Initial characterization of shade avoidance response suggests functional diversity between Populus phytochrome B genes


Author for correspondence:

Abhijit Karve

Tel: +1 864 650 3395



  • Shade avoidance signaling involves perception of incident red/far-red (R/FR) light by phytochromes (PHYs) and modulation of downstream transcriptional networks. Although these responses are well studied in Arabidopsis, little is known about the role of PHYs and the transcriptional responses to shade in the woody perennial Populus.
  • Tissue expression and subcellular localization of Populus PHYs was studied by quantitative real-time PCR (qRT-PCR) and protoplast transient assay. Transgenic lines with altered PHYB1 and/or PHYB2 expression were used in phenotypic assays and transcript profiling with qRT-PCR. RNA-Seq was used to identify transcriptional responses to enriched FR light.
  • All three PHYs were differentially expressed among tissue types and PHYBs were targeted to the nucleus under white light. Populus PHYB1 rescued Arabidopsis phyB mutant phenotypes. Phenotypes of Populus transgenic lines and the expression of candidate shade response genes suggested that PHYB1 and PHYB2 have distinct yet overlapping functions. RNA-Seq analysis indicated that genes associated with cell wall modification and brassinosteroid signaling were induced under enriched FR light in Populus.
  • This study is an initial attempt at deciphering the role of Populus PHYs by evaluating transcriptional reprogramming to enriched FR and demonstrates functional diversity and overlap of the Populus PHYB1 and PHYB2 in regulating shade responses.


In addition to acting as a key energy source for photosynthesis, light provides plants with important spatial and temporal signals. Plants can detect the presence of neighboring vegetation, in part, by measuring the relative ratio of red (R) to far-red (FR) light incident on the leaves (Ballare et al., 1989). In an open canopy, plants are exposed to relatively equal fluxes of R and FR radiation (Holmes & Smith, 1975). However, in a closed canopy, the selective absorbance of R wavelengths relative to FR by the neighboring vegetation results in a substantially lower R/FR ratio under the canopy (Cummings, 1963; Quail, 1998). For example, light passing through a single Arabidopsis leaf decreases in its R/FR from 1.2 to 0.2 (Franklin, 2008). Under continuous FR enrichment, plants undergo a series of morphological changes, which enable them to effectively tolerate or avoid shading from neighboring vegetation. Shade-avoiding species undergo a suite of developmental changes when subjected to low R/FR, collectively known as shade avoidance syndrome (SAS) (Smith & Whitelam, 1997). Morphologically, SAS is characterized by a rapid elongation of stems and leaves, upward reorientation of leaves and early flowering (Franklin, 2008).

Phytochromes (PHYs) are the only photoreceptors that are reversibly photochromic biliproteins and maximally absorb R and FR light (Franklin & Whitelam, 2004). In addition to their direct role in germination, seedling establishment, flowering, dormancy, nyctinasty and stomatal development (Mathews, 2010 and references therein), PHYs are uniquely suited for proximity detection owing to their capacity to interconvert between an active R-absorbing Pfr form and an inactive FR light-absorbing Pr form. The photoconversion of phytochrome from its Pr to Pfr form triggers translocation from the cytoplasm to the nucleus where phytochrome controls the expression of a number of genes that in turn suppress elongation (Sakamoto & Nagatani, 1996; Whitelam & Devlin, 1997; Ni et al., 1999). Under open canopies, phytochrome exists predominantly in the active Pfr form as a result of a higher R/FR. By contrast, the lower R/FR characteristic of closed canopies converts phytochrome to its inactive Pr form, thereby removing the Pfr-mediated inhibition of elongation (Devlin et al., 2003).

In most plant species, PHYs are encoded by a small multigene family (Bae & Choi, 2008). The better-studied PHY families include Arabidopsis with five PHY gene members (PHYA to PHYE; Sharrock & Quail, 1989; Mathews et al., 1995), maize with six members (PHYA1, PHYA2, PHYB1, PHYB2, PHYC1 and PHYC2; Sheehan et al., 2004) and tomato with at least five members (PHYA, PHYB1, PHYB2, PHYE, PHYF; Alba et al., 2000). Of the five PHYs that exist in Arabidopsis, PHYA is light-labile and predominates in etiolated seedlings where it mediates FR-high irradiance responses in de-etiolating seedlings (Smith & Whitelam, 1990). The other Arabidopsis PHYs (PHYB to PHYE) are light-stable and have distinct but overlapping functions throughout plant development (Bae & Choi, 2008).

Arabidopsis mutants deficient in phyB display a constitutive shade avoidance phenotype characterized by elongated seedling hypocotyls, longer petioles, smaller leaves and accelerated flowering when grown under white light (WL; i.e. high R/FR; Reed et al., 1993). PHYB-deficient mutants of other species such as rapeseed and cucumber also show a similar shade avoidance phenotype (Devlin et al., 1992; Lopezjuez et al., 1992). PHYB is therefore implicated as the major PHY mediating responses to canopy shade (Franklin et al., 2003). However, both PHYD and PHYE have also been shown to mediate SAS. Although phyD and phyE single mutants do not display the elongated appearance and early flowering response displayed by phyB mutants grown under high R/FR ratio, the phyBphyD and phyBphyE double mutants show extreme hypocotyl and petiole elongation (Devlin et al., 1999). Furthermore, an investigation with phyBphyDphyE triple mutants indicates that these three PHYs act redundantly to regulate some aspects of SAS such as leaf morphology and flowering time (Franklin et al., 2003). Since phyC alone, or in combination with other PHYs, does not show any aberration attributed to the perception of R/FR ratio, it likely does not play a direct role in SAS (Franklin et al., 2003).

