Calmodulin7: recent insights into emerging roles in plant development and stress.

KEY MESSAGE: Analyses of the function of Arabidopsis Calmodulin7 (CAM7) in concert with multiple regulatory proteins involved in various signal transduction processes. Calmodulin (CaM) plays various regulatory roles in multiple signaling pathways in eukaryotes. Arabidopsis CALMODULIN 7 (CAM7) is a unique member of the CAM family that works as a transcription factor in light signaling pathways. CAM7 works in concert with CONSTITUTIVE PHOTOMORPHOGENIC 1 and ELONGATED HYPOCOTYL 5, and plays an important role in seedling development. Further, it is involved in the regulation of the activity of various Ca2+-gated channels such as cyclic nucleotide gated channel 6 (CNGC6), CNGC14 and auto-inhibited Ca2+ ATPase 8. Recent studies further indicate that CAM7 is also an integral part of multiple signaling pathways including hormone, immunity and stress. Here, we review the recent advances in understanding the multifaceted role of CAM7. We highlight the open-ended questions, and also discuss the diverse aspects of CAM7 characterization that need to be addressed for comprehensive understanding of its cellular functions.

MYC2 influences salicylic acid biosynthesis and defense against bacterial pathogens in Arabidopsis thaliana.

Arabidopsis MYC2 is a basic helix-loop-helix transcription factor that works both as a negative and positive regulator of light and multiple hormonal signaling pathways, including jasmonic acid and abscisic acid. Recent studies have suggested the role of MYC2 as a negative regulator of salicylic acid (SA)-mediated defense against bacterial pathogens. By using myc2 mutant and constitutively MYC2-expressing plants, we further show that MYC2 also positively influences SA-mediated defense; whereas, myc2 mutant plants are resistant to virulent pathogens only, MYC2 over-expressing plants are hyper-resistant to multiple virulent and avirulent strains of bacterial pathogens. MYC2 promotes pathogen-induced callose deposition, SA biosynthesis, expression of PR1 gene, and SA-responsiveness. Using bacterially produced MYC2 protein in electrophoretic mobility shift assay (EMSA), we have shown that MYC2 binds to the promoter of several important defense regulators, including PEPR1, MKK4, RIN4, and the second intron of ICS1. MYC2 positively regulates the expression of RIN4, MKK4, and ICS1; however, it negatively regulates the expression of PEPR1. Pathogen inoculation enhances MYC2 association at ICS1 intron and RIN4 promoter. Mutations of MYC2 binding site at ICS1 intron or RIN4 promoter abolish the associated GUS reporter expression. Hyper-resistance of MYC2 over-expressing plants is largely light-dependent, which is in agreement with the role of MYC2 in SA biosynthesis. The results altogether demonstrate that MYC2 possesses dual regulatory roles in SA biosynthesis, SA signaling, pattern-triggered immunity (PTI), and effector-triggered immunity (ETI) in Arabidopsis.

Description of the Methylene Amidogene Radical and Its Anion with an Economical Treatment of Correlation Effects Using Density Functional Theory Orbitals.

The ground and low-lying excited state electronic structural properties (such as equilibrium geometries, harmonic frequencies, excitation energies, barrier energy, and so on) of the methylene amidogene radical (H2CN) and its anion (H2CN-) have been studied using the CASCI (complete active space configuration interaction) and SSMRPT (state-specific multireference Møller-Plesset perturbation theory) methods with density function theory (DFT) orbitals. Here, the span of the active orbitals have been obtained from Kohn-Sham DFT using B3LYP exchange-correlation functionals in the CASCI (DFT-CASCI) approximation to describe nondynamic correlation associated with electronic degeneracies. The DFT-SSMRPT protocol provides an attractive way to deal with both dynamical and nondynamical correlation effects in strongly correlated systems such as H2CN and H2CN-. The present work clearly indicates that the electronic absorption band near 35,050 cm-1 corresponds to the B̃2A1 ← X̃2B2 transition. DFT-SSMRPT findings are in close agreement with high-level theoretical estimates. It is concluded that the transition at 1725 cm-1 could be due to the CN stretching of the trans-HCNH isomer which is originally assigned to the CN stretch of H2CN in the experiment. The present results confirm most of the previous vibrational assignments. It is not possible to definitively assign a transition to the 35,600 cm-1 band with the present estimations, suggesting further experiment is urgently called for.

