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Publications

2017
Shor E, Paik I, Kangisser S, Green R, Huq E. Phytochrome interacting factors (PIFs) mediate metabolic regulation of the circadian system in Arabidopsis. New Phytologist. 215 (1) :217-228.
Possart A, Xu T, Paik I, Hanke S, Keim S, Hermann H-M, Wolf L, Hiss M, Becker C, Huq E, et al. Characterization of PIFs from the moss Physcomitrella patens illustrates conservation of phytochrome signaling modules in land plants. Plant Cell. 29 :310-330.
2016
Zhu L, Xin R, Huq E. A Protein-Based Genetic Screening Uncovers Mutants Involved in Phytochrome Signaling in Arabidopsis. Front. Plant Sci. 7 :1086.
Zhu L, Xin R, Bu Q, Shen H, Dang J, Huq E. A negative feedback loop between PHYTOCHROME INTERACTING FACTORs and HECATE proteins fine tunes photomorphogenesis in Arabidopsis. The Plant Cell [Internet]. 28 (4) :855-874. Publisher's VersionAbstract

The Phytochrome Interacting Factors (PIFs), a small group of basic helix-loop-helix (bHLH) transcription factors, repress photomorphogenesis both in the dark and light. Light signals perceived by the phytochrome family of photoreceptors induce rapid degradation of PIFs to promote photomorphogenesis. Here we show that HECATE (HEC) proteins, another small group of HLH proteins, antagonistically regulate PIFs to promote photomorphogenesis. HEC1 and HEC2 heterodimerize with PIF family members. PIF1, HEC1 and HEC2 genes are spatially and temporally co-expressed, and HEC2 is localized in the nucleus. hec1, hec2 and hec3 single and hec1 hec2 double mutants showed hyposensitivity to light-induced seed germination, accumulation of chlorophyll and carotenoids, hallmark processes oppositely regulated by PIF1. HEC2 inhibits PIF1 target gene expression by directly heterodimerizing with PIF1 and preventing DNA binding and transcriptional activation activity of PIF1. Conversely, PIFs directly activate the expression of HEC1 and HEC2 in the dark and light reduces the expression of these HECs possibly by degrading PIFs. HEC2 is partially degraded in the dark through the ubiquitin/26S-proteasome pathway and is stabilized by light. HEC2 overexpression also reduces the light-induced degradation of PIF1. Taken together, these data suggest that PIFs and HECs constitute a negative feedback loop to fine tune photomorphogenesis in Arabidopsis thaliana.

Sharma N, Xin R, Kim D-H, Sung S, Lange T, Huq E. NO FLOWERING IN SHORT DAY (NFL) is a bHLH transcription factor thatpromotes flowering specifically under short-day conditions in Arabidopsis. Development [Internet]. 143 (4) : 682-690. Publisher's Version
2015
Xu X, Paik I, Zhu L, Huq E. Illuminating progress in phytochrome-mediated light signaling pathways. Trends in Plant Science. 20 (10) :641-650.Abstract

Light signals regulate a plethora of plant responses throughout their life cycle, especially the red and far-red regions of the light spectrum perceived by the phytochrome family of photoreceptors. However, the mechanisms by which phytochromes regulate gene expression and downstream responses remain elusive. Several recent studies have unraveled the details on how phytochromes regulate photomorphogenesis. These include the identification of E3 ligases
that degrade PHYTOCHROME INTERACTING FACTOR (PIF) proteins, key negative regulators, in response to light, a better view of how phytochromes inhibit another key negative regulator, CONSTITUTIVELY PHOTOMORPHOGENIC 1 (COP1), and an understanding of why plants evolved multiple negative regulators to repress photomorphogenesis in darkness. These advances will surely fuel future research on many unanswered questions that have intrigued plant photobiologists for decades.

