Characterization of Maize Phytochrome-Interacting …Characterization of Maize Phytochrome-Interacting Factors in Light Signaling and Photomorphogenesis1 Guangxia Wu,a Yongping Zhao,a

Characterization of Maize Phytochrome-InteractingFactors in Light Signaling and Photomorphogenesis1

Guangxia Wu,a Yongping Zhao,a Rongxin Shen,b Baobao Wang,a Yurong Xie,a Xiaojing Ma,a

Zhigang Zheng,b and Haiyang Wangb,2,3

aBiotechnology Research Institute, Chinese Academy of Agricultural Sciences, Beijing 100081, ChinabState Key Laboratory for Conservation and Utilization of Subtropical Agro-bioresources, South ChinaAgricultural University, Guangzhou 510642, China

ORCID IDs: 0000-0001-5615-7910 (B.W.); 0000-0001-6476-2467 (Y.X.); 0000-0001-6903-7512 (X.M.); 0000-0002-1302-5747 (H.W.).

Increasing planting density has been an effective means of increasing maize (Zea mays ssp. mays) yield per unit of land area overthe past few decades. However, high-density planting will cause a reduction in the ratio of red to far-red incident light, whichcould trigger the shade avoidance syndrome and reduce yield. The molecular mechanisms regulating the shade avoidancesyndrome are well established in Arabidopsis (Arabidopsis thaliana) but poorly understood in maize. Here, we conducted aninitial functional characterization of the maize Phytochrome-Interacting Factor (PIF) gene family in regulating light signaling andphotomorphogenesis. The maize genome contains seven distinct PIF genes, which could be grouped into three subfamilies:ZmPIF3s, ZmPIF4s, and ZmPIF5s. Similar to the Arabidopsis PIFs, all ZmPIF proteins are exclusively localized to the nucleus andmost of them can form nuclear bodies upon light irradiation. We show that all of the ZmPIF proteins could interact withZmphyB. Heterologous expression of each ZmPIF member could partially or fully rescue the phenotype of the Arabidopsis pifqmutant, and some of these proteins conferred enhanced shade avoidance syndrome in Arabidopsis. Interestingly, all ZmPIFproteins expressed in Arabidopsis are much more stable than their Arabidopsis counterparts upon exposure to red light.Moreover, the Zmpif3, Zmpif4, and Zmpif5 knockout mutants generated via CRISPR/Cas9 technology all showed severelysuppressed mesocotyl elongation in dark-grown seedlings and were less responsive to simulated shade treatment. Takentogether, our results reveal both conserved and distinct molecular properties of ZmPIFs in regulating light signaling andphotomorphogenesis in maize.

Maize (Zea mays ssp. mays) has become the highestproducing crop globally (FAO statistics, http://www.fao.org/faostat). Average maize yields in the UnitedStates increased over sevenfold from the 1930s to 2010(Mansfield and Mumm, 2014). This increase can belargely attributed to the synergistic improvement ofgenetic gain and management practices (Tollenaarand Lee, 2002). In particular, increasing plantingdensity has been a key factor for the increased maizeyields in the central Corn Belt in the United States(from 30,000 plants ha21 in the 1930s to approximately

70,000 plants ha21 in 2010; Mansfield and Mumm,2014). A series of studies by Duvick and others haveshown that newer maize hybrids are better adaptedto high-density planting than older ones, mainly dueto a series of morphological changes associated withgrain production efficiency, including reduced leafangle and tassel branch number, and increased tol-erance to various biotic and abiotic stresses associ-ated with high-density planting, such as drought,shading, pathogens, and insects (Duvick, 2005a,2005b; Lauer et al., 2012; Mansfield and Mumm,2014). Optimal plant architecture, such as reducedplant and ear height and increased culm strength,could contribute to lodging resistance; elevated leafangle and smaller tassel sizes allow plants to inter-cept sunlight more efficiently at high planting den-sities; the reduced anthesis-silking interval couldhelp to synchronize male and female flowering, thusimproving kernel setting (Duvick, 2005a, 2005b;Lauer et al., 2012; Mansfield and Mumm, 2014).Numerous studies have established that phyto-

chromes (phys), the major photoreceptors of red andfar-red light signals in plants, play critical roles in reg-ulating plant growth and development throughouttheir life cycle, from seed germination, seedling dee-tiolation, and vegetative growth to flowering and seedsetting (Franklin and Quail, 2010). Especially, a role ofphys in conferring the developmental plasticity in

1This work was supported by the National Natural Science Foun-dation of China (31601319), the Beijing Natural Science Foundation(6174050), and the China Postdoctoral Science Foundation(2015M581211).

2Author for contact: [email protected] author.The author responsible for distribution of materials integral to the

findings presented in this article in accordance with the policy de-scribed in the Instructions for Authors (www.plantphysiol.org) is:Haiyang Wang ([email protected]).

H.W. conceived the original research plans and supervised theexperiments; G.W. performedmost of the experiments; Y.Z. providedtechnical assistance to G.W.; R.S., B.W., Y.X., X.M., and Z.Z. per-formed some of the experiments; G.W. and H.W. analyzed the dataand wrote the article with contributions of all the authors.

www.plantphysiol.org/cgi/doi/10.1104/pp.19.00239

Plant Physiology�, October 2019, Vol. 181, pp. 789–803, www.plantphysiol.org � 2019 American Society of Plant Biologists. All Rights Reserved. 789

https://plantphysiol.orgDownloaded on November 4, 2020. - Published by Copyright (c) 2020 American Society of Plant Biologists. All rights reserved.

http://orcid.org/0000-0001-5615-7910

http://orcid.org/0000-0001-5615-7910

http://orcid.org/0000-0001-6476-2467

http://orcid.org/0000-0001-6476-2467

http://orcid.org/0000-0001-6903-7512

http://orcid.org/0000-0001-6903-7512

http://orcid.org/0000-0002-1302-5747

http://orcid.org/0000-0002-1302-5747

http://orcid.org/0000-0001-5615-7910

http://orcid.org/0000-0001-6476-2467

http://orcid.org/0000-0001-6903-7512

http://orcid.org/0000-0002-1302-5747

http://www.fao.org/faostat

http://www.fao.org/faostat

http://crossmark.crossref.org/dialog/?doi=10.1104/pp.19.00239&domain=pdf&date_stamp=2019-09-24

mailto:[email protected]

http://www.plantphysiol.org

mailto:[email protected]

http://www.plantphysiol.org/cgi/doi/10.1104/pp.19.00239

https://plantphysiol.org

response to the changing light environments hasbeen well documented in a number of plant species(Gururani et al., 2015). For example, in response toanticipation of shading (reduced red to far-red lightratios [R:FR]) or actual shading (reduced R:FR plusreduced photoactive irradiation) at high plantingdensities, plants trigger a series of adaptive responses,including the promotion of hypocotyl (or stem) andpetiole elongation, reduced branching, reorientation ofthe growth direction of leaf or branch, and early flow-ering, collectively known as the shade avoidance syn-drome (SAS; Franklin, 2008). Despite the fact that theseresponses are believed to increase the chance of indi-vidual success, they are detrimental to yield productionof crops at high planting densities. Thus, understandingthe molecular mechanism governing SAS in plants,particularly in crops, will be essential to guide thebreeding of shade-tolerant cultivars suitable for high-density planting.

A consensus pathway of phy regulating SAS hasbeen recently established in the model dicot plant spe-cies Arabidopsis (Arabidopsis thaliana). Arabidopsis hasfive phys, phyA to phyE. phyA is the major photore-ceptor for perceiving far-red light, whereas phyB tophyE are themajor photoreceptors for sensing red light,with phyB playing a dominant role (Franklin andQuail,2010). Phys are synthesized in cytoplasm in the inactivered light-absorbing (Pr) form, which are changed to theactive far-red light-absorbing (Pfr) form by red lightirradiation. The activated phys are translocated to thenucleus to form nuclear bodies (Van Buskirk et al., 2012;Klose et al., 2015). In the nucleus, phys interact directlywith a set of basic helix-loop-helix (bHLH) transcrip-tion factors called PHYTOCHROME INTERACTINGFACTORs (PIFs). In Arabidopsis, the AtphyB singlemutant displays various traits reminiscent of a consti-tutive shade avoidance response, such as elongatedhypocotyls and petioles, acceleration of flowering, andhigher apical dominance under high R:FR, indicatingthat it is the major photoreceptor for canopy shade.Recent studies showed that under high R:FR condi-tions, active phyB can promote the degradation of PIFproteins (PIF3, PIF4, PIF5, and probably PIF1) orphosphorylation of PIF7, whereas low R:FR signalsreduce the levels of active phyB, thus promoting sta-bilization of PIF proteins (PIF3, PIF4, and PIF5) or de-phosphorylation of PIF7, which in turn stimulateschanges in the downstream transcriptional network(such as up-regulation of auxin biosynthetic genes) andinduces growth responses to shade (Lorrain et al., 2008;Leivar et al., 2012;Mansfield andMumm, 2014;Mizunoet al., 2015; Xie et al., 2017).

There is a paucity of evidence to support a role ofphys in regulating SAS in maize. The maize genomecontains six phy genes: PHYA1, PHYA2, PHYB1,PHYB2, PHYC1, and PHYC2 (Sheehan et al., 2004).Similar to the phyB mutant in Arabidopsis, the maizephyB1 phyB2 double mutant also displays constitutiveshade avoidance responses, such as increased plantheight, elongated internodes, and tendency to lodge

(Kebrom et al., 2006, 2010; Sheehan et al., 2007). Simi-larly, the maize elongated mesocotyl1 mutant, whichcarries a lesion in the ZmHY2 gene encoding phyto-chromobilin synthase, also shows pronounced elonga-tion of the mesocotyl and fails to deetiolate under redor far-red light conditions (Sawers et al., 2002). Recentstudies also showed that overexpression of ZmPhyA1causes increased plant and ear height in maize (Yuet al., 2018) and that phy-mediated signaling is in-volved in the suppression of axillary bud outgrowth inresponse to canopy shade in maize, as in Arabidopsisand rice (Oryza sativa; Kebrom et al., 2006, 2010;Whipple et al., 2011).