Underlying the morphological changes associated with SAS are the physiological changes mediated by phytohormones. Mutants defective in metabolism or signaling of several phytohormones, including brassinosteroid, auxin, ethylene and GA, either have a reduced FR-light-induced elongation response or can suppress the constitutive shade-avoiding phyB mutant phenotype (Kim et al., 1998; Kanyuka et al., 2003; Pierik et al., 2004; Hisamatsu et al., 2005). It has been suggested that transcriptional networks associated with light quality changes (low R/FR) connect with phytohormone networks to regulate cell division and expansion (Sorin et al., 2009). Multiple studies have used a transcriptomics approach to identify Arabidopsis genes that respond to low R/FR. Devlin et al. (2003) identified 301 shade-responsive genes in Arabidopsis, including genes involved in hormone signaling and novel shade-regulated transcription factors. Based on the expression data, they further substantiated that Arabidopsis PHYB plays a major role in mediating hypocotyl elongation response, whereas PHYA moderates PHYB action.

Although much of our knowledge of the role of PHYs in SAS comes from studies with Arabidopsis, our understanding of the molecular networks associated with SAS in woody perennials is relatively limited. The study of SAS is particularly important in the model tree species Populus trichocarpa, given the potential of Populus as a bioenergy crop and its distribution as a keystone species in some forest ecosystems (Whitham et al., 2008). Despite its recent genome duplication (Tuskan et al., 2006), the Populus genome encodes only three PHY genes, PHYA, PHYB1 and PHYB2 (Howe et al., 1998), with no known homologs for PHYC, PHYD or PHYE.

Here we report the initial characterization of SAS from Populus in the context of transcriptional response, morphology and PHYB function. We first show the tissue expression and subcellular localization of the three Populus PHYs. We then use quantitative real-time PCR (qRT-PCR) and RNA-Seq to identify the downstream targets of SAS in Populus. In order to validate the role of individual PHYBs in SAS, we use transgenic lines overexpressing Populus PHYB1 and PHYB2 in Arabidopsis mutant phyB and wildtype (WT) Populus deltoides background. Our results indicate that PHYB1 and PHYB2 are involved in the Populus shade avoidance response.

Materials and Methods

Plant material and growth conditions

Seeds of Arabidopsis thaliana (L.) Heynh (ecotype Ler) were obtained from the Arabidopsis Biological Resource Center. Seeds of the phyB (phyB-1 in Ler background, Koornneef et al., 1980) mutant were provided by Dr Robert Sharrock (University of Montana). Seeds were imbibed at 4°C with distilled water for 4 d to ensure uniform germination; cold-treated seeds were resuspended in 0.05% agarose and planted in pots containing Fafard 3B mix (Fafard, Agawam, MA, USA). Arabidopsis plants were grown in a chamber under the following conditions: 22°C, 60% relative humidity, a 12 h photoperiod and 300 μmol m−2s−1 light intensity. Dormant cuttings of Populus trichocarpa Torr. & A.Gray (clone 93-968) or P. deltoides W.Bartram ex Marshall WV94 were planted with ProMix BX potting mix ( containing vermiculite and perlite. The cuttings were pretreated with rooting hormone (0.3% IBA) before planting. All the cuttings had approximately the same diameter (0.5 cm), were 16–20 cm long and had two to three buds above the soil. The Populus cuttings were grown in a Conviron BDW walk-in growth chamber (Conviron, Canada) under the following conditions: 22°C, 60% relative humidity, 16 h photoperiod and 300 μmol m−2s−1 light intensity. All plants were watered as needed and fertilized weekly according to species-specific requirements: 250 ppm N for Populus (11.4 g/L 20-10-20, Jack's Professional, Allentown, PA, USA) and 125 ppm N for Arabidopsis (11.8 ml/L 10-10-10, Vigoro, Sylacauga, AL, USA). For measuring the tissue expression of PHYs, seeds of P. trichocarpa were surface sterilized and grown on woody plant medium (Caisson Labs, North Logan, UT, USA) supplemented with 0.5% sucrose and 0.75 phytagar either under constant light or wrapped in aluminum foil for 5 d in a growth chamber maintained at 22°C, 60% relative humidity and 300 μmol m−2s−1 light intensity.

Far-red light treatment

All of the light treatments for Populus were performed in a growth chamber maintained at 22°C, 60% relative humidity and 300 μmol m−2s−1 of photosynthetically active radiation (PAR). One-month-old Populus cuttings growing under WL were subjected to FR light enrichment with 730 nm LEDs (Quantum devices, Barneveld, WI, USA) for the specified time (WL + FR, low R/FR), whereas control cuttings were incubated in the same chamber without the addition of FR irradiance for the same amount of time (WL, high R/FR). The fluence rates were measured using Li250 quantum photometer (Li-Cor, Lincon, NE, USA). The ratio of R/FR was 1.25 under WL control and 0.07 under WL + FR and conditions. For 454 analysis and, real time quantitative reverse transcription PCR (qRT-PCR) tissue was harvested 1 and 4 h after WL or WL + FR irradiance, flash-frozen in liquid nitrogen and then stored at −80°C until further processing. All the light treatment experiments were replicated in two separate and independent growth chamber experiments, resulting in two biological replicates unless otherwise specified.