Zhu L, Bu Q, Xu X, Paik I, Hunag X, Hoecker U, Deng XW, Huq E. CUL4 forms an E3 ligase with COP1 and SPA to promote light-induced degradation of PIF1. Nature Communications. 6 :7245.Abstract

Plants undergo contrasting developmental programs in dark and light. Photomorphogenesis, a light-adapted programme is repressed in the dark by the synergistic actions of CUL4COP1–SPA E3 ubiquitin ligase and a subset of basic helix-loop-helix transcription factors called phytochrome interacting factors (PIFs). To promote photomorphogenesis, light activates the phytochrome family of sensory photoreceptors, which inhibits these repressors by poorly understood mechanisms. Here, we show that the CUL4COP1–SPA E3 ubiquitin ligase is necessary for the light-induced degradation of PIF1 in Arabidopsis. The light-induced ubiquitylation and subsequent degradation of PIF1 is reduced in the cop1, spaQ and cul4 backgrounds. COP1, SPA1 and CUL4 preferentially form complexes with the phosphorylated forms of PIF1 in response to light. The cop1 and spaQ seeds display strong hyposensitive response to far-red light-mediated seed germination and light-regulated gene expression. These data show a mechanism by which an E3 ligase attenuates its activity by degrading its cofactor in response to light.

Mulekar JJ, Huq E. Arabidopsis Casein Kinase 2 α4 subunit regulates various developmental pathways in a functionally overlapping manner. Plant Science. 236 :295-303.Abstract

Casein kinase 2 (CK2) is an essential and well-conserved Ser/Thr kinase that regulates proteins in a post-translational manner. CK2 has been shown to affect a large number of developmental processes acrosseukaryotes. It is a tetrameric protein composed of a dimer of alpha (catalytic) and beta (regulatory)subunit each. In our previous study we showed that three of the four CK2 subunits in Arabidopsisact in a functionally redundant manner to regulate various developmental pathways. In this study weconstructed two independent CK2 4 RNAi lines in the CK2 alpha triple mutant background. Throughfunctional characterization of these RNAi lines we show that the fourth subunit in Arabidopsis alsofunctions redundantly in regulating ABA response, lateral root formation and flowering time. CK2 4-GFP localizes to the chloroplast in transgenic Arabidopsis seedlings, consistent with the presence of achloroplast localization signal at the amino-terminus of CK2 4 subunit. Taken together, our results sug-gest a functionally overlapping role for the CK2 4 subunit in regulating various developmental processesin plants