Given the central roles of PIF proteins in integratingmultiple signaling pathways (light, temperature, hor-mones, biotic and abiotic stresses, etc.), thus optimiz-ing plant growth and development tailored to itsenvironments (Paik et al., 2017), there is strong interestto exploit them for biotechnological applications toproduce improved crops that can be better adapted toadverse environmental conditions. A number of pre-vious studies have attempted to identify the maize PIFgenes and conducted functional analyses of some ofthe members (Kumar et al., 2016; Shi et al., 2018; Gaoet al., 2019). For example, Gao et al. (2019) identifiedsix maize PIF genes (named ZmPIF1–ZmPIF6) andshowed that ZmPIF1 and ZmPIF3 might regulate re-sponse to salt or drought stresses in rice, while Shiet al. (2018) showed that overexpression of ZmPIF4confers a constitutive shade avoidance response inArabidopsis. Despite the progress made, systematiccharacterization of the ZmPIF gene family in regulat-ing phy-mediated light signaling and SAS remainsforthcoming.

Here, we cloned seven potential ZmPIF genes andsystematically analyzed their molecular properties(expression patterns, protein localization and stability,and interaction with phys). We show that all sevenZmPIFs possess conserved function with their Arabi-dopsis counterparts in rescuing the phenotypes of theArabidopsis pifq mutant. In addition, we performed aninitial characterization of these ZmPIFmembers in lightsignaling and photomorphogenesis in maize. Our re-sults establish a foundation for future dissection ofZmPIFs in regulating plant architecture and SAS inmaize and might provide potential targets for geneticimprovement of maize for adapting to high-densityplanting.

RESULTS

Cloning and Phylogenetic Analysis of the ZmPIF Genes

In order to identify all genes potentially encoding PIFproteins in maize, we first performed BLAST analysisusing the bHLH domain of Arabidopsis PIF3 (AtPIF3)in the data set (Gramene gene model set 5b1 forRefgen_v3-translations) in MaizeGDB (https://www.maizegdb.org/). A total of 200 hits with an E-value

790 Plant Physiol. Vol. 181, 2019

Wu et al.


https://www.maizegdb.org/

https://www.maizegdb.org/


cutoff of 1e-4 were kept, and self-BLAST was per-formed on the resulting sequence list to eliminate all theredundant sequences. In total, 123 proteins were kept,and the corresponding sequences were downloaded.Each sequence was manually checked to determine thepresence of the complete bHLH domain and the con-served active phyB-binding (APB) domain. Finally,seven potential PIF homologs were identified in themaize B73 genome (Supplemental Table S1), andthe DNA fragments were amplified and cloned into thepGADT7 vector (Clontech) for sequencing. We suc-cessfully cloned the full-length cDNAs for all sevenputative ZmPIF genes via reverse transcription (RT)PCR using a lab-owned B73 inbred line, indicatingthat these genes are all actively transcribed. Sequence

analysis showed that the cloned cDNA sequences offiveZmPIFswere 100% identical to the predicted cDNAsequences deposited in the AGP B73v3 and/or AGPB73v4 version. However, the sequences of the clonedcDNAs of GRMZM2G165042 and GRMZM2G016756differed from the predicted cDNA sequences depositedin the AGP B73v3 and/or AGP B73v4 database. Toexamine whether the differences might be caused byalternative splicing, as previously reported for theArabidopsis PIF6 gene (Penfield et al., 2010), we ran-domly picked 20 cDNA clones for sequencing analysis.Indeed, we found that both GRMZM2G165042 andGRMZM2G016756 had two alternative splicing vari-ants (named isoform a and isoform b, respectively), butno clone matching the predicted cDNA sequences was

Figure 1. Characterization of the ZmPIFgene family. A, Phylogenetic analysisandmotif comparisons of the ZmPIFandAtPIF proteins. The phylogenetic treewas constructed based on their full-length amino acid sequences using themaximum likelihood method (left). Thepresence of active phyA-binding (APA),APB, and bHLH motifs is depicted asboxes and shown on the right. Bar 5100 amino acid residues. B, Expressionanalysis of ZmPIFs in various tissues ofmaize seedlings. Different tissues ofthree-leaf stage seedlings of the maizeinbred line B73were harvested and thenused to perform RT-quantitative PCR(qPCR) analysis. The mRNA level ofmaize Tubulin5was used as a reference.Data are means and SD of three inde-pendent biological replicates. Asterisksindicate significant differences com-pared with leaf tissue using Student’st test (**, P , 0.001). C, Coleoptile; L,leaf; M, mesocotyl; R, root.

Plant Physiol. Vol. 181, 2019 791

Phytochrome-Interacting Factors in Maize


http://www.plantphysiol.org/cgi/content/full/pp.19.00239/DC1


found (Supplemental Fig. S1). This result suggeststhat the predicted cDNA for GRMZM2G165042 andGRMZM2G016756 might be inaccurate.

Phylogenetic analysis revealed that these sevenZmPIFs could be grouped into three clades and wererenamed according to their respective clades (Fig. 1A,left). GRMZM2G062541 was named ZmPIF3.3, as itwas grouped into the same clade with the previousreported proteins ZmPIF3.1 (GRMZM2G115960) andZmPIF3.2 (GRMZM2G387528), and these three pro-teins share homology with AtPIF3 (Fig. 1A). Thepreviously reported ZmPIF4 (GRMZM5G865967)and ZmPIF5 (GRMZM2G165042; Shi et al., 2018)were renamed ZmPIF4.1 and ZmPIF4.2, respectively,as they were clustered together in the phylogenetictree (Fig. 1A), and GRMZM5G865967 has higher se-quence similarity to AtPIF4 (88% identity) than toAtPIF5 (41% identity). Similarly, GRMZM2G065374and GRMZM2G016756 were named ZmPIF5.1 andZmPIF5.2, respectively, as they were grouped intothe same clade and GRMZM2G016756 has highersequence similarity to AtPIF5 (61% identity) than toAtPIF4 (47% identity; Fig. 1A). In addition, analysisof ZmPIF protein sequence motifs using MEME(Bailey et al., 2009) showed that all seven ZmPIFproteins have a highly conserved bHLH domainand APBmotif, while ZmPIF3s (ZmPIF3.1, ZmPIF3.2,and ZmPIF3.3) also have an active phyA-bindingmotif at their N termini, similar to AtPIF1 andAtPIF3 (Fig. 1A, right).

Expression Profiles of ZmPIF Genes and ProteinSubcellular Localization

In order to better understand the function of eachZmPIF gene, their differential tissue expression profileswere investigated in 10-d-old maize seedlings. Usingtranscriptomic data from maize B73 (Sekhon et al.,2011), an expression heat map was constructed for allseven ZmPIFs in different tissues from various devel-opmental stages (Supplemental Fig. S2). Our RT-qPCRassay showed that the expression patterns of theseseven ZmPIF genes were highly similar, all with a rel-atively high expression level in leaf and lower expres-sion levels in root, mesocotyl, and coleoptile (Fig. 1B),which was in line with the transcriptomic data of maizeB73 (Supplemental Fig. S2).

To examine whether the transcript levels of ZmPIFsare regulated by light, 7-d-old dark-grown maizeB73 seedlings were transferred to white light (WL) forvarious times or retained in darkness as controls.Notably, the expression levels of all ZmPIF genes fluc-tuated during the time course examined, but in gen-eral, ZmPIF3.1, ZmPIF3.2, and ZmPIF3.3 showed asimilar profile, being rapidly down-regulated bylight treatment (Supplemental Fig. S3, top). ZmPIF4sand ZmPIF5s shared a similar profile, being up-regulated by light following prolonged light exposure(2–3 h; Supplemental Fig. S3, middle and bottom).

These results suggest that the homologous genes in thesame clade may more likely perform similar functions.

In order to detect the localization of these ZmPIFproteins, we expressed GFP fusion of each ZmPIFprotein in Nicotiana benthamiana leaf cells. Resemblingthe typical localization of AtPIFs, all seven ZmPIFs lo-calized to the nucleus, and four of them (ZmPIF3.1,ZmPIF3.2, ZmPIF3.3, and ZmPIF4.2) formed obviousnuclear bodies, whereas ZmPIF4.1, ZmPIF5.1, andZmPIF5.2 showed uniform nuclear localizationwithoutdistinct nuclear bodies in N. benthamiana leaf cells(Fig. 2).

Figure 2. ZmPIF proteins localize to the nucleus. The expression con-structs Pro-35S:ZmPIFs-GFP were individually cotransformed with anuclear protein marker construct (Pro35S:mRFP-AHL22; Xiao et al.,2009) into N. benthamiana leaves. The N. benthamiana leaveswere incubated for 48 h in the dark after transformation and thentransferred to WL for 3 to 6 h prior to imaging using a confocal mi-croscope. Bars 5 20 mm.


Wu et al.