For hypocotyl elongation assays, Arabidopsis seeds were surface-sterilized and grown vertically on 1 × MS agar plates (modified basal medium with Gamborg vitamins; PhytoTechnology Laboratories, Shawnee Mission, KS, USA) at pH 5.7, with 0.5% sucrose, at 22°C under constant light (60 μmol m−2 s−1, R/FR > 1.25). For the FR light treatment, 7-d-old seedlings were transferred under FR LEDs within the same growth chamber for 24 h (R/FR = 0.05).

Cloning and plasmid constructs

Poplar transformation work was performed by ArborGen, Inc. (Summerville, SC, USA). Full-length PdPHYB1 and PdPHYB2 sequences were amplified using P. deltoides gene-specific primers (Supporting Information, Table S1) from a P. deltoides clone WV94 cDNA library and cloned into the Invitrogen Gateway entry vector pCR8/GW-TOPO (Invitrogen). LRII Clonase II (Invitrogen) was used to transfer the desired gene fragment from the entry vector to the final binary destination vector pAGW60 (Fig. S1). Agrobacterium tumefaciens (strain EHA105), harboring the plasmids containing individual or double constructs, were used to infect the petiole explants as described previously (Dinus et al., 1995). The transformants were selected on the plant growth medium supplemented with 50 mg ml−1 of kanamycin. Rooted plantlets were transferred to soil and hardened off before light experiments. To generate phyBB1 and phyBB2, A. tumefaciens (strain GV3101) harboring binary vector with either PdPHYB1 or PdPHYB2 constructs was used to transform Arabidopsis phyB mutant plants using the floral dip method (Clough & Bent, 1998).

For transient gene expression in protoplasts, full-length PtPHYA, PtPHYB1 and PtPHYB2 were amplified using gene-specific primers (Table S1) from the P. trichocarpa leaf cDNA library and cloned in pENTR-D-TOPO vector (Invitrogen) using the manufacturer's protocol. Individual genes were then subcloned into the pEarleyGate 103 (Earley et al., 2006) plant expression vector as an N-terminal GFP-histidine tagged construct using the LR clonase II enzyme (Invitrogen). ABF4-GFP was provided by Dr Brandon Moore (Clemson University).

Protoplast transient expression and microscopy

Protoplasts were isolated from Arabidopsis leaves as previously described (Karve & Moore, 2009). These were transfected using the polyethylene glycol 4000 (Sigma-Aldrich) protocol (Yoo et al., 2007) and 6–12 μg of midi-prep plasmid DNA isolated using a Plasmid Midi Kit (Qiagen). After transfection, protoplasts were incubated continuously in the dark or under constant WL for 12 h. At the end of incubation period, protoplasts were visualized by fluorescent confocal microscopy and imaged using DFC300FX colored camera attached to a Leica DM IRE2 LSM (Leica, Wetzlar, Germany).

RNA extraction, library preparation and 454 sequencing

RNA was extracted using a Spectrum™ Plant Total RNA isolation kit (Sigma-Aldrich) according to the protocol provided. After on-column DNAse treatment, RNA quality was assessed using an Experion™ RNA StdSens Analysis Kit (Bio-Rad). RNA samples with an OD260 : OD280 ratio ≥ 1.8 of the same treatment type for all biological replicates were pooled, resulting in three pooled samples, a WL time point zero control, a 1 h WL + FR light and a 4 h WL + FR light sample. The mRNA purification was performed for each of the three samples using the Dynabeads® mRNA purification kit (Invitrogen). Purified mRNA from each of the three samples was used to synthesize a cDNA library using the cDNA Rapid Library Preparation protocol provided by Roche. After the libraries were synthesized, library quantity was determined using a 96-well plate Fluoroskan Ascent (Labsystems, Finland) on an Agilent Bioanalyzer High Sensitivity DNA chip (Agilent, Santa Clara, CA, USA). A GS FLX Titanium emPCR Lib-L SV kit (Roche) was used to do an emulsion titration to determine the amount of library to be added to the large-volume emulsions, and a GS FLX Titanium emPCR Lib-L LV kit (Roche) was used to populate the beads. The resulting beads were sequenced using a GS Titanium sequencing kit XLR70 on a Genome Sequencer FLX Instrument (Roche).

The raw expressed sequence tag (EST) sequences generated by transcriptome sequencing were processed and clustered as described by Yang et al. (2011). Specifically, the ESTs were trimmed for vector, adaptor/linker, polyA or T tails and then tagged by IDs corresponding to respective control and treatments. The reference Ptrichocarpa transcript sequences (GeneCatalog_frozen20080522;) were pooled with the tagged EST sequences and clustered using SCLUST implemented within the TGICL software (Pertea et al., 2003) using 97% identity and 80% sequence coverage criteria. The number of ESTs clustered with annotated Populus transcripts was counted separately for the control and individual treatments.

qRT-PCR analysis

The relative expression levels of several candidate shade response genes were compared across treatments using relative qRT-PCR (Table S2). Total RNA was extracted from fully expanded leaves, flash-frozen in liquid nitrogen and kept at –80°C using a Spectrum™ Plant Total RNA Extraction Kit (Sigma-Aldrich). On-column DNase I treatment (Sigma-Aldrich) was performed according to the provided protocol to remove any potential genomic DNA contamination. One microgram of RNA was used for cDNA synthesis. The cDNA synthesis was carried out using a SuperScript® III First-Strand Synthesis SuperMix for qRT-PCR (Invitrogen) according to the protocol provided.