A Fock space coupled cluster based probing of the single- and double-ionization profiles for the poly-cyclic aromatic hydrocarbons and conjugated polyenes.

Sequential formation of a poly-cyclic aromatic hydrocarbon (PAH) dication in the H I regions of the interstellar medium (ISM) is proposed to be a function of internal energy of the doubly ionized PAHs, which, in turn, is dependent on the single- and double-ionization potentials of the system. This sets a limit on the single- and double-ionization energies of the system(s) that can further undergo sequential absorption of two photons, leading to a dication (PAH+2). Here, we report the single-ionization (I+1) and double-ionization (I+2) energies and the I+2/I+1 ratio for some selected PAHs and conjugated polyenes obtained using the Fock space coupled cluster technique, enabling simultaneous consideration of several electronic states of different characters. The I+2 to I+1 ratio bears a constant ratio, giving allowance to determine I+2 from the knowledge of single-ionization (I+1) and vice versa. Our observations are in good agreement with the established literature findings, confirming the reliability of our estimates. The measured single- and double-ionization energies further demonstrate that the sequential formation and fragmentation of a PAH dication in the H I regions of the ISM for systems such as benzene and conjugated polyenes such as ethylene and butadiene are quite unlikely because I+2-I+1 for such system(s) is higher than the available photon energy in the H I regions of the ISM. Present findings may be useful to understand the formation and underlying decay mechanisms of multiply charged ions from PAHs and related compounds that may accentuate the exploration of the phenomenon of high-temperature superconductivity.

Functional interrelation of MYC2 and HY5 plays an important role in Arabidopsis seedling development.

Arabidopsis MYC2 bHLH transcription factor plays a negative regulatory role in blue light (BL)-mediated seedling development. HY5 bZIP protein works as a positive regulator of multiple wavelengths of light and promotes photomorphogenesis. Both MYC2 and HY5, belonging to two different classes of transcription factors, are the integrators of multiple signaling pathways. However, the functional interrelations of these two transcription factors in seedling development remain unknown. Additionally, whereas HY5-mediated regulation of gene expression has been investigated in detail, the transcriptional regulation of HY5 itself is yet to be understood. Here, we show that HY5 and MYC2 work in an antagonistic manner in Arabidopsis seedling development. Our results reveal that HY5 expression is negatively regulated by MYC2 predominantly in BL, and at various stages of development. On the other hand, HY5 negatively regulates the expression of MYC2 at various wavelengths of light. In vitro and in vivo DNA-protein interaction studies suggest that MYC2 binds to the E-box cis-acting element of HY5 promoter. Collectively, this study demonstrates a coordinated regulation of MYC2 and HY5 in blue-light-mediated Arabidopsis seedling development.

Investigation of Multiple-Bond Dissociation Using Brillouin-Wigner Perturbation with Improved Virtual Orbitals.

An improved virtual orbital complete active space configuration interaction reference function-based multireference Brillouin-Wigner perturbation approach (IVO-BWMRPT) that avoids the numerical divergence because of the intruder problem by focusing on obtaining a single root of the many-body Hamiltonian is used to compute dissociation energy surfaces and spectroscopic constants of C2, N2, and CN radicals. For such correlated molecules, the computational demand to delineate the wavefunction exactly is quite challenging. The IVO-BWMRPT method, a convenient and effective way to handle quasidegenerate situations, accurately captures different correlations and is capable of treating the variation of multiconfigurational nature of wave functions that occur during the multiple-bond breaking processes. Spectroscopic constants extracted from the computed surface are in good agreement with experimental or benchmark results, indicating that the components of the IVO-BWMRPT scheme must perform in harmony for providing a well-behaved and consistent description of all computed bond lengths.

Taming the excited states of butadiene, hexatriene, and octatetraene using state specific multireference perturbation theory with density functional theory orbitals.