ZmPIF Proteins Physically Interact with ZmphyB

Since all seven ZmPIF proteins possess a putativeAPB motif, which is necessary for interacting withphyB, we performed a yeast two-hybrid (Y2H) assay totest this possibility. Surprisingly, we found that ZmPIFsinteracted with ZmphyB1 under all conditions exam-ined (darkness or light treatment, presence or absenceof phycocyanobilin [PCB]; Fig. 3; Supplemental Fig. S4).A similar interactionwas observed betweenAtPIF3 andAtphyB (Fig. 3; Supplemental Fig. S4). However, novisible interactions were observed between ZmPIFproteins and full-length ZmphyB2 in yeast under thesame conditions (Fig. 3). Western-blot analysis showedthat the ZmphyB2 protein is normally expressed inyeast (Supplemental Fig. S5), suggesting that ZmphyB1and ZmphyB2 may have distinct affinities for interact-ing with ZmPIFs.We next performed luciferase complementation

imaging (LCI) and bimolecular fluorescence comple-mentation (BiFC) assays to test the interaction betweenZmPIFs and ZmphyBs. The LCI assay showed that allcombinations of ZmPIFs with either ZmphyB1 orZmphyB2 could reconstitute the functional LUC

activity in plant cells (Fig. 4, A and B), but the negativecontrols could not reconstitute the LUC activity in plantcells (Supplemental Fig. S6). The interactions betweenZmPIFs with ZmphyB1 and ZmphyB2 were also con-firmed using BiFC assay in the nucleus of N. ben-thamiana leaf cells (Fig. 4, C and D; Supplemental Fig.S7). Additionally, Y2H and LCI assays showed that allmaize PIF proteins could also interact with AtphyB(Supplemental Fig. S8), indicating that the interactionbetween phyB and PIF is conserved betweenmaize andArabidopsis.

ZmPIFs Complement the Arabidopsis pifqMutant Phenotype

To further examine whether ZmPIFs could function-ally complement the Arabidopsis pifqmutant phenotype,we overexpressed each ZmPIF member (ZmPIF-OE) inthe pifq mutant background. The transcript levelsof ZmPIFs in the transgenic lines were determinedusing RT-qPCR to verify successful transformation(Supplemental Fig. S9). Since the Arabidopsis pifq

Figure 3. ZmPIF-ZmphyB interaction analyzed by Y2H assay. The Y2H assay shows that ZmPIFs interact with ZmphyB1, but notwith ZmphyB2, in yeast. Full-length ZmphyB1 and ZmphyB2 were fused with the DNA-binding domain (BD) as the baits. Eachfull-length ZmPIFmemberwas fusedwith the activation domain (AD) as the prey. The interaction betweenAtPIF3 and AtphyBwasused as a positive control. Empty vectors were used as negative controls. Yeast cells (AH109) coexpressing the indicated com-binations of constructs were grown on nonselective (synthetic dextrose [SD]-T-W) or selective (SD-T-W-H) mediumwith 1 mM 3-aminotriazole (3AT) in the presence (1PCB) or absence (2PCB) of 25 mM phycocyanobilin (PCB) under continuous red light (Rc[R]; 4 mmol photons m22 s21) or far-red light (FR; 3 mmol photons m22 s21) or in darkness (D) for 3 d.













mutant showed a constitutive photomorphogenic-like phenotype with shortened hypocotyls and openapical hooks in darkness (Leivar et al., 2008b), theZmPIF-OE transgenic lines were checked for hypocotyllength in dark-grown seedlings. The phenotypic anal-ysis showed that most of the dark-grown ZmPIF-OE

seedlings exhibited significantly elongated hypocotylsand partially closed apical hooks compared with pifq,and this effect was most robust in the ZmPIF4.1-OElines (Fig. 5, A–F). Interestingly, dark-grown ZmPIF5-OE transgenic seedlings exhibited an exaggerated api-cal hook phenotype, reminiscent of the Arabidopsis

Figure 4. ZmPIFs interact with ZmphyB1and ZmphyB2 in plant cells. A and B, LCIassay showing the ZmPIFs-ZmphyB in-teraction in N. benthamiana leaf cells.EachZmPIFmemberwas fused to cLUC.The full-length ZmphyB1 or ZmphyB2was fused to nLUC. TheN. benthamianaleaves were infiltrated with the indi-cated combinations, incubated for 48 hin the dark, and then transferred to WLfor 3 to 6 h prior to photographing us-ing an in vivo imaging system. Emptyvectors were used as negative controls(Supplemental Fig. S6). C and D, BiFCassay showing the ZmPIFs-ZmphyB in-teraction in N. benthamiana leaf cells.Full-length ZmPIF and ZmphyB proteinswere fused to the split N-terminal (nYFP[yellow fluorescent protein]) or C-terminal(cYFP) fragments of YFP, respectively. TheN. benthamiana leaves infiltrated with thecombinations were adapted in darknessfor 48 h and then exposed to light for 3 to6 h before examination with a confocalmicroscope. Red fluorescence protein(RFP) served as the internal control.The empty vectors were used as neg-ative controls (Supplemental Fig. S7).Bars 5 20 mm.


Wu et al.





PIF5-overexpressing lines (Fig. 5C). We therefore in-vestigated whether ZmPIF5 is involved in the ethylenesignaling pathway regulating hook opening, as is thecase for AtPIF5 (Khanna et al., 2007). As expected,treatment with ACC aggravated the triple responsephenotype of the ZmPIF5-OE seedlings (SupplementalFig. S10, middle). By contrast, treatment with AgNO3abolished the triple response phenotype observed forthe ZmPIF5-OE seedlings (Supplemental Fig. S10, bot-tom). These observations provide additional support forthe functional conservation betweenAtPIF5 and ZmPIF5.To further confirm the regulation of ZmPIFs on hy-

pocotyl elongation-related genes, we measured thetranscript levels of XTH15 and IAA19, two repre-sentative genes involved in dark-induced hypocotylelongation (Zhang et al., 2013a). Consistent with the

morphological phenotype, the expression levels ofboth XTH15 and IAA19 were dramatically elevated inthe ZmPIFs overexpression lines (Fig. 5, G and H).Taken together, these data clearly demonstrate thatthese ZmPIFs possess a conserved function with AtPIFsin repressing photomorphogenesis.

ZmPIF Proteins Are Slowly Degraded in ArabidopsisSeedlings in Response to Red Light

In Arabidopsis, light-activated phys accumulate inthe nucleus, where they directly interact with PIFs,leading to light-induced phosphorylation and degra-dation of PIF proteins (PIF3, PIF4, PIF5, and probablyPIF1 as well; Ni et al., 2013). Therefore, we examined

Figure 5. ZmPIFs rescue the phenotypeof Arabidopsis pifq mutant seedlings. Ato C, The phenotype of dark-grown Ara-bidopsis pifq mutant seedlings is partiallycomplemented by ZmPIF overexpression.Columbia-0 (Col-0) and pifq mutant aswell as pifq mutant seedlings express-ing Pro-35S:ZmPIFs-GFP were grownin darkness for 4 d. Bars 5 1 mm. D toF, Quantification of hypocotyl length ofthe seedlings shown in A to C. Datarepresent means from at least 20 plants,and the error bars are SD. Asterisks in-dicate significant differences comparedwith pifq mutant plants using Student’st test (**, P , 0.001). G and H, ZmPIFoverexpression restores the expressionof PIF-dependent genes in the Arabi-dopsis pifq mutants. The expression ofmarker genes including XTH15 andIAA19was analyzed by RT-qPCR in 4-ddark-grown seedlings of Col-0, pifq,and pifq expressing Pro-35S:ZmPIFs-GFP. ACT2 was used as the internalcontrol. Data represent means and SD

of three independent biological rep-licates. Asterisks indicate significantdifferences compared with pifq plantsusing Student’s t test (**, P , 0.001).








whether these ZmPIF proteins have similar degrada-tion kinetics to the AtPIFs in etiolated Arabidopsisseedlings irradiated with Rc. As shown in Figure 6A, allZmPIFs:GFP proteins accumulated in the nucleus anddid not form nuclear bodies before Rc treatment. In-terestingly, fluorescent nuclear bodies were detectedfor ZmPIF3.1:GFP, ZmPIF3.2:GFP, ZmPIF3.3:GFP, andZmPIF4.2:GFP fusion proteins after a short high Rcexposure (R 1min) and remained visible even after alow Rc exposure for 1 h (R 1h), a behavior reminiscent of

that of AtPIF7 (Leivar et al., 2008a; Fig. 6A). However,the ZmPIF4.1:GFP, ZmPIF5.1:GFP, and ZmPIF5.2:GFPfusion proteins did not form nuclear bodies upon Rctreatment and appearedmuchmore stable and similar totheir accumulation in the nucleus in the dark (Fig. 6A).Consistent with this, western-blot analysis showed thatdegradation of ZmPIFswasmuch slower (up to 6 or 24 hin the light; Fig. 6B) than degradation of the ArabidopsisPIF proteins. These results suggest that these ZmPIFproteins exhibit distinct properties in light-induced nu-clear body formation and are more stable than the Ara-bidopsis PIF proteins in response to red light.

Knockout of ZmPIFs by CRISPR/Cas9 Causes ShortMesocotyls in Maize under Darkness

To investigate the in vivo function of these ZmPIFgenes in regulating maize growth and development,we knocked out the endogenous ZmPIF genes in themaize wild-type inbred line ZC01 using ClusteredRegularly Interspaced Short Palindromic Repeats/Cas9 (CRISPR/Cas9) technology. To overcome func-tional redundancy, homologous ZmPIF genes of thesame subgroup were constructed in the same CRISPR/Cas9 vector system for multiple target site cleavage(Supplemental Fig. S11). We successfully obtainedZmpif3.1/Zmpif3.2/Zmpif3.3 triple knockout, Zmpif4.1/Zmpif4.2 double knockout, and Zmpif5.1 singleknockout mutants. Results of sequencing analysis ofthese ZmPIF knockout mutant lines are shown inSupplemental Figure S12. All the homozygous ZmPIFsmaize mutants exhibit shorter mesocotyls than the wildtype in etiolatedmaize seedlings (Fig. 7, A and B), whichwere similar to the short-hypocotyl phenotype of theArabidopsis pifmutant. Remarkably, the mesocotyl washardly detectable in the etiolated seedlings of Zmpif3striple knockout mutants (Fig. 7A). Tissue section analy-sis showed that the mesocotyl cells of the Zmpifs lineswere significantly shorter than those of wild-type plantsunder dark conditions (Fig. 7, C and D). Several cellelongation-related genes were also detected to be down-regulated in theZmpifs lines as comparedwithwild-typeplants (Fig. 7E). These data suggest that ZmPIFs functionas repressors of photomorphogenesis and positivelyregulate mesocotyl growth in dark-grown maize seed-lings through promoting cell elongation.