Sheerin DJ, Menon C, Zur Oven-Krockhaus S, Enderle B, Zhu L, Johnen P, Schleifenbaum F, Stierhof Y-D, Huq E, Hiltbrunner A. Light-Activated Phytochrome A and B Interact with Members of the SPA Family to Promote Photomorphogenesis in Arabidopsis by Reorganizing the COP1/SPA Complex. Plant Cell. 27 (1) :189-201.Abstract
Phytochromes function as red/far-red photoreceptors in plants and are essential for light-regulated growth and development. Photomorphogenesis, the developmental program in light, is the default program in seed plants. In dark-grown seedlings, photomorphogenic growth is suppressed by the action of the CONSTITUTIVELY PHOTOMORPHOGENIC1 (COP1)/SUPPRESSOR OF phyA-105 (SPA) complex, which targets positive regulators of photomorphogenic growth for degradation by the proteasome. Phytochromes inhibit the COP1/SPA complex, leading to the accumulation of transcription factors promoting photomorphogenesis; yet, the mechanism by which they inactivate COP1/SPA is still unknown. Here, we show that light-activated phytochrome A (phyA) and phytochrome B (phyB) interact with SPA1 and other SPA proteins. Fluorescence resonance energy transfer-fluorescence lifetime imaging microscopy analyses show that SPAs and phytochromes colocalize and interact in nuclear bodies. Furthermore, light-activated phyA and phyB disrupt the interaction between COP1 and SPAs, resulting in reorganization of the COP1/SPA complex in planta. The light-induced stabilization of HFR1, a photomorphogenic factor targeted for degradation by COP1/SPA, correlates temporally with the accumulation of phyA in the nucleus and localization of phyA to nuclear bodies. Overall, these data provide a molecular mechanism for the inactivation of the COP1/SPA complex by phyA- and phyB-mediated light perception.
2014
Mulekar JJ, Huq E. Expanding roles of protein kinase CK2 in regulating plant growth and development. J Exp Bot. 65 (11) :2883-93.Abstract
Protein kinase CK2 (formerly known as casein kinase II) is a ubiquitious Ser/Thr kinase present in all eukaryotes. The α (catalytic) and β (regulatory) subunits of CK2 exist both as a tetrameric holoenzyme and as monomers in eukaryotic cells. CK2 has been implicated in multiple developmental and stress-responsive pathways including light signalling and circadian clock in plants. Recent studies using CK2 knockout and dominant negative mutants in Arabidopsis have uncovered new roles for this enzyme. CK2 substrates that have been identified so far are primarily transcription factors or regulatory proteins. CK2-mediated phosphorylation of these factors often results in alteration of the protein function including changes in the DNA-binding affinity, dimerization, stability, protein-protein interactions, and subcellular localization. CK2 has evolved as an essential housekeeping kinase in plants that modifies protein function in a dynamic way. This review summarizes the current knowledge of the role of CK2 in plant development.
Krzymuski M, Cerdán PD, Zhu L, Vinh A, Chory J, Huq E, Casal JJ. Phytochrome A antagonizes PHYTOCHROME INTERACTING FACTOR 1 to prevent over-activation of photomorphogenesis. Mol Plant. 7 (9) :1415-28.Abstract
Phytochrome A (phyA) is crucial to initiate the early steps of the transition between skoto- and photomorphogenesis upon light exposure and to complete this process under far-red light (typical of dense vegetation canopies). However, under prolonged red or white light, phyA mutants are hyper-photomorphogenic in many respects. To investigate this issue, we analyzed the late response of the transcriptome of the phyA mutant to red light. Compared to the wild-type (WT), hyper-responsive genes outnumbered the genes showing reduced response to red light in phyA. A network analysis revealed the co-expression of PHYTOCHROME INTERACTING FACTOR 1 (PIF1) with those genes showing hyper-promotion by red light in phyA. The enhanced responses of gene expression, cotyledon unfolding, hypocotyl growth, and greening observed in the phyA mutant compared to the WT were absent in the phyA pif1 double mutant compared to pif1, indicating that the hyper-photomorphogenic phenotype of phyA requires PIF1. PIF1 directly binds to gene promoters that displayed PIF1-mediated enhanced response to red light. Expression of mutant PIF1 deficient in interactions with phyA and phyB enhanced the long-term growth response to red light but reduced the expression of selected genes in response to red light. We propose that phytochrome-mediated degradation of PIF1 prevents over-activation of photomorphogenesis during early seedling development.
Xu X, Paik I, Zhu L, Bu Q, Huang X, Deng XW, Huq E. PHYTOCHROME INTERACTING FACTOR1 Enhances the E3 Ligase Activity of CONSTITUTIVE PHOTOMORPHOGENIC1 to Synergistically Repress Photomorphogenesis in Arabidopsis. Plant Cell. 26 (5) :1992-2006.Abstract