To compute the electronic excitation energies, a state-specific multireference Møller-Plesset perturbation theory (SSMRPT) with a complete active space configuration interaction reference function constructed using the orbitals obtained by the density functional theory (DFT) is presented as an accurate, as well as computationally affordable, and efficient protocol at the level of second order. The global hybrid B3LYP (Becke, 3-parameter, Lee-Yang-Parr) functional has been used to generate orbitals. The present method, called DFT-SSMRPT, uses perturbers that are individual Slater determinants and accounts for the coupling between the nondynamical and dynamical correlation effects. We have applied the new method to compute excitation energies in conjugated systems of π-electrons such as trans-1,3-butadiene, trans,trans-1,3,5-hexatriene, and all-trans-1,3,5,7-octatetraene. The ordering of the excited states is correctly reproduced by the DFT-SSMRPT calculations. The relative ordering of low-lying excited 1Bu and 1Ag states alters when the length of the polyene changes. The results match reasonably well with the literature including experimental and best theoretical findings. The accuracy of the method is sufficient to discern the energy gap between the close low-lying singlet and triplet states. The DFT-SSMRPT appears as an affordable computational ab initio avenue for a qualitatively correct description of excitation energies.

Simplified Treatment of Electronic Structures of the Lowest Singlet and Triplet States of Didehydropyrazines.

Experiments on the lowest lying singlet and triplet states of didehydropyrazine isomers (that focus on energy gaps, geometries, and vibrational frequencies) have been carried out computationally by implementing the improved virtual orbital-based multireference (MR) ab initio methods. Pyrazines possess a reasonable MR nature making their description challenging with the conventional quantum chemical approaches. Although wave functions of the diradicals usually have two dominant configurations, a larger reference space is warranted to consistently and accurately describe pyrazine diradicals indicating the complex nature of the systems. Present calculations predict a singlet ground state for ortho- and para-pyrazines, while a triplet ground state is suggested for the meta isomer. The adiabatic singlet-triplet energy gap for the para form is found to be notably higher (by 28.4 kcal/mol) compared to ortho (2.0 kcal/mol) and meta isomers (-5.1 kcal/mol). Accurate and reliable computations are imperative for forecasting the state-ordering in such diradicals. The structural properties obtained from the present calculations lend strong support toward future experimental explorations on these systems.

Ab Initio Probing of the Ground State of Tetraradicals: Breakdown of Hund's Multiplicity Rule.

The electronic structure of organic σ-type polyradical including 2,4,6-tridehydropyridine radical cation (246-TDHP) and three isomers of tetradehydrobenzene (TDHB) have been studied using a computationally robust and cost-effective second-order multireference perturbative model which provides a balanced treatment of nondynamic and dynamic contributions to the electron correlation problem in the ground or excited electronic states which are imperative for predicting structural properties (e.g., ground state multiplicity, energy gaps between high-spin and low-spin states, etc.) of polyradicals. Energy gaps are useful to capture insight into the degree of interaction between the radical sites. An important finding of this study is that the tetraradicals considered here possess singlet ground states, contrary to Hund's rule. Present findings are in close agreement with the available high-level ab initio estimates at attainable cost implying that a perturbative description of the systems is adequate. The impact of N+ on the nature of ground state for the 246-TDHP have also analyzed. The singlet-triplet energy gaps for 1245- and 1234-TDHB are smaller than for o-benzyne mainly due to the ring strain. 1235-TDHB is 14.42 and 11.05 kcal/mol lower in energy than 1245- and 1234-isomers, respectively. IVO-SSMRPT predicts 1A1-3B2 and 1A1-5B2 gaps of 25.84 and 105.15 kcal/mol, respectively for the 246-TDHP cation.

Fock-space multireference coupled cluster calculations of Auger energies of noble gas elements using relativistic spinors.

We report the Auger and Coster-Kronig transition energies (related to double ionization potentials) of noble gas elements obtained using the Fock-space multireference coupled cluster (FSMRCC) method with relativistic spinors. The resulting Auger and Coster-Kronig lines are found to be in agreement with the experimental data and with other reference theoretical estimates. To the best of our knowledge, no prior report of relativistic calculations is available