ZmPIFs Act as Positive Regulators in Response to Shade

Light quality is a crucial environmental factor thatinfluences hypocotyl elongation. To investigate whetherZmPIFs are involved in regulating hypocotyl growthin Arabidopsis under low R:FR conditions, we ana-lyzed the hypocotyl responses of Col-0, pifq, andZmPIF-OE plants to simulated shade conditions. Hy-pocotyl measurements showed that the Arabidopsistransgenic plants overexpressing ZmPIF3.1, ZmPIF3.2,ZmPIF3.3, and ZmPIF4.2 appeared to be similar to pifq

Figure 6. Red light induces slow degradation of ZmPIF proteins inArabidopsis. A, Epifluorescence imaging of GFP fluorescence in hy-pocotyl cell nuclei of Arabidopsis transgenic seedlings expressingZmPIF-GFP as indicated. Seedlings were grown in the dark for 4 d andthen maintained in darkness (D), exposed to high Rc of 198 mmolphotons m22 s21 for 1 min (R 1min), or exposed to low Rc of 4 mmolphotons m22 s21 for 1 h (R 1h). Samples were fixed in 4% (v/v) para-formaldehyde and examined using a fluorescence microscope. Bar 510mm. B, Immunoblot analysis of ZmPIF protein stability in response tored light. Seedlings of transgenic lines expressing ZmPIFs-GFP fusionproteins were grown for 4 d in the dark (D) or exposed to Rc (4 mmolphotons m22 s21) for 1, 3, 6, or 24 h. ZmPIF fused to a GFP tag wasdetected in total protein extracts by immunoblot using anti-GFP anti-bodies. Protein extracts from pifq were included as a control. The de-tection of Actin using anti-Actin antibodies is shown as a loadingcontrol.


Wu et al.





under simulated shade (WL1FR); however, theZmPIF4.1-OE, ZmPIF5.1-OE, and ZmPIF5.2-OE plantshad significantly longer hypocotyls than pifq undershade (Fig. 8, A and B). These results suggest thatZmPIF4.1, ZmPIF5.1, and ZmPIF5.2may have strongereffects on promoting hypocotyl growth than ZmPIF3sand ZmPIF4.2 in response to shade in Arabidopsis. Inaddition, we noted that the ZmPIF4.1-OE transgeniclines displayed a constitutive shade avoidance re-sponse, resulting in early flowering and exaggeratedpetiole elongation compared with Col-0 and the pifqmutant under long-day conditions (Supplemental Fig.S13). This phenotype was reminiscent of the AtPIF4overexpression plants (Kumar et al., 2012).To further investigate the potential roles of ZmPIFs in

this process in maize, we conducted a similar simulatedshade treatment with the maize ZmPIFs knockout

mutants. As expected, the wild-type maize seedlingsexposed to such simulated shade conditions displayedlonger first leaf sheaths characteristic of the shadeavoidance response, but this response was attenuatedin the Zmpifs mutants (Fig. 8, C and D). Interestingly,the extent of this reduction in responsiveness was moreobvious in the Zmpif3s triple knockout mutant than inthe Zmpif4s double knockout mutant and the Zmpif5.1single knockout mutant (Fig. 8, C and D), indicatingthat ZmPIF3s are the dominant positive regulators ofearly shade response in maize.

DISCUSSION

Phys are the predominant photoreceptors that sensechanges in light quality in an ambient environment to

Figure 7. Knockoutmutants of ZmPIFs show inhibitedmesocotyl elongation in etiolated seedlings. A, Phenotypes of the etiolatedwild-type (WT) and Zmpif3s, Zmpif4s, and Zmpif5.1 knockout mutant seedlings grown for 7 d after germination in darkness. Redarrowheads indicate the coleoptilar nodes between the mesocotyl and coleoptile. The seedlings were digitally extracted forcomparison. Bar 5 2 cm. B, Mesocotyl lengths of the etiolated seedlings represented in A. The values are means 6 SD (n $ 10).Asterisks indicate significant differences comparedwith wild-type plants using Student’s t test (**, P, 0.001). C,Methylene Blue-stained longitudinal sections of etiolated wild-type and Zmpif3s, Zmpif4s, and Zmpif5.1 knockout mutant mesocotyls. Bars 550 mm. D, Cell lengths of the mesocotyl of the etiolated seedlings represented in C. The values are means6 SD (n$ 30). Asterisksindicate significant differences compared with wild-type plants using Student’s t test (**, P , 0.001). E, Expression analysis ofthree cell expansion-related genes (ZmEXPA3, ZmEXPB4, and ZmEXPB6) in the mesocotyls of wild-type and Zmpif3s, Zmpif4s,and Zmpif5.1mutant seedlings grown for 2 d after germination in darkness. The 2-d-oldwhole seedlings of Zmpif3smutantswereharvested for expression analysis instead of mesocotyls, as the mesocotyls of Zmpif3smutants were too short to be harvested. Thetranscript levels were first normalized to Tubulin5. Data are means and SD of three independent biological replicates. Asterisksindicate significant differences compared with wild-type plants using Student’s t test (**, P , 0.001).







regulate plant growth and development. Several Ara-bidopsis PIF proteins, including PIF3, PIF4, PIF5, andPIF7, have been shown to play a particularly prominentrole in shade avoidance by directly activating the ex-pression of targeting genes, including the auxin syn-thesis genes (TAA1 and YUC), a cell wall-associatedgene (XTH15), and transcription factors such as ATHB-2, PIF3-LIKE1 (PIL1), and LONGHYPOCOTYL IN FAR-RED1 (Zhang et al., 2013b). We recently showed that, inresponse to simulated shade, accumulation of AtPIFproteins increases and they directly repress the ex-pression of MIR156s, thus releasing the downstreamSPL gene family of transcription factors to regulatevarious aspects of shade avoidance responses (Xie et al.,2017). In this study, we conducted an initial character-ization of the molecular properties and functions ofZmPIFs in regulating light signaling and photomor-phogenesis in maize. As previously reported (Kumaret al., 2016), we identified seven potential ZmPIFs inmaize, which share the conserved bHLH domain andAPB motif. However, only members of the ZmPIF3ssubgroup (ZmPIF3.1, ZmPIF3.2, and ZmPIF3.3) alsohave a phyA-binding motif, similar to AtPIF1 andAtPIF3 (Fig. 1A, right), suggesting that ZmPIF3s mightbe closer to AtPIF1 and AtPIF3 on an evolutionaryscale. We found that all ZmPIF genes are constitutivelyexpressed in most tissues examined, with relativelyhigher expression in leaves, an expression pattern alsosimilar to that of AtPIFs (Jeong and Choi, 2013).

We collected several lines of evidence to show thatthese ZmPIF proteins share several similar molecularproperties with the Arabidopsis PIFs. First, we showedthat, like the AtPIFs, all ZmPIF proteins are capable ofphysically interacting with the full-length ZmphyB1and AtphyB in both yeast and plant cells (Figs. 3 and 4;Supplemental Fig. S8). Although no interaction wasdetected between ZmPIFs and the full-length ZmphyB2in yeast cells (Fig. 3), interaction between them wasdetected in plant cells using both LCI and BiFC assays(Fig. 4). Surprisingly, in contrast to the earlier reportshowing that the AtphyB (NT)-AtPIF3 interaction is redlight inducible, we detected interaction betweenZmPhyB1 and AtphyB (full length) with ZmPIFs underboth darkness and light conditions in our Y2H assay(Fig. 3; Supplemental Fig. S8A). We speculate that thismight be due to different lengths of phyB constructs(N-terminal fragment versus full length) or differentconfigurations of phyB fusions with the GAL4 DNA-binding domain (GBD) used in these assays (phyB-GBDversus GBD-phyB; Ni et al., 1999). Second, weshowed that all of these ZmPIFs are targeted to thenucleus like the Arabidopsis PIFs, and most of them(ZmPIF3.1, ZmPIF3.2, ZmPIF3.3, and ZmPIF4.2) areable to form nuclear bodies upon exposure to light(Figs. 2 and 6A). Third, we showed that all of theseZmPIFs can complement the mutant phenotypes ofthe pifq mutant to different degrees (Fig. 5) and thatsome of the ZmPIF overexpression transgenic plants(ZmPIF4.1-OE,ZmPIF5.1-OE, andZmPIF5.2-OE) displayan enhanced shade avoidance response phenotype in

Figure 8. ZmPIFs act as positive regulators in response to shade. A,Heterologous expression of ZmPIFs confers increased responses tosimulated shade in Arabidopsis seedlings. Visible phenotypes of Col-0,pifq mutant, and ZmPIFs-OE seedlings (in the pifq background) weregrown in constant WL (28 mmol photons m22 s21) for 2 d from germi-nation and then retained in WL (high R:FR 5 6.48) or transferred toWL1FR (low R:FR 5 0.145) for an additional 5 d. The seedlings weredigitally extracted for comparison. Bars 5 2 mm. B, Quantification ofhypocotyl lengths for Col-0, pifq mutant, and ZmPIFs-OE seedlings inthe pifq background grown as in A. Data represent mean values and SD

from at least 20 seedlings. Letters indicate significant differences bytwo-way ANOVA. C, Knockout of ZmPIFs by CRISPR/Cas9 reducessensitivity to the simulated shade treatment in maize seedlings. Seed-lings of the wild type (WT) and Zmpif mutant lines were grown for 3 dunder continuous WL (35 mmol photons m22s21) and then transferredto light/dark cycles (10 h of light/14 h of dark) for an additional 7 d. Thelight conditions were WL either supplemented with far-red light (lowR:FR of 0.121) or not (high R:FR of 7.7). Red arrowheads indicate thelengths of the first leaf sheaths. The seedlingswere digitally extracted forcomparison. Bar 5 2 cm. D, Quantification of first leaf lengths of theseedlings shown in C. Data represent means 6 SD (n $ 6). Letters in-dicate significant differences by two-way ANOVA.


