig. 3N](#fig-3){ref-type="fig"}), similar to reported patterns of eDR5::*LUC* ([@ref-32]) suggesting that it may play a role in defining these patterns. The *YUCCA8* and *9* genes were expressed in a more diffuse pattern in the leaves starting from the leaf margins ([Figs. 3K](#fig-3){ref-type="fig"} and [3L](#fig-3){ref-type="fig"}), similar to previously reported patterns of eDR5::*GUS* and *Ptaa1*::*TAA1*::*GUS* ([@ref-45]). They were also expressed in secondary roots ([Figs. 3O](#fig-3){ref-type="fig"} and [3P](#fig-3){ref-type="fig"}) but not in the petioles or the shoot apical meristem. In summary, these genes are expressed in the main organs where shade induction of eDR5*::LUC* expression is observed: all four are expressed in leaves and *YUCCA2* and *5* also in the shoot apex.

![*YUCCA* genes are required for shade avoidance.\
(A--C) Hypocotyl (A, C) or petiole (B) measurements of short day grown plants with (dark red) or without (blue) EOD-FR pulses. Means of *n* = 17 − 137 plants ± SEM are shown. Representative data from one of three experiments is shown. (D) Petiole lengths of plants grown in long day high (red, simulated sun) or low (dark red, simulated shade) R:FR conditions. Means of *n* = 48 − 116 petioles ± SEM are shown. (E) Induction of eDR5::LUC2 expression in 15 day-old wild type and *yucca2, 5, 8, 9* mutants moved from short day (8L:16D) conditions to darkness (blue line) or treated with a 30 minute EOD-FR pulse (dark red line). Shading indicates 95% confidence interval. Time 0 indicates the beginning of the EOD-FR treatment. Fourteen Col and 10 *yucca2589* plants were measured. For growth conditions see "seedling stage WID-FR analysis" (A, C) and "analysis of juvenile plants under EOD-FR" (B, E), and "analysis of juvenile plants under high and low R:FR" (D) in Materials and Methods.](peerj-04-2574-g004){#fig-4}

![Hypocotyl length of additional lines in simulated sun and shade.\
Four independent experiments were performed with a total of 35--150 plants per treatment/genotype combination. For growth conditions see "seedling stage high and low R:FR analysis" in Materials and Methods.](peerj-04-2574-g005){#fig-5}

![Adult wild-type and *yucca* mutant lines.\
The mutant lines did not show severe morphological defects, although some showed reduced fertility. For growth conditions see "analysis of juvenile plants under high and low R:FR" in Materials and Methods. These plants were only grown in high R:FR.](peerj-04-2574-g006){#fig-6}

*AtHB-2* is not required for *YUCCA* induction
----------------------------------------------

The HD-zip transcription factor *AtHB-2* is strongly induced by shade and affects both shade-avoidance traits and auxin-responsive processes ([@ref-6]; [@ref-4]; [@ref-31]; [@ref-43]). We were therefore curious if *athb-2* mutations would affect *YUCCA* induction. However, we found full induction of *YUCCA2, 5, 8,* and *9* in *athb-2* mutants ([Fig. 2C](#fig-2){ref-type="fig"}). Although not statistically significant the induction appears higher in *athb-2* than in wild type, perhaps hinting at a compensatory feedback loop. *AtHB-2* may primarily affect auxin transport, as previously proposed ([@ref-31]) but is not required for *YUCCA* expression.

*YUCCA* genes 2, 5, 8, and 9 are required for EOD-FR and low R:FR stimulation of auxin signaling and cell elongation
--------------------------------------------------------------------------------------------------------------------