CONSTITUTIVE PHOTOMORPHOGENIC1 (COP1) is a RING/WD40 repeat-containing ubiquitin E3 ligase that is conserved from plants to humans. COP1 forms complexes with SUPPRESSOR OF PHYTOCHROME A (SPA) proteins, and these complexes degrade positively acting transcription factors in the dark to repress photomorphogenesis. Phytochrome-interacting basic helix-loop-helix transcription factors (PIFs) also repress photomorphogenesis in the dark. In response to light, the phytochrome family of sensory photoreceptors simultaneously inactivates COP1-SPA complexes and induces the rapid degradation of PIFs to promote photomorphogenesis. However, the functional relationship between PIFs and COP1-SPA complexes is still unknown. Here, we present genetic evidence that the pif and cop1/spa Arabidopsis thaliana mutants synergistically promote photomorphogenesis in the dark. LONG HYPOCOTYL5 (HY5) is stabilized in the cop1 pif1, spa123 pif1, and pif double, triple, and quadruple mutants in the dark. Moreover, the hy5 mutant suppresses the constitutive photomorphogenic phenotypes of the pifq mutant in the dark. PIF1 forms complexes with COP1, HY5, and SPA1 and enhances the substrate recruitment and autoubiquitylation and transubiquitylation activities of COP1. These data uncover a novel function of PIFs as the potential cofactors of COP1 and provide a genetic and biochemical model of how PIFs and COP1-SPA complexes synergistically repress photomorphogenesis in the dark.
Bu Q, Lv T, Shen H, Luong P, Wang J, Wang Z, Huang Z, Xiao L, Engineer C, Kim TH, et al. Regulation of drought tolerance by the F-box protein MAX2 in Arabidopsis. Plant Physiol. 164 (1) :424-39.Abstract
MAX2 (for MORE AXILLARY GROWTH2) has been shown to regulate diverse biological processes, including plant architecture, photomorphogenesis, senescence, and karrikin signaling. Although karrikin is a smoke-derived abiotic signal, a role for MAX2 in abiotic stress response pathways is least investigated. Here, we show that the max2 mutant is strongly hypersensitive to drought stress compared with wild-type Arabidopsis (Arabidopsis thaliana). Stomatal closure of max2 was less sensitive to abscisic acid (ABA) than that of the wild type. Cuticle thickness of max2 was significantly thinner than that of the wild type. Both of these phenotypes of max2 mutant plants correlate with the increased water loss and drought-sensitive phenotype. Quantitative real-time reverse transcription-polymerase chain reaction analyses showed that the expression of stress-responsive genes and ABA biosynthesis, catabolism, transport, and signaling genes was impaired in max2 compared with wild-type seedlings in response to drought stress. Double mutant analysis of max2 with the ABA-insensitive mutants abi3 and abi5 indicated that MAX2 may function upstream of these genes. The expression of ABA-regulated genes was enhanced in imbibed max2 seeds. In addition, max2 mutant seedlings were hypersensitive to ABA and osmotic stress, including NaCl, mannitol, and glucose. Interestingly, ABA, osmotic stress, and drought-sensitive phenotypes were restricted to max2, and the strigolactone biosynthetic pathway mutants max1, max3, and max4 did not display any defects in these responses. Taken together, these results uncover an important role for MAX2 in plant responses to abiotic stress conditions.
Zhu L, Huq E. Suicidal co-degradation of the phytochrome interacting factor 3 and phytochrome B in response to light. Mol Plant. 7 (12) :1709-11.
2012
Mulekar JJ, Bu Q, Chen F, Huq E. Casein kinase II α subunits affect multiple developmental and stress-responsive pathways in Arabidopsis. Plant J. 69 (2) :343-54.Abstract
Casein kinase II (formerly known as CK2), a ubiquitous Ser/Thr kinase, plays critical roles in all higher organisms including plants. The CK2 holoenzyme consists of two catalytic α subunits and two regulatory β subunits. The Arabidopsis genome has four α subunit and four β subunit genes, and members of both the α and β subunit families have been shown to be localized in the cytoplasm, nucleus and also in chloroplasts. However, the biological roles of CK2 subunits have not been fully characterized yet. Here we identified T-DNA insertion mutants in three α subunit genes (α1, α2 and α3) and made double and triple mutants. The CK2 α1α2α3 triple mutants displayed reduced CK2 activity compared with wild-type seedlings. Phenotypic characterization showed that CK2 α1α2α3 triple mutants are late flowering under both long- and short-day conditions. Genes encoding floral integrators are differentially regulated in the triple mutant compared with the wild-type plants. CK2 α1α2α3 triple mutants also displayed reduced hypocotyl growth, smaller cotyledon size and a reduced number of lateral roots compared with wild-type seedlings under light. Abscisic acid-induced blockage of seed germination and cotyledon greening is reduced in CK2 α subunit mutants in an additive manner. Moreover, CK2 α subunit mutants are also hyposensitive to a NaCl-induced blockage of seed germination. Taken together, these data suggest that CK2 α subunits affect diverse developmental and stress responsive pathways in Arabidopsis.
Mulekar JJ, Huq E. Does CK2 affect flowering time by modulating the autonomous pathway in Arabidopsis?. Plant Signal Behav. 7 (2) :292-4.Abstract