Wu et al.





response to simulated shade (Fig. 8, A and B). Itis particularly interesting that the ZmPIF4.1 over-expression plants displayed a strong constitutive SASphenotype, similar to its Arabidopsis counterpartAtPIF4 (Supplemental Fig. S13A), which has beenshown to be a strong promoting factor of elongatedgrowth in response to shade or elevated tempera-ture (Lorrain et al., 2008; Kumar et al., 2012; Sunet al., 2012). Also noteworthy, dark-grown ZmPIF5-OE transgenic seedlings exhibited an exaggeratedapical hook phenotype similar to the ArabidopsisPIF5-overexpressing lines (Fig. 5C; Supplemental Fig.S10). Fourth, using the CRISPR/Cas9 technology, wecreated a set of ZmPIFs knockout mutants (Zmpif3striple mutant, Zmpif4s double mutant, and Zmpif5.1single mutant) and, as expected, we found that theseknockouts all displayed reduced mesocotyl pheno-types in dark-grown seedlings, with the strongestphenotype being observed with the Zmpif3s tripleknockouts (Fig. 7). We further showed that theseknockouts all exhibited a compromised elongation re-sponse to simulated shade treatment (Fig. 8C). Together,these data suggest that ZmPIFs play a conserved func-tion with the Arabidopsis PIFs in regulating seedlingphotomorphogenesis and the shade avoidance response.Our results also provided evidence suggesting that

these ZmPIFs may differ in several aspects from theAtPIFs. First, it has been shown that, upon brief lightexposure, all AtPIFs are able to form nuclear bodieswhere they interact with activated phys (Bauer et al.,2004; Al-Sady et al., 2006), but we found that only theZmPIF3.1:GFP, ZmPIF3.2:GFP, ZmPIF3.3:GFP, andZmPIF4.2:GFP fusion proteins, but not others, formedvisible nuclear bodies in Arabidopsis seedlings after ashort exposure to high intensity of red light (Fig. 6A, R1min), hinting that other ZmPIFs (ZmPIF4.1, ZmPIF5.1,and ZmPIF5.2) may have distinct interacting propertieswith Atphys. Second, previous studies showed thatAtPIFs (PIF1, PIF3, and PIF5) have a relatively shorthalf-life (less than 5 min) when exposed to red light(Shen et al., 2007, 2008). However, we found that all theZmPIFs:GFP fusion proteins are muchmore stable thantheir Arabidopsis counterparts and they remained vis-ible even after prolonged exposure to low Rc (1, 3, 6,and 24 h), a behavior reminiscent of that of AtPIF7(Leivar et al., 2008a; Fig. 6B). There are several possibleexplanations for the observed slow degradation ofZmPIFs in Arabidopsis. One possibility is that, as het-erologously expressed proteins, these ZmPIFs mightnot be efficiently recognized by the E3 ubiquitin ligasesresponsible for targeted degradation of AtPIFs, or itmay reflect the intrinsic property of ZmPIFs beingmore stable proteins. In this regard, it is worth indi-cating that light-induced phosphorylation of AtPIF3at multiple sites is necessary for the recruitment ofthe Light-Response Bric-a-Brack E3 ubiquitin ligasesto target both PIF3 and phyB for degradation invivo (Ni et al., 2013, 2014). Another possibility is thatlight-induced phosphorylation of ZmPIFs might be im-paired inArabidopsis, leading to their slowdegradation.

More detailed studies are required to address thesepossibilities.Besides participating in the phy pathway, recent

studies demonstrated that Arabidopsis PIFs serve abroader function, as a signaling hub that integratesenvironmental signals with multiple phytohormonebiosynthetic or signaling pathways (Leivar and Quail,2011; Pham et al., 2018). Consistent with this notion,recent studies showed that OsPIL1/OsPIL13 (a PIF4homolog) plays a role in regulating the drought stressresponse by reducing internode elongation in rice(Todaka et al., 2012). Similarly, it was shown in anearlier study that overexpression of ZmPIF1 andZmPIF3 (renamed ZmPIF3.1 and ZmPIF3.2 in thisstudy) in rice also enhances drought tolerance (Gaoet al., 2015, 2018). Furthermore, it was shown thatZmPIF4 (renamed ZmPIF4.1 in this study) physicallyinteracts with the Arabidopsis DELLA protein RE-PRESSOR OF GA1-3 (RGA), indicating a potential in-teraction between ZmPIF4 and aGA signaling pathwayon plant growth (Shi et al., 2018). We suspect that theseZmPIFs are likely to play important roles in regulatingdiverse aspects of plant growth and development, aswell as in response to a wide range of biotic and abioticstresses, through mediating signaling cross talk andintegration with various hormone signaling pathways.Considering that there may be functional redundancyamong ZmPIF members, further studies using addi-tional single and various combinations of higher ordermutants may help to answer this question.Given that the phyB-PIF signaling module is highly

conserved in plant architecture regulation and stressresponses in plants, it was considered to be a preferredcandidate target for crop improvement (Sawers et al.,2005; Wang and Wang, 2015). Previous attempts toimprove crop shade tolerance by overexpressing phyBin several crops have demonstrated promise, despitesome undesirable side effects (Gururani et al., 2015;Carriedo et al., 2016). It will be worthwhile to test theeffect of overexpressing ZmphyB in future studies toattenuate SAS in maize. Alternatively, manipulatingthe downstream factors of phy (such as ZmPIFs) mayalso help to attenuate the effect of SAS in maize. In thisstudy, we found that knocking out ZmPIFs in maizecould substantially reduce the shade avoidance inmaize seedlings. Next, we will examine whether theseadultZmpifs plants and their singlemutants also exhibitattenuated SAS. Additionally, our previous studyrevealed a direct functional link between the phyB-PIFmodule and SPL factors in mediating shade avoidanceresponses in Arabidopsis (Xie et al., 2017) and specu-lated that this link may also operate in maize and othercereal crops (Wei et al., 2018). These downstream SPLfactors might represent valuable targets for optimizingplant architecture for high-density planting. With therapid development of genome/functional genome re-search and gene-editing technology in crops, we believethat more effective strategies will surface tomodify SASwhen a better understanding of light signaling mecha-nisms is achieved in crops.








MATERIALS AND METHODS

Plant Materials and Growth Conditions

The Arabidopsis (Arabidopsis thaliana) mutant used in this study was the pifqquadruple mutant (pif1, pif3, pif4, pif5; Leivar et al., 2008b), and the Arabidopsisecotype Col-0 was used as the wild-type control. All the Arabidopsis transgenicplants used in this studywere generated in the pifq background. TheAtPIF5-OE(35S:PIF5-HA) transgenic line was kindly provided by Dr. Jiaqiang Sun (Nozueet al., 2007). Surface-sterilized seeds were sown on one-half-strengthMurashigeand Skoog medium (1% [w/v] Suc and 0.8% [w/v] agar, pH 5.8) and stratifiedfor 3 d at 4°C. Adult plants were grown in soil under long-day conditions (16 hof light/8 h of dark) at 22°C.

Nicotiana benthamiana seedswere directly sown into the soil and grown in thegreenhouse for about 1 month. Maize (Zea mays ssp.mays) inbred line seedlingswere grown in growth chambers under WL conditions (14 h of light/10 h ofdark, light intensity of 200mmol photonsm22 s21) at 28°C. Various tissues of 10-d-old maize B73 seedlings were used for RNA extraction to detect the tissueexpression patterns of ZmPIFs. For detection of the mRNA expression ofZmPIFs in response to light, B73 seedlings were grown in darkness for 7 d andthen transferred to WL for various times or retained in darkness as controls.

Total RNA Extraction and RT-qPCR Assays

Total RNA was isolated with Trizol reagent (Invitrogen), and reverse tran-scription reactions were performed using the manufacturer’s instructions of thefirst-strand cDNA synthesis kit (Tiangen Biotech). RT-qPCR was performedusing the SYBR Green SuperReal PreMix Plus (Tiangen Biotech) on an AppliedBiosystems 7500 real-time PCR detection system. The expression levels of Tu-bulin5 and ACT2 were used as internal controls for RT-qPCR in maize andArabidopsis, respectively. The expression levels of genes were calculated usingthe relative 2–DDCt method (Saleh et al., 2008). The sequences of the primers arelisted in Supplemental Table S2.

Phylogenetic Analysis

The full-length amino acid sequences of PIF in maize and Arabidopsis werealigned using the ClustalW program with default parameters in the alignmentwindow of MEGA6 software (Tamura et al., 2013). A phylogenetic tree wasconstructed using the PIF sequences of maize and Arabidopsis using a maxi-mum likelihood method, the Jones-Taylor-Thornton model, and partial dele-tion parameters.