Amplification reactions (20 μl) were carried out using iQ™ SYBR® Green Supermix with ROX according to instructions provided by Bio-Rad Laboratories. PCR amplification reactions were performed in triplicate under the conditions described previously in Weston et al. (2011). Gene expression was normalized by the endogenous control gene, Populus ubiquitin 11 (PtUBQ11, Brunner et al., 2004). The forward and reverse primers used in the qRT-PCR were designed using Primer3 Express (Rozen & Skaletsky, 2000). All PCR amplification reactions were performed on the RNA extracted from the tissue of two independent biological replicates. A biological replicate is defined as an independent experiment with pooled tissue samples from three or more plants or seedlings. Relative mRNA abundance was calculated using the Pfaffl method (Pfaffl, 2001). For each cDNA, three technical replicates were assayed and the values averaged. The efficiency of each primer pair was assessed by use of a dilution series. Across the biological replicates, threshold cycles for all products from the cDNAs fell within the valid range of the standard curves. Values > 1 represent up-regulation relative to the reference group and values < 1 represent down-regulation relative to the reference group.


Tissue expression and subcellular localization of P. trichocarpa PHYs

We examined the relative expression of individual P. trichocarpa PHY (PtPHY) transcripts among various Populus tissue types by qRT-PCR. The PtPHYA was highest in seedlings, especially etiolated seedlings grown under dark conditions (c. 60-fold), relative to the root PHYA expression. PtPHYA expression was also apparent in male and female flowers (Fig. 1). Among PtPHYBs, PtPHYB1 was most abundant in female flowers followed by shoot apex and was not detected in roots. PtPHYB2 was slightly more abundant in xylem, followed by shoot apex and then female flowers, although the overall fold change was relatively low (< 1.5) among these three tissue types.

Figure 1.

Tissue expression of Populus trichocarpa phytochrome family genes determined by quantitative real-time PCR. The data were expressed as fold-change relative to the leaf expression of individual gene family member. Bars represent the mean fold change ± SD of two biological replicates.

In Arabidopsis, PHYs form homo- and heterodimers that are localized to the nucleus where they regulate gene expression. To study PtPHY localization we expressed GFP-PtPHY in Arabidopsis mesophyll protoplasts. As a nuclear marker, we transfected the protoplasts with ABF4-GFP (Fig. 2a) (Choi et al., 2005). Both GFP-PHYB1 and PHYB2 showed a similar nuclear localization pattern to ABF4-GFP under WL (Fig. 2c,d). As Arabidopsis PHYA is light-labile, we incubated the protoplasts transfected with GFP-PHYA in the dark. Unlike GFP-PHYBs, GFP-PHYA was largely dispersed in the cytoplasm (Fig. 2b). However, when the protoplasts were moved to constant WL the intensity of the GFP fluorescence decreased drastically. No such change in the fluorescence was observed for the expression of GFP-PHYB1 and GFP-PHYB2 (data not shown).

Figure 2.

Subcellular localization of Populus trichocarpa phytochrome (PHY) family proteins. (a) AtABF4-GFP; (b) GFP-PtPHYA; (c) GFP-PtPHYB1; (d) GFP-PHYB2. N-terminal GFP fusion proteins were cloned and expressed in leaf protoplasts and then imaged with a laser scanning confocal microscope. GFP fluorescence is pseudocolored green. Chlorophyll autofluorescence from a long-pass filter is shown pseudocolored red. AtABF4-GFP is used as a marker for nuclear localization.

Effect of low R/FR light treatment on the expression of early shade response genes

The timing of early transcriptional responses to shade in Populus was assessed by a simulated shade experiment. One-month-old rooted Populus lines grown under constant WL (high R/FR) were either maintained under WL or were transferred to WL enriched with FR light (WL + FR, low R/FR) from 15 min to 4 h. In Arabidopsis, transcript abundance of PHYB, ATHB2 and HFR1 show rapid induction by low R : FR exposure (Steindler et al., 1999; Salter et al., 2003). Our results confirm these findings under our experimental system (Fig 3) and show that the Populus orthologs of these genes are also responsive to WL + FR. These responses were specific to the WL + FR treatment, as gene expression from WL control plants did not significantly change through time (Fig. S2). Specifically, both PtPHYBs, PtATHB2 and PtHFR1, were slightly induced within 15 min of WL + FR and were significantly induced after 1 h of WL + FR relative to WL control conditions (P < 0.001; Fig. 3). After 4 h of WL + FR treatment conditions, the transcript abundances of PtPHYA and PtPHYB2 were near control values, whereas those of PtPHYB1, PtHFR1 and PtATHB2 were still significantly induced relative to WL control (P < 0.001; Fig. 3). PHYA transcript abundance was relatively unresponsive compared with the PtPHYBs, except after 1 h of WL + FR treatment, where a significant induction was observed (Fig. 3).

Figure 3.

Effect of supplemental far red (FR) light on the expression of shade response genes in wildtype Populus trichocarpa leaf tissue. The transcripts of phytochrome (PHY) gene family members and expression of Populus orthologs of Arabidopsis genes (see Supporting Information Table S2 for details of the candidate genes) were measured by quantitative real-time PCR and expressed relative to the white light (WL, control) after 15 min, 1 and 4 h of exposure to WL supplemented with FR light (WL+FR); Bars represent means ± SD of three replicate experiments. Student's t-tests were used to determine the significance of differences in the transcript abundance from the respective WL control (*, < 0.001).