CK2 (Casein Kinase II), a ubiquitous Ser/Thr kinase, affects multiple developmental and stress response pathways in Arabidopsis, including flowering time under both long- and short-day conditions through the photoperiod and autonomous pathways. CK2 phosphorylates central clock components, CCA1 and LHY, to modulate circadian clock that regulates flowering time through the photoperiod pathway. However, how CK2 regulates flowering time through the autonomous pathway is still unknown. Analyses of phosphorylation sites using several prediction softwares show that most of the autonomous pathway components have multiple CK2 phosphorylation sites. CK2 might phosphorylate any or all of these components to modulate their activity/stability resulting in altered expression of FLC that drives flowering time through the autonomous pathway.
Sung S, Huq E, Chen JZ. International plant molecular biology: a bright future for green science. Genome Biol. 13 (11) :323.Abstract
A report on the 10th International Congress of Plant Molecular Biology, Jeju, South Korea, October 21-26, 2012.
Shen H, Zhu L, Bu Q-Y, Huq E. MAX2 affects multiple hormones to promote photomorphogenesis. Mol Plant. 5 (3) :750-62.Abstract
Ubiquitin-26S proteasome system (UPS) has been shown to play central roles in light and hormone-regulated plant growth and development. Previously, we have shown that MAX2, an F-box protein, positively regulates facets of photomorphogenic development in response to light. However, how MAX2 controls these responses is still unknown. Here, we show that MAX2 oppositely regulates GA and ABA biosynthesis to optimize seed germination in response to light. Dose-response curves showed that max2 seeds are hyposensitive to GA and hypersensitive to ABA in seed germination responses. RT-PCR assays demonstrated that the expression of GA biosynthetic genes is down-regulated, while the expression of GA catabolic genes is up-regulated in the max2 seeds compared to wild-type. Interestingly, expression of both ABA biosynthetic and catabolic genes is up-regulated in the max2 seeds compared to wild-type. Treatment with an auxin transport inhibitor, NPA, showed that increased auxin transport in max2 seedlings contributes to the long hypocotyl phenotype under light. Moreover, light-signaling phenotypes are restricted to max2, as the biosynthetic mutants in the strigolactone pathway, max1, max3, and max4, did not display any defects in seed germination and seedling de-etiolation compared to wild-type. Taken together, these data suggest that MAX2 modulates multiple hormone pathways to affect photomorphogenesis.
2011
Bu Q, Castillon A, Chen F, Zhu L, Huq E. Dimerization and blue light regulation of PIF1 interacting bHLH proteins in Arabidopsis. Plant Mol Biol. 77 (4-5) :501-11.Abstract
Phytochrome Interacting Factor 1 (PIF1), a basic helix-loop-helix (bHLH) protein, functions as a negative regulator of various facets of photomorphogenesis. To indentify PIF1-interacting proteins, we performed yeast two-hybrid screening using PIF1 as a bait and identified a group of proteins including PIF1 itself, PIF3 and long hypocotyl in far-red 1 (HFR1), an atypical HLH protein. Directed yeast two-hybrid interaction assays showed that PIF1 can form heterodimers with all other PIFs as well as with HFR1. PIF1 and PIF3 interacted with each other in both in vitro and in vivo co-immunoprecipitation assays. PIF1-PIF3 heterodimer also bound to a G-box DNA sequence element in vitro. To understand the biological significance of these interactions, a pif1pif3 double mutant was obtained and characterized. Analyses of the single and double mutants showed that PIF3 plays a prominent role in repressing photomorphogenesis under continuous blue light conditions. pif1 and pif3 showed additive phenotypes more prominently under discontinuous blue light conditions. Similar to PIF1, PIF3 was also rapidly phosphorylated, poly-ubiquitylated and degraded in response to blue light. PIF3 also interacted with phytochromes in response to blue light. A PIF3 mutant defective in interaction with both phyA and phyB displayed reduced degradation under blue light, suggesting that phy-interaction was necessary for the blue light-induced degradation of PIF3. Taken together, these data suggest a combinatorial control of photomorphogenesis by bHLH proteins in response to light in Arabidopsis.
Zhu L, Huq E. Mapping functional domains of transcription factors. Methods Mol Biol. 754 :167-84.Abstract
Transcription factors are modular in nature in all organisms. In general, they have a DNA binding domain, one or more transcription activation and/or repressor domain, and often a dimerization domain. In many cases, transcription factors also have other protein-protein interaction domain(s). Mapping these functional domains in transcription factors is critical in understanding their molecular function. In this chapter, protocols for mapping the DNA binding domain and the transcription activation domain of a bHLH class of transcription factor are described. In principle, these protocols can be applied to other classes of transcription factors for mapping their functional domains.

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