Subcellular Localization Analysis

Coding sequences of each ZmPIF gene without the terminator were clonedinto the pCambia1305 binary vector through the XbaI site with the CaMV35Spromoter and a GFP tag to generate the Pro-35S:ZmPIFs-GFP vectors (primersare shown in Supplemental Table S2), and all binary vectors were introducedinto Agrobacterium tumefaciens strain GV3101. For subcellular localizationanalysis of ZmPIFs, the A. tumefaciens GV3101 cells harboring Pro-35S:ZmPIFs-GFP and a nuclear protein marker construct (Pro-35S:mRFP-AHL22; Xiao et al.,2009) were coinjected into N. benthamiana leaf epidermal cells. GFP and RFPsignals in transient transformed plants were observed using confocal micros-copy (Zeiss LSM710) after 2 to 3 d.

Y2H Assay

The pGADT7 vector containing the GAL4 activation domain (GAD) and thepGBKT7 vector containing the GBD were obtained from Clontech. Full-lengthcoding regions of ZmphyB1, ZmphyB2, and AtphyB were cloned into thepGBKT7 vector at the NdeI and EcoRI sites to generate GAL4-BD-ZmphyB1,GAL4-BD-ZmphyB2, and GAL4-BD-AtphyB, respectively. For GAL4-AD-PIFs,the coding region of eachZmPIFmemberwas cloned into the pGADT7 vector atthe EcoRI site. The BD- and AD-fused plasmids were cotransformed into theyeast strain AH109 according to the manufacturer’s instructions (ClontechYeast Protocols Handbook). The phyB-ZmPIF interaction assay with PCB wasperformed as described by Shimizu-Sato et al. (2002), with minor modification.Briefly, the positive yeast colonies were first selected on SD-Leu-Trp mediumand then plated on SD-Leu-Trp-His medium with or without 25 mM PCB (Sci-entific Frontier) under continuous far-red light (3 mmol photonsm22 s21) or red

light (4 mmol photons m22 s21) or in darkness for 3 d. The LacZ activity assaywas performed to quantify protein-protein interactions according to the man-ufacturer’s instructions, using o-nitrophenyl b-D-galactopyranoside as thesubstrate. The sequences of the primers are listed in Supplemental Table S2.

LCI Assays

The vectors for the LCI assay (pCAMBIA1300-nLUC and pCAMBIA1300-cLUC) were described previously in Chen et al. (2008). The full-length codingregions of ZmphyB1, ZmphyB2, and AtphyB were fused to the pCAMBIA1300-nLUC vector at the SalI/KpnI sites to generate ZmphyB1-nLUC and ZmphyB2-nLUC, respectively. The full-length coding regions of ZmPIFswere fused to thepCAMBIA1300-cLUC vector at the SalI/KpnI sites to generate the corre-sponding ZmPIFs-cLUC vectors. LCI assays were performed as described byChen et al. (2008). Briefly, both the nLUC- and cLUC-fused proteins weretransformed into the A. tumefaciens strain EHA105 and infiltrated into N. ben-thamiana leaves with the indicated combinations. The negative controls (theempty vectors and split-LUC constructs) were supplemented with an equalamount of the control strain harboring the 35S:GUS reporter gene to confirmsuccessful transfection. Samples were incubated in darkness for 48 h after theinfiltration and then transferred to WL for 3 to 6 h. The luciferase activity wasphotographed using the NightShade LB985 Plant Imaging System (BertholdTechnologies) after spraying with 20 mg mL21 potassium luciferin (Gold Bio-tech). These experiments were independently repeated at least three times. Thesequences of the primers are listed in Supplemental Table S2.

BiFC Assays

The vectors for BiFC assays (p2YN, p2YC, pSPYNE-35S, and pSPYCE-35S)were described previously (Walter et al., 2004; Yang et al., 2007). Full-lengthcoding sequences of ZmPIFs were fused in-frame with the N terminus of YFP.Full-length coding sequences of ZmphyB1 and ZmphyB2 were fused in-framewith the C terminus of YFP. ZmPIF3s and ZmPIF4s were inserted to p2YNvector at the PacI/SpeI sites, and ZmPIF5.1 and ZmPIF5.2 were inserted topSPYNE-35S vector at the SalI site. ZmphyB1 and ZmphyB2 were inserted top2YC and pSPYCE-35S vectors at the SalI site. The N. benthamiana leaves werecoinfiltrated with A. tumefaciens EHA105 cells carrying the indicated plasmidpairs. TheN. benthamiana plantswere grown in the dark for 48 h after infiltrationand then exposed to light for 3 to 6 h before analysis by confocal microscopy(Zeiss LSM710). The primers used in the BiFC assays are listed in SupplementalTable S2.

Arabidopsis Transformation and Phenotypic Analysis

For Arabidopsis transformation, the A. tumefaciens GV3101 cells harboringvarious Pro-35S:ZmPIFs-GFP constructs were separately transformed into theArabidopsis pifq mutant plants using the floral dip method (Clough and Bent,1998) to generate various ZmPIFs-OE/pifq lines. More than 20 independent linesof each transformation were selected with hygromycin, and two independenttransgenic lines were verified by western-blot analysis for further studies.

For dark-grown seedlings, seeds were irradiatedwith continuousWL for 6 hat 22°C to stimulate germination and then placed at 22°C for 4 d under darkconditions before phenotypic analysis of seedlings. For analyzing the responsesto simulated shade conditions, seedlings were grown in WL (28 mmol photonsm22 s21, R:FR of 6.48) for 2 d at 22°C and then kept in WL or transferred to WLwith supplemental continuous far-red light (WL1FR, R:FR of 0.145) for anadditional 5 d before measurements were taken. Hypocotyl lengths weremeasured from at least 20 seedlings using the ImageJ software. Data were an-alyzed using Student’s t test (Statistical Analysis System package, version 8.01;SAS Institute).

Epifluorescence Microscopy

For fluorescence microscopy analyses, seedlings were grown in the dark for4 d and then maintained in darkness, given a 1-min high Rc pulse (R 1min), orexposed to a low Rc for 60 min of 4 mmol photons m22 s21 (R 1h). The Rc wasprovided by the Percival LED-41HL2 growth chamber. Etiolated seedlingsexposed to Rc over time as indicated were collected under dim-green safelightand fixed in 4% (v/v) paraformaldehyde butter as described by Zuo et al.(2012). Then the whole seedling was immersed with one drop of 49,6-dia-mino-phenylindole (Vector Laboratories; catalog no. HT-1200) and examined


Wu et al.









with a Zeiss Axio Observer microscope. Representative cells were recorded byphotography with a digital Zeiss camera system.

Protein Extraction and Immunoblots

For western-blot analysis, Arabidopsis seedlings were grown in the dark for5 d and transferred to Rc (4 mmol photons m–2 s–1) for various durations. Twohundred milligrams of seedling powder was homogenized in a hot extractionbuffer as described by Bauer et al. (2004), and heated for 5 min at 95°C. Thesupernatant was used for further experiments. Aliquots from each samplecontaining equal amounts of protein were subjected to PAGE as described byAl-Sady et al. (2006). The GFP tag was detected by western-blot assay using ananti-GFP antibody (Abmart). As a loading control, Actin was detected using ananti-Actin antibody (Abmart).

Generation and Mutation Analysis of CRISPR/Cas9Knockout Lines in Maize

To generate ZmPIFs knockout transgenic lines, the expression vector of thedual-single guide RNA (sgRNA)/Cas9-mediated targeted deletion was con-structed as described by Zhao et al. (2016) with minor modifications. Briefly, aseries of target sites located on all the exons of each gene were analyzed usingthe SnapGene Viewer 3.2 software according to the reported criteria of 59-GG-(N)18-NGG-39. All the target sites were additionally BLASTed for specificity intheMaizeGDB database. Finally, two sgRNAs that specifically target sequencesof individual ZmPIF genes were selected (Supplemental Table S3). The differentforward primers ZmPIFs-sgR-F1 and ZmPIFs-sgR-F2 paired with the samereverse primer sgR-R were used to assemble sgRNA1 and sgRNA2 fragmentsthrough overlap-PCR, respectively. These sgRNA fragments driven by themaize ubiquitin U6-1 promoter were cloned into the CPB vector (Zhao et al.,2016) using the HindIII restriction site and an In-fusion HD Cloning Kit(TaKaRa). To overcome functional redundancy, the homologous ZmPIF genesof the same subgroup were combined in a single CRISPR/Cas9 vector system(Supplemental Fig. S11). Each construct was confirmed by PCR and sequencinganalysis. All the constructs were introduced into the strain EHA105 andtransformed into the immature embryo of a recipient maize inbred line ZC01using the conventional A. tumefaciens-mediated approach.

The genotypes of selected knockout transgenic lineswere confirmedbyDNAsequencing using specific primers listed in Supplemental Table S2. The targetregion of each ZmPIF gene was amplified from ZC01 and the transgenic lines,and then PCR products were either directly sequenced or cloned into the pCRTA clone vector (Transgene) and sequenced. The mutated sequences of eachZmPIF gene in transgenic lines were revealed by alignment of sequences be-tween ZC01 and the transgenic lines.

Phenotypic Analysis of Maize Zmpifs Knockout Mutantsand Simulated Shade Treatment

All the homozygousZmpifsmaizemutants and the correspondingwild-typeinbred maize line (ZC01) were uniformly buried at a 2-cm depth in plastic pots(83 8 cm) filled with soil. The same planting density of four kernels per plasticpot was used for all experiments. To measure the mesocotyls, the seedlingswere grown in constant darkness at 28°C for 10 d. For simulated shade treat-ment, maize seeds were germinated and grown in a growth chamber (PercivalLED-41HL2) at 28°C for 3 d under continuous WL (35 mmol photons m22 s21

photosynthetically active radiation; R:FR of 7.7) and then transferred to light/dark cycles (10 h of WL/14 h of dark) with WL1FR (low R:FR of 0.121) or withWL (high R:FR of 7.7) for an additional 7 d before measurement.