Global gene expression profiling of SAS in Populus

We used an RNA-Seq approach to identify conserved and novel transcript targets involved in the Populus SAS response. RNA was isolated from leaves of P. trichocarpa under the same treatment conditions as those used for the qRT-PCR experiment described earlier. A total of 471 636 reads were generated from both the WL control and WL + FR experimental libraries. RNA-Seq analysis identified 10 224 transcripts in either the experimental or the control Populus-treated leaves (Table S3) out of the total 17 500 annotated Populus genes found previously in the leaf transcriptome (Philippe et al., 2010). From the Populus data we identified orthologs of some of the genes reported as FR induced in Arabidopsis (Table S4). Although the goal of the RNA-Seq experiment was gene discovery and not transcript quantification, we found a surprisingly high Pearson correlation (r > 0.8) between RNA-Seq and qRT-PCR (Table S4, Fig. S3).

Relative to WL, the WL + FR light treatment had 357 transcripts exhibiting induction > 3 loge2-fold (Table S5). Out of these, 120 and 198 were induced after 1 and 4 h of WL + FR light, respectively, and 39 were induced in both 1 and 4 h samples (Fig. S4). A total of 155 transcripts were significantly suppressed (< −3 loge2-fold) after the WL + FR light treatment (Table S6). Out of these, 68 were suppressed after 1 h and 48 were repressed after 4 h of WL + FR light treatment (Fig. S4). A total of 39 transcripts were suppressed in both 1 and 4 h WL + FR samples. In addition, a total of 80 transcripts were induced from 1 to 4 h, whereas 172 were suppressed from 1 to 4 h. The PageMan (Usadel et al., 2006) bioinformatics software was used to link these changes in transcriptional reprogramming to core metabolism, signaling and cellular processes. Under- and overrepresented groups were determined using Wilcoxon rank summary test statistics. Within 1 h of exposure to WL + FR light, genes associated with carbohydrate metabolism were down-regulated, especially those genes that are functionally associated with starch metabolism. This enrichment analysis also showed up-regulation of genes associated with cell wall modification and brassiosteroid metabolism/signaling after 1 h of WL + FR treatment. Furthermore, this up-regulation continued even after 4 h of WL + FR treatment (Fig. 4). Jasmonic acid biosynthesis was repressed from 1 to 4 h of WL + FR treatment.

Figure 4.

PageMan analysis of prominent categories of far red (FR) light response genes in Populus trichocarpa. Over- and underrepresented categories were pseudocolored blue and red, respectively. An unpaired Wilcoxon rank-sum test was used to determine if the median fold change within a particular ontological group was the same as the median fold change of all genes not in that group. CHO, carbohydrate metabolism; LRR, leucine rich repeat; RGP, reversibly glycosylated polypeptide.

PdPHYB1 complements the Arabidopsis phyB phenotype

In order to test whether both Populus PHYBs have retained the FR light signaling function of AtPHYB, we transformed the previously characterized (Reed et al., 1993) Arabidopsis phyB mutant with P. deltoides PdPHYB1 (phyBB1) or PdPHYB2 (phyBB2) driven by the Arabidopsis UBQ5 promoter (Fig. 5a). The expression of PHYB1 or PHYB2 was confirmed by qRT-PCR and five independent lines were identified for phyBB1OE and four for phyBB2OE. Based on qRT-PCR expression for PHYB1 or PHYB2 in T3 transgenic lines, three lines with comparable target gene expression patterns were selected for further phenotypic analyses with data presented for one of the three lines (Fig. 5b). When grown under WL, the Arabidopsis phyB plants had elongated leaf petioles, larger rosettes and reduced rosette leaf number compared with the WT grown parental Ler plants (Fig. 5a, Table 1). Interestingly, phyBB1 resembled Ler in rosette diameter, petiole length as well as leaf area, while the phyBB2 seedlings resembled the phyB mutant line for all measured parameters.

Figure 5.

Complementation analysis of Arabidopsis phyB mutant. (a) Four-week-old Arabidopsis thaliana plants grown in a growth chamber under white light: Ler, wildtype Landsberg erecta; phyB, Arabidopsis PHYB mutant line; phyBB1OE, phyB mutant-expressing Populus PHYB1; phyBB2OE, phyB mutant-expressing Populus PHYB2. (b) Transcript expression of PdPHYB1and PdPHYB2 by semiquantitative reverse transcription-PCR. AtUBQ5 was amplified as a control for template amounts.

Table 1. Phenotypic comparison of the Arabidopsis thaliana phyB mutant and transgenic lines expressing PdPHYB1 or PdPHYB2
GenotypeRosette diameter (cm)Leaf numberPetiole length (cm)
  1. Data represent means ± SD. Student's t-tests were used to determine the significance of differences from phyB (a, P < 0.001) and Ler (b, P < 0.001).

Ler5.46 ± 0.26a12.60 ± 0.89a0.49 ± 0.03a
phyB 8.90 ± 0.57b9.00 ± 1.00b2.05 ± 0.48b
phyB B1OE 4.30 ± 0.34a12.80 ± 0.84a0.48 ± 0.15a
phyB B2OE 9.36 ± 1.23b9.20 ± 0.84b1.68 ± 0.42b

We then subjected the phyBB1 and phyBB2, along with Ler and phyB seedlings, to a seedling hypocotyl elongation assay. One-week-old Arabidopsis seedlings grown under WL were either maintained under WL or transferred to a growth chamber supplemented with FR (WL + FR) for 24 h as previously described (Devlin et al., 2003). The Ler seedlings under WL + FR light had longer seedling hypocotyls relative to those maintained under WL (Fig. 6), suggesting a normal SAS. Under WL conditions (high R/FR), phyB mutants had longer seedling hypocotyls than Ler; however, WL + FR light did not induce hypocotyl elongation in phyB mutant seedlings. The lack of elongation response in phyB seedlings is consistent with the role of phyB as a far-red light sensor. Like the parental phyB line, seedlings of phyBB2 had longer seedling hypocotyls under control WL and were relatively insensitive to WL + FR-induced hypocotyl elongation. By contrast, the hypocotyl length of phyBB1 seedlings was comparable to Ler seedlings under WL. Further, like Ler, phyBB1 seedlings also showed hypocotyl elongation response under WL + FR (Fig. 6). Thus, the phenotypic response of phyBB1 and phyBB2 to WL + FR light indicates that PdPHYB1 but not PdPHYB2 functionally complements AtPHYB.

Figure 6.

Effect of far-red (FR) light treatment on the hypocotyl length of transgenic Arabidopsis thaliana seedlings expressing PdPHYB1 or PdPHYB2. Seedlings grown for 7 d under white light (WL) were either maintained under WL (closed bars) or transferred to WL + FR (gray bars) for 24 h. Bars represent the mean hypocotyl length of two experiments ± SD (= 35). Asterisks (*) indicate that the mean hypocotyl length was significantly different from WL at < 0.001.

Populus PHYB1 and PHYB2 regulate distinct yet overlapping responses to shade

In order to verify our findings from the complementation of Arabidopsis mutants, we generated transgenic P. deltoides lines with altered PHYB1 and PHYB2 expression. Three independent transgenic homozygous overexpression lines each for PdPHYB1, PdPHYB2 and PdPHYB1/B2 were isolated. Under controlled growth conditions (WL), all the PHYBOE lines looked similar to WT plants. Out of these identified three lines per construct, two lines were selected based on expression of the target genes (Fig. S5) and subjected to further phenotypic analysis using PHYB1OE#2, PHYB2OE#2 and PHYB1B2OE#1.

The WT Populus deltoides clone ‘WV94’ (ArborGen, Summerville, SC, USA) and transgenic overexpression lines were tested in a shade response assay. One-month-old cuttings grown under WL (high R/FR) within growth chamber conditions were kept under either WL or WL supplemented with FR (WL + FR, low R/FR) to attain a R/FR ratio of 0.07 for 5 d. Total plant height and petiole length were measured at the beginning and the end of the assay. The WL + FR treatment resulted in increased plant height (116%) and petiole length (87%) in relative attenuated in PtPHYB1OE lines, with only a 68% increase over WL under the WL + FR condition (Fig. 7a). The stem elongation response of PHYB2OE and PHYB1B2OE lines were not significantly different from WT WV94 under WL + FR. Based on measurements of plant height, we expected PdPHYB1OE to be less responsive to WL + FR-light-induced petiole elongation and PHYB2OE to behave as WV94 (Fig. 7b). However, WL + FR-induced petiole elongation was suppressed not only in PtPHYB1OE but also in PHYB2OE and PHYB1B2OE. Furthermore, this suppression was most prominent in the PHYB1B2OE transgenic lines rather than in PHYB1 or PHYB2 transgenic lines alone, suggesting a possible overlap between PHYB1 and PHYB2 in regulating the FR petiole elongation response.

Figure 7.

Effect of increased levels of PdPHYB1 (PHYB1OE), PdPHYB2 (PHYB2OE), or both (PHYB1B2OE), on Populus deltoides plant height (a) and petiole length (b) by low red/far-red (R/FR) light. Bars represent the percentage increase in plant height or petiole length over the WL. Data represent means ± SD of three independent experiments of = 5. WT, Populus deltoides WV94. Asterisks (*) indicate that the percentage change was significantly different from WT at < 0.001.

Gene expression changes in identified shade-responsive genes (Table S4) were further investigated in the overexpression lines. These genes are the Populus orthologs of Arabidopsis genes that are known to be up-regulated by FR light (Devlin et al., 2003; Sessa et al., 2005; Roig-Villanova et al., 2006) or that have been identified as being up-regulated within 1 h of WL + FR treatment in our RNA-Seq analysis (Figs 3b, S3). All the gene transcripts studied were up-regulated after 1 h of low R/FR light treatment (WL + FR) in WT WV94 (Fig. 8). The transcripts of PdPRTSP, PdNPH1, and PdHFR1 were suppressed relative to the WL in PHYB1OE, PHYB2OE and PHYB1B2OE after the WL + FR light treatment, indicating the possible involvement of both PHYB1 and PHYB2 in transcriptional regulation of these genes. The PdNPH3, PdLRX3 and PdBRI1 transcripts were significantly suppressed in PHYB1OE as well as in the PHYB1B2OE line after light treatment (Fig. 8). Interestingly, PdNPH3 and PdBRI1 were induced by the WL + FR light treatment for the PHYB2OE line, whereas PdLRX3 remained unchanged after WL + FR treatment for the PdPHYB2OE line. Additionally, PdCKX5 transcript abundance was induced (suppressed?) under WL within the PHYB1OE line, and was also induced by WL + FR for the PHYB2OE and PHYB1B2OE lines. Both PdECA2 and PdATHB2 were up-regulated by WL + FR treatment in PdPHYB1OE lines. Of these, PdECA2 was only slightly induced (Relative quantity, RQ = 1.3), whereas PdATHB2 was suppressed by WT + FR in the PHYB2OE line. In general, most of the transcriptional responses in the PHYB1B2OE lines were either intermediate or resembled PHYB1OE. These results suggest that, as in Arabidopsis, Populus FR-enriched light responses are under complex regulation from a signaling cascade that includes PHYB. Furthermore, WT responses of ECA2 in all the transgenic overexpression lines studied suggests a possible role of another phytochrome, likely PHYA, in its regulation.