To determine the relative importance of *YUCCA* genes for EOD-FR or shade-mediated increases in auxin signaling and subsequent hypocotyl and petiole elongation, we constructed a quadruple mutant with insertions disrupting *YUCCA2, 5, 8,* and *9* (*yucQd)* and compared this to *yucca5, 8, 9 (yucT)* and *yucca3, 5, 7 8, 9 (yucQt)* mutant strains. The *yucT* and *yucQt* strains behaved similarly, partially reducing hypocotyl and petiole EOD-FR responses ([Figs. 4A](#fig-4){ref-type="fig"} and [4B](#fig-4){ref-type="fig"}), similar to previous studies of *yucca1, 4* or *yucQt* lines ([@ref-28]; [@ref-45]; [@ref-50]). In contrast, the quadruple mutant line completely disrupted EOD-FR in hypocotyls ([Fig. 4C](#fig-4){ref-type="fig"}) and low R:FR growth responses in petioles ([Fig. 4D](#fig-4){ref-type="fig"}). In separate experiments we also compared hypocotyl low R:FR response in the *yucQd* strain to *yucca2, yucca5, yucca8,* and *yucca9* single mutants, a *yucca1, 4* double mutant strain, and a *yucca2, 5, 9* triple mutant strain ([Fig. 5](#fig-5){ref-type="fig"}). In this assay all strains were shade responsive except for *yucQd* ([Fig. 5](#fig-5){ref-type="fig"}). Across these different experiments the only consistent non-responder to low R:FR and EOD-FR is the *yucQd.* The difference between the *yucQd* mutant and the *yucT* and *yucQt* combinations is that the *yucQd* mutant is the only line missing the function of all four of the EOD-FR / low R:FR inducible *YUCCA* genes. Therefore, this result shows that *YUCCA2, 5, 8,* and *9* act additively and together are required for the shade avoidance response. In growing the mutant lines for these studies we did not observe any severe morphological defects, although *yucQd* had reduced fertility ([Fig. 6](#fig-6){ref-type="fig"}).

The failure of the *yucQd* mutant to show a morphological shade avoidance response suggested that induction of eDR5*::LUC2* by EOD-FR was likely also diminished. To investigate this possibility, the eDR5*::LUC2* construct was transformed into the *yucQd* strain and wild-type plants. We found that EOD-FR induction of eDR5*::LUC2* expression was essentially abolished in the *yucQd* mutant (juvenile plants; [Fig. 4E](#fig-4){ref-type="fig"}). Thus, *YUCCA2, 5, 8,* and *9* are required for increased auxin signaling in response to EOD-FR and shade for the subsequent induction of hypocotyl and petiole elongation.

Conclusions
===========

The phenotypic plasticity exhibited by plants in response to shade from other plants is visually striking and is of agronomic importance. Accumulating evidence has led to a model whereby inactivation of phytochromes in shade allows accumulation of PIF transcription factors that upregulate *YUCCA* transcription and a concomitant increase in auxin biosynthesis. Given this model it has been something of a conundrum that multiple *yucca* mutants retain a significant (albeit reduced) shade avoidance response, leaving open the possibility of a parallel, *YUCCA-*independent pathway. By creating a multiple mutant that removes all of the shade-inducible *YUCCA* genes we demonstrate that *YUCCAs* are essential for measurable shade avoidance responses in the hypocotyl and also the petiole.

We thank Yunde Zhao and Youfa Cheng for sharing seed and reagents prior to publication. We thank Judy Callis and John Labavitch for helpful discussions on this project. Some seed stocks were obtained from the ABRC.

Additional Information and Declarations
=======================================

Melissa Pytlak is an employee of PASCO scientific, Roseville, California and Andreah Wallace is an employee of Clontech Laboratories, Mountain View, California

[Patricia Müller-Moulé](#author-1){ref-type="contrib"} and [Julin N. Maloof](#author-8){ref-type="contrib"} conceived and designed the experiments, performed the experiments, analyzed the data, wrote the paper, prepared figures and/or tables, reviewed drafts of the paper.

[Kazunari Nozue](#author-2){ref-type="contrib"} performed the experiments, analyzed the data, prepared figures and/or tables, reviewed drafts of the paper.

[Melissa L. Pytlak](#author-3){ref-type="contrib"} conceived and designed the experiments, performed the experiments, reviewed drafts of the paper.

[Christine M. Palmer](#author-4){ref-type="contrib"} and [Andreah D. Wallace](#author-6){ref-type="contrib"} performed the experiments, reviewed drafts of the paper.

[Michael F. Covington](#author-5){ref-type="contrib"} and [Stacey L. Harmer](#author-7){ref-type="contrib"} contributed reagents/materials/analysis tools, reviewed drafts of the paper.

The following information was supplied regarding data availability:

Raw data and code for making figures is at GitHub: <https://github.com/MaloofLab/Mueller-Moule-PeerJ-2016>.