Mesocotyl Semithin Sections

Tomeasure the length of themesocotyl cells, themesocotylmiddle portion of10-d-old maize etiolated seedlings was cut into 1 3 1 3 0.5-mm pieces. Thespecimens were treated according to the method of Kong et al. (2016). Thespecimens were cut to sections of 1 mm thickness on a microtome (LeicaRM2155), and the sections were stained with 0.5% (w/v) Toluidine Blue andobserved with a Leica DMLB microscope. The epidermal cells on the centralregion of the mesocotyl were observed using a Nikon Eclipse 80i upright mi-croscope on the bright-field setting. Two fields were observed for each meso-cotyl, and ;20 to 30 cells per field were measured under 103 magnification.

The average length of measured cells from three mesocotyls was used to rep-resent cell length for each genotype.

Statistical Analysis

Todetermine the significantdifferences among thevariousgenotypes treatedwith or without far-red light, the method of Brady et al. (2015) for two-wayANOVA with interaction was used by performing the aov function in the statspackage in R version 3.5.0. The Tukey’s honestly significant difference methodwas used for all pairwise comparisons, with P values corrected for multiplecomparisons to control against type I errors. Student’s t test was adopted toevaluate the significant differences in hypocotyl length of the ZmPIF-OEtransgenic Arabidopsis plants in dark conditions and in mesocotyl length ofthe etiolated Zmpifs knockout maize seedlings.

Accession Numbers

Sequences of the maize genes analyzed in this work are available at http://ensembl.gramene.org/genome_browser/index.html/: ZmPIF3.1 (GRMZM2G115960),ZmPIF3.2 (GRMZM2G387528), ZmPIF3.3 (GRMZM2G062541), ZmPIF4.1(GRMZM5G865967), ZmPIF4.2 (GRMZM2G165042), ZmPIF5.1 (GRMZM2G065374),ZmPIF5.2 (GRMZM2G016756), ZmphyB1 (GRMZM2G124532), ZmphyB2(GRMZM2G092174), ZmEXPB4 (GRMZM2G154178) , ZmEXPB6(GRMZM2G176595), ZmEXPA3 (GRMZM2G074585) , and Tubulin5(GRMZM2G099167). Sequences of the Arabidopsis genes analyzed in this workare available at TAIR under the following accession numbers: AtPIF1(AT2G20180), AtPIF3 (AT1G09530), AtPIF4 (AT2G43010), AtPIF5 (AT3G59060),AtPIF6 (AT3G62090), AtPIF7 (AT5G61270), AtPIF8 (AT4G00050), AtphyB(AT2G18790), XTH15 (AT4G14130), IAA19 (AT3G15540), and ACT2 (AT3G18780).

Supplemental Data

The following supplemental materials are available.

Supplemental Figure S1. Diagram showing the annotated and actualexon-intron structure (a- and b-isoforms) of GRMZM2G165042 andGRMZM2G016756.

Supplemental Figure S2. A heat map illustrating the expression levels ofthe seven ZmPIF genes in different tissues from various developmentalstages.

Supplemental Figure S3. Light regulation of the ZmPIF genes.

Supplemental Figure S4. Quantitative Y2H liquid assay.

Supplemental Figure S5. Western-blot analysis of ZmphyB2 protein inyeast cells.

Supplemental Figure S6. GUS histochemistry staining showing propertransformation of negative controls in the LCI assay.

Supplemental Figure S7. Negative controls for the BiFC assay.

Supplemental Figure S8. ZmPIFs physically interact with AtphyB.

Supplemental Figure S9. Expression analysis of the ZmPIFs transgenes inthe selected Arabidopsis transgenic lines.

Supplemental Figure S10. Effects of ZmPIF5 overexpression on ethylene-related responses.

Supplemental Figure S11. Vector construction for genome editing of theZmPIF genes.

Supplemental Figure S12. Sequence analysis of the target sites in theZmpifs knockout mutant lines.

Supplemental Figure S13. Adult phenotypes of the Arabidopsis ZmPIFs-OE plants.

Supplemental Table S1. Characteristics of the ZmPIF gene family.

Supplemental Table S2. Primers used in this study.

Supplemental Table S3. The targeted genes and two sgRNA target se-quences with PAM.







http://ensembl.gramene.org/genome_browser/index.html/

http://ensembl.gramene.org/genome_browser/index.html/


















ACKNOWLEDGMENTS

We thank Dr. Hongbing Wei (South China Agricultural University) as wellas Dr. Yang Liu and Dr. Mengdi Ma (Chinese Academy of AgriculturalSciences) for critical reading and comments on the article.

Received February 25, 2019; accepted July 18, 2019; published July 26, 2019.

LITERATURE CITED

Al-Sady B, Ni W, Kircher S, Schäfer E, Quail PH (2006) Photoactivatedphytochrome induces rapid PIF3 phosphorylation prior to proteasome-mediated degradation. Mol Cell 23: 439–446

Bailey TL, Boden M, Buske FA, Frith M, Grant CE, Clementi L, Ren J, LiWW, Noble WS (2009) MEME SUITE: Tools for motif discovery andsearching. Nucleic Acids Res 37: W202–W208

Bauer D, Viczián A, Kircher S, Nobis T, Nitschke R, Kunkel T, PanigrahiKCS, Adám E, Fejes E, Schäfer E, et al (2004) Constitutive photomor-phogenesis 1 and multiple photoreceptors control degradation of phy-tochrome interacting factor 3, a transcription factor required for lightsignaling in Arabidopsis. Plant Cell 16: 1433–1445

Brady SM, Burow M, Busch W, Carlborg Ö, Denby KJ, Glazebrook J,Hamilton ES, Harmer SL, Haswell ES, Maloof JN, et al (2015) Reassessthe t test: Interact with all your data via ANOVA. Plant Cell 27:2088–2094

Carriedo LG, Maloof JN, Brady SM (2016) Molecular control of crop shadeavoidance. Curr Opin Plant Biol 30: 151–158

Chen H, Zou Y, Shang Y, Lin H, Wang Y, Cai R, Tang X, Zhou JM (2008)Firefly luciferase complementation imaging assay for protein-proteininteractions in plants. Plant Physiol 146: 368–376

Clough SJ, Bent AF (1998) Floral dip: A simplified method forAgrobacterium-mediated transformation of Arabidopsis thaliana. PlantJ 16: 735–743

Duvick DN (2005a) The contribution of breeding to yield advances inmaize (Zea mays L.). Adv Agron 86: 83–145

Duvick DN (2005b) Genetic progress in yield of United States maize (Zeamays L.). Maydica 50: 193–202

Franklin KA (2008) Shade avoidance. New Phytol 179: 930–944Franklin KA, Quail PH (2010) Phytochrome functions in Arabidopsis de-

velopment. J Exp Bot 61: 11–24Gao Y, Jiang W, Dai Y, Xiao N, Zhang C, Li H, Lu Y, Wu M, Tao X, Deng

D, et al (2015) A maize phytochrome-interacting factor 3 improvesdrought and salt stress tolerance in rice. Plant Mol Biol 87: 413–428

Gao Y, Wu M, Zhang M, Jiang W, Ren X, Liang E, Zhang D, Zhang C,Xiao N, Li Y, et al (2018) A maize phytochrome-interacting factorsprotein ZmPIF1 enhances drought tolerance by inducing stomatal clo-sure and improves grain yield in Oryza sativa. Plant Biotechnol J 16:1375–1387

Gao Y, Ren X, Qian J, Li Q, Tao H, Chen J (2019) The phytochrome-interacting family of transcription factors in maize (Zea mays L.): Iden-tification, evolution, and expression analysis. Acta Physiol Plant 41: 8

Gururani MA, Ganesan M, Song PS (2015) Photo-biotechnology as a toolto improve agronomic traits in crops. Biotechnol Adv 33: 53–63

Jeong J, Choi G (2013) Phytochrome-interacting factors have both sharedand distinct biological roles. Mol Cells 35: 371–380

Kebrom TH, Burson BL, Finlayson SA (2006) Phytochrome B repressesTeosinte Branched1 expression and induces sorghum axillary bud out-growth in response to light signals. Plant Physiol 140: 1109–1117

Kebrom TH, Brutnell TP, Finlayson SA (2010) Suppression of sorghumaxillary bud outgrowth by shade, phyB and defoliation signallingpathways. Plant Cell Environ 33: 48–58

Khanna R, Shen Y, Marion CM, Tsuchisaka A, Theologis A, Schäfer E,Quail PH (2007) The basic helix-loop-helix transcription factor PIF5 actson ethylene biosynthesis and phytochrome signaling by distinct mech-anisms. Plant Cell 19: 3915–3929

Klose C, Viczián A, Kircher S, Schäfer E, Nagy F (2015) Molecularmechanisms for mediating light-dependent nucleo/cytoplasmic parti-tioning of phytochrome photoreceptors. New Phytol 206: 965–971

Kong DX, Li YQ, Wang ML, Bai M, Zou R, Tang H, Wu H (2016) Effects oflight intensity on leaf photosynthetic characteristics, chloroplast struc-ture, and alkaloid content of Mahonia bodinieri (Gagnep.) Laferr. ActaPhysiol Plant 38: 120

Kumar I, Swaminathan K, Hudson K, Hudson ME (2016) Evolutionarydivergence of phytochrome protein function in Zea mays PIF3 signaling.J Exp Bot 67: 4231–4240

Kumar SV, Lucyshyn D, Jaeger KE, Alós E, Alvey E, Harberd NP, WiggePA (2012) Transcription factor PIF4 controls the thermosensory activa-tion of flowering. Nature 484: 242–245

Lauer JG, Bijl CG, Grusak MA, Baenziger PS, Boote K, Lingle S, Carter T,Kaeppler S, Boerma R, Eizenga G, et al (2012) The scientific grandchallenges of the 21st century for the Crop Science Society of America.Crop Sci 52: 1003–1010

Leivar P, Quail PH (2011) PIFs: Pivotal components in a cellular signalinghub. Trends Plant Sci 16: 19–28

Leivar P, Monte E, Al-Sady B, Carle C, Storer A, Alonso JM, Ecker JR,Quail PH (2008a) The Arabidopsis phytochrome-interacting factor PIF7,together with PIF3 and PIF4, regulates responses to prolonged red lightby modulating phyB levels. Plant Cell 20: 337–352

Leivar P, Monte E, Oka Y, Liu T, Carle C, Castillon A, Huq E, Quail PH(2008b) Multiple phytochrome-interacting bHLH transcription factorsrepress premature seedling photomorphogenesis in darkness. Curr Biol18: 1815–1823

Leivar P, Tepperman JM, Cohn MM, Monte E, Al-Sady B, Erickson E,Quail PH (2012) Dynamic antagonism between phytochromes and PIFfamily basic helix-loop-helix factors induces selective reciprocal re-sponses to light and shade in a rapidly responsive transcriptional net-work in Arabidopsis. Plant Cell 24: 1398–1419

Lorrain S, Allen T, Duek PD, Whitelam GC, Fankhauser C (2008)Phytochrome-mediated inhibition of shade avoidance involves degra-dation of growth-promoting bHLH transcription factors. Plant J 53:312–323

Mansfield BD, Mumm RH (2014) Survey of plant density tolerance in U.S.maize germplasm. Crop Sci 54: 157

Mizuno T, Oka H, Yoshimura F, Ishida K, Yamashino T (2015) Insight intothe mechanism of end-of-day far-red light (EODFR)-induced shadeavoidance responses in Arabidopsis thaliana. Biosci Biotechnol Biochem79: 1987–1994

Ni M, Tepperman JM, Quail PH (1999) Binding of phytochrome B to itsnuclear signalling partner PIF3 is reversibly induced by light. Nature400: 781–784

Ni W, Xu SL, Chalkley RJ, Pham TN, Guan S, Maltby DA, BurlingameAL, Wang ZY, Quail PH (2013) Multisite light-induced phosphorylationof the transcription factor PIF3 is necessary for both its rapid degrada-tion and concomitant negative feedback modulation of photoreceptorphyB levels in Arabidopsis. Plant Cell 25: 2679–2698

Ni W, Xu SL, Tepperman JM, Stanley DJ, Maltby DA, Gross JD,Burlingame AL, Wang ZY, Quail PH (2014) A mutually assured de-struction mechanism attenuates light signaling in Arabidopsis. Science344: 1160–1164

Nozue K, Covington MF, Duek PD, Lorrain S, Fankhauser C, Harmer SL,Maloof JN (2007) Rhythmic growth explained by coincidence betweeninternal and external cues. Nature 448: 358–361

Paik I, Kathare PK, Kim J-I, Huq E (2017) Expanding roles of PIFs in signalintegration from multiple processes. Mol Plant 10: 1035–1046

Penfield S, Josse EM, Halliday KJ (2010) A role for an alternative splicevariant of PIF6 in the control of Arabidopsis primary seed dormancy.Plant Mol Biol 73: 89–95

Pham VN, Kathare PK, Huq E (2018) Phytochromes and phytochromeinteracting factors. Plant Physiol 176: 1025–1038

Saleh A, Alvarez-Venegas R, Avramova Z (2008) An efficient chromatinimmunoprecipitation (ChIP) protocol for studying histone modificationsin Arabidopsis plants. Nat Protoc 3: 1018–1025

Sawers RJ, Linley PJ, Farmer PR, Hanley NP, Costich DE, Terry MJ,Brutnell TP (2002) Elongated mesocotyl1, a phytochrome-deficientmutant of maize. Plant Physiol 130: 155–163

Sawers RJ, Sheehan MJ, Brutnell TP (2005) Cereal phytochromes: Targetsof selection, targets for manipulation?. Trends Plant Sci 10: 138–143

Sekhon RS, Lin H, Childs KL, Hansey CN, Buell CR, de Leon N,Kaeppler SM (2011) Genome-wide atlas of transcription during maizedevelopment. Plant J 66: 553–563

Sheehan MJ, Farmer PR, Brutnell TP (2004) Structure and expression ofmaize phytochrome family homeologs. Genetics 167: 1395–1405

Sheehan MJ, Kennedy LM, Costich DE, Brutnell TP (2007) Sub-functionalization of PhyB1 and PhyB2 in the control of seedling andmature plant traits in maize. Plant J 49: 338–353


Wu et al.



Shen H, Zhu L, Castillon A, Majee M, Downie B, Huq E (2008) Light-induced phosphorylation and degradation of the negative regulatorPHYTOCHROME-INTERACTING FACTOR1 from Arabidopsis dependupon its direct physical interactions with photoactivated phytochromes.Plant Cell 20: 1586–1602

Shen Y, Khanna R, Carle CM, Quail PH (2007) Phytochrome induces rapidPIF5 phosphorylation and degradation in response to red-light activa-tion. Plant Physiol 145: 1043–1051

Shi Q, Zhang H, Song X, Jiang Y, Liang R, Li G (2018) Functional char-acterization of the maize phytochrome-interacting factors PIF4 and PIF5.Front Plant Sci 8: 2273

Shimizu-Sato S, Huq E, Tepperman JM, Quail PH (2002) A light-switchable gene promoter system. Nat Biotechnol 20: 1041–1044

Sun J, Qi L, Li Y, Chu J, Li C (2012) PIF4-mediated activation of YUCCA8expression integrates temperature into the auxin pathway in regulatingArabidopsis hypocotyl growth. PLoS Genet 8: e1002594

Tamura K, Stecher G, Peterson D, Filipski A, Kumar S (2013) MEGA6:Molecular Evolutionary Genetics Analysis version 6.0. Mol Biol Evol 30:2725–2729

Todaka D, Nakashima K, Maruyama K, Kidokoro S, Osakabe Y, Ito Y,Matsukura S, Fujita Y, Yoshiwara K, Ohme-Takagi M, et al (2012) Ricephytochrome-interacting factor-like protein OsPIL1 functions as a keyregulator of internode elongation and induces a morphological responseto drought stress. Proc Natl Acad Sci USA 109: 15947–15952

Tollenaar M, Lee EA (2002) Yield potential, yield stability and stress tol-erance in maize. Field Crops Res 75: 161–169

Van Buskirk EK, Decker PV, Chen M (2012) Photobodies in light signal-ing. Plant Physiol 158: 52–60

Walter M, Chaban C, Schütze K, Batistic O, Weckermann K, Näke C,Blazevic D, Grefen C, Schumacher K, Oecking C, et al (2004) Visual-ization of protein interactions in living plant cells using bimolecularfluorescence complementation. Plant J 40: 428–438

Wang H, Wang H (2015) Phytochrome signaling: time to tighten up theloose ends. Mol Plant 8: 540–551

Wei H, Zhao Y, Xie Y, Wang H (2018) Exploiting SPL genes to improvemaize plant architecture tailored for high-density planting. J Exp Bot 69:4675–4688

Whipple CJ, Kebrom TH, Weber AL, Yang F, Hall D, Meeley R, SchmidtR, Doebley J, Brutnell TP, Jackson DP (2011) grassy tillers1 promotesapical dominance in maize and responds to shade signals in the grasses.Proc Natl Acad Sci USA 108: E506–E512

Xiao C, Chen F, Yu X, Lin C, Fu YF (2009) Over-expression of an AT-hookgene, AHL22, delays flowering and inhibits the elongation of the hy-pocotyl in Arabidopsis thaliana. Plant Mol Biol 71: 39–50

Xie Y, Liu Y, Wang H, Ma X, Wang B, Wu G, Wang H (2017) Phytochrome-interacting factors directly suppress MIR156 expression to enhanceshade-avoidance syndrome in Arabidopsis. Nat Commun 8: 348

Yang X, Baliji S, Buchmann RC, Wang H, Lindbo JA, Sunter G, BisaroDM (2007) Functional modulation of the geminivirus AL2 transcrip-tion factor and silencing suppressor by self-interaction. J Virol 81:11972–11981

Yu HQ, Sun FA, Lu FZ, Feng WQ, Zhang YY, Liu BL, Yan L, Fu FL, Li WC(2018) Positive regulation of phytochrome A on shade avoidance inmaize. Pak J Bot 50: 1433–1440

Zhang J, Stankey RJ, Vierstra RD (2013a) Structure-guided engineering ofplant phytochrome B with altered photochemistry and light signaling.Plant Physiol 161: 1445–1457

Zhang Y, Mayba O, Pfeiffer A, Shi H, Tepperman JM, Speed TP, QuailPH (2013b) A quartet of PIF bHLH factors provides a transcriptionallycentered signaling hub that regulates seedling morphogenesis throughdifferential expression-patterning of shared target genes in Arabidopsis.PLoS Genet 9: e1003244

Zhao Y, Zhang C, Liu W, Gao W, Liu C, Song G, Li WX, Mao L, Chen B,Xu Y, et al (2016) An alternative strategy for targeted gene replacementin plants using a dual-sgRNA/Cas9 design. Sci Rep 6: 23890

Zuo ZC, Meng YY, Yu XH, Zhang ZL, Feng DS, Sun SF, Liu B, Lin CT(2012) A study of the blue-light-dependent phosphorylation, degrada-tion, and photobody formation of Arabidopsis CRY2. Mol Plant 5:726–733





Documents

Characterization of Maize Phytochrome-Interacting …Characterization of Maize Phytochrome-Interacting Factors in Light Signaling and Photomorphogenesis1 Guangxia Wu,a Yongping Zhao,a