Figure 8.

Effect of increased levels of PdPHYB1 (PHYB1OE), PdPHYB2 (PHYB2OE), or both (PHYB1B2OE), on the expression of candidate shade response genes of Populus deltoides. See Supporting Information Table S2 for gene annotation and information on individual genes. One-month-old Populus deltoides lines grown under white light (WL) were either maintained under WL or transferred to WL + FR for 1 h. Bars represent fold change in the gene transcript over the WL control of two biological replicates; error bars represent the SE. WT, Populus deltoides WV-94.


Shade-avoiding species such as Arabidopsis and Populus undergo complex developmental and physiological changes that result in the SAS phenotype. Although much of our understanding of these complex changes comes from the study of SAS in Arabidopsis, we know very little about SAS in woody perennial species. In this study, we provide an initial characterization of the Populus SAS response. Previous work in Arabidopsis has identified PHYB is a key mediator of SAS; thus, the main objective of this study was to ascertain the role of Populus PHYB1 and PHYB2 in SAS.

In Arabidopsis, transcripts of the individual PHY gene family members accumulate in different plant organs (Clack et al., 1994). These expression patterns have previously been linked to the function of at least some of the Arabidopsis PHYs (Aukerman et al., 1997). Using a similar approach, we investigated the transcript abundance of Populus PHY members across different tissue types. PtPHYA was the most abundant phytochrome in dark-grown seedlings; however, its expression was drastically reduced in light-grown seedlings. The same is true for tomato and Arabidopsis PHYA, which are highly up-regulated in dark-grown seedlings compared with seedlings grown under constant light (Adam et al., 1996; Canton & Quail, 1999). Among the different plant tissues studied, PtPHYB2 was expressed predominantly in the leaf, shoot apex, xylem and female flower. On the other hand, PtPHYB1 was expressed in all tissue types except the root and phloem, with the highest expression in female flowers. In Arabidopsis, PHYB, PHYD and PHYE regulate flowering in response to light quality (Halliday et al., 1994; Aukerman et al., 1997; Devlin et al., 1998). According to current genome annotation (JGI version 2.2), the Populus genome does not encode PHYD and PHYE members; however, expression of both Populus PHYB1 and PHYB2 is evident in flowers and the shoot apex, suggesting a role in regulating flower development. Although AtPHYA accounts for > 80% of all PHYs in etiolated Arabidopsis seedlings, the amount of AtPHYA drops considerably and becomes significantly less abundant than AtPHYB in photosynthetically active Arabidopsis tissue (Sharrock & Clack, 2002). This change in relative abundance of AtPHYA and AtPHYB has important developmental consequences, particularly for SAS (Franklin & Quail, 2010). Our data support the notion that a similar regulatory mechanism exists in Populus.

Similar to Arabidopsis, tomato and tobacco PHYBs, both Populus PHYBs were localized to the nucleus under constant WL, suggesting a direct role in gene regulation. Consistent with the previous findings that PHYA is the light-labile PHY (Shanklin et al., 1987) we did not see any GFP fluorescence for the WL-incubated protoplast transfected with GFP-PHYA. It has been proposed that light-induced degradation of PHYA constitutes an important mechanism enabling de-etiolated plants to compete effectively for light in shaded environments (Mathews, 2006; Franklin & Quail, 2010).

The presence of duplicated PHYB in Populus raises questions about their individual roles in plant growth and development as well as in mediating SAS. In Arabidopsis, overexpression of PHYB results in attenuation of SAS responses such as hypocotyl elongation (Wagner et al., 1991). By contrast, the phyB mutant displays a constitutive SAS phenotype. When either PdPHYB1or PdPHYB2 was expressed in the Arabidopsis phyB mutant, only PdPHYB1 complemented the mutant phenotype. Further, transgenic Populus lines overexpressing PdPHYB1 were less sensitive to WL + FR-induced stem and leaf petiole elongation. This raises questions about the biological role of PHYB2. However, our PdPHYB2-overexpressing Populus lines showed reduced sensitivity to WL + FR-induced petiole elongation. One possibility is that, as in tomato and tobacco, Populus PHYB1 and PHYB2 have distinct functions during seedling development but function more or less redundantly in the mature plant (Sheehan et al., 2007). However, lack of complementation of the Arabidopsis phyB by PdPHYB2 raises doubts about its direct role in modulating elongation growth in response to WL + FR. Interestingly, a quantitative trait locus analysis in Populus suggests a possible role for PHYB2 in regulating bud set and dormancy (Frewen et al., 2000; Ingvarsson et al., 2008). Based on our phenotypic analysis we speculate that PHYB2 may have a minor role in SAS.

In addition to the observed phenotypes, PdPHYB2 and PdPHYB1 exhibited similar regulation of some shade-responsive genes, including PdPRTSP, PdNPH1 and PdHFR1. Furthermore, PdATHB2 expression was suppressed only in the PHYB2OE line but not in PHYB1OE after WL + FR light treatment, whereas others SAS genes, such as NPH3, were induced within PHYB2OE. These data suggest an overlap for PHYB1 and PHYB2 in mediating SAS in Populus, although a gene knockdown strategy is needed to substantiate this suggestion. One possibility is that PHYB1 acts as a primary player in SAS, whereas PHYB2 fine-tunes the initial responses to adjust rapidly to changing light conditions. The contrasting responses of PHYB1B2OE to stem and petiole elongation support this notion. However, it is impossible to unambiguously assign an individual function to PHYB1 and PHYB2 before single, double and triple (including PHYA) knockout lines have been characterized. Still, it is evident that both PHYB1 and PHYB2 may play a role in SAS in Populus. It is also possible that PHYB2 regulates other distinct responses such as bud flushing in Populus and that this pathway diverges from the SAS signaling downstream for the candidate genes studied.

The difference in PHYB1 and PHYB2 functionality can also be attributed to differences in amino acid sequence. PtPHYB1 and PtPHYB2 are 92% identical at the amino acid level. Although all the residues in a functional PHYB reported elsewhere (Bae & Choi, 2008) are conserved in both PtPHYB1 and PtPHYB2 (Fig. S4), other key substitutions might have functional significance for Populus PHYBs. Specifically, PtPHYB2 has two single amino acid changes at V980I and S1161T (Fig. S6). The light response between diverse Arabidopsis ecotypes have previously been attributed to a single amino acid change in AtPHYB (Filiault et al., 2008), including one of the two amino acid changes observed in PtPHYB2. In Arabidopsis, PHYB forms heterodimers with other type II PHYs (Sharrock & Clack, 2004). It has been proposed that these heterodimers mediate responses differently from the homodimers (Sheehan et al., 2004). Although the Populus genome does not encode any type II PHYs, it is possible that PHYB1 and PHYB2 may form heterodimers, which in turn may lead to different regulatory responses. The phenotypic responses of PHYB1B2OE support this possibility.

In Arabidopsis SAS is characterized by a balance between transcriptional regulation of both positive and negative regulators (Sessa et al., 2005). It has been proposed that the rapid change in gene expression of both the positive and negative regulators ensures a rapid reprogramming of the plant towards light conditions optimal for growth (Ruberti et al., 2011). Our RNA-Seq analysis identified 357 transcripts as up-regulated and 155 as down-regulated by low R/FR treatment, including positive and negative regulators of FR light signaling (Tables S5, S6). Interestingly, the bioinformatics enrichment analysis for functional categories of these genes suggests that genes associated with cell wall biosynthesis and remodeling were up-regulated under both conditions. Rapid shoot elongation during SAS is primarily accomplished by cellular expansion; this in turn is facilitated by loosening of the cell wall by the action of several cell wall-modifying enzymes, such as expansins and hydrolases, and synthesis of cell wall precursors such as xyloglucan and hemicellulose (Sasidharan et al., 2010).

It has been proposed that the elongation during shading in Arabidopsis is realized by cross-talk among auxin, GA, ethylene and brassinosteroid signaling (Vandenbussche et al., 2005). To this end we observed an up-regulation of homologs of several of these hormone biosynthesis and signaling genes, including IAA16, PIN3 and AFB3 (auxin-associated), RGL2 (GA-associated) and ETR2 and EIN2 (ethylene-associated) within 1 h of WL + FR light treatment (Table S2). However, we did not detect a significant trend in the up-regulation of these pathways using PageMan analysis. The lack of significant pathway up-regulation can be, in part, a result of the initial annotation of the Populus genome, as the PageMan analysis relies heavily on defined annotation to classify functional responses. However, we did see a rapid up-regulation of brassinosteroid biosynthesis and signaling using the PageMan enrichment analysis (Fig. 4). This was further confirmed by qRT-PCR for BRI1, which encodes for a brassinosteroid receptor. The PageMan analysis also indicated a severe down-regulation of jasmonate biosynthesis and signaling between 1 and 4 h of WL + FR light treatment. In addition to modulating plant growth, in Arabidopsis, SAS also affects plant defense responses in part by reducing the sensitivity to jasmonate (Moreno et al., 2009). Thus, the observed down-regulation of jasmonate in Populus appears to be an auxiliary SAS response. It should be noted that our gene expression assays were performed on leaves to ease comparison to the Arabidopsis literature and cannot be used as a direct proxy for pathway analysis within elongating stems.

The molecular analysis of shade response signaling in woody perennial species is still in its infancy. Here we have identified the transcriptional responses to WL-enriched FR light in Populus. We have also tried to designate a function to individual PHY genes from Populus. Our results have provided a list of select candidate SAS-related genes for future study and genetic manipulation of Populus. Future experiments using transgenic and mutant PHY Populus lines will shed light on the role of individual PHYs in SAS.


This research was sponsored by the Genomic Science Program, US Department of Energy, Office of Science, Biological and Environmental Research, as part of the Plant-Microbe Interfaces Scientific Focus Area ( Oak Ridge National Laboratory is managed by UT-Battelle LLC, for the US Department of Energy under contract DE-AC05-00OR22725.