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- Materials and Methods
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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.
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- Materials and Methods
- Supporting Information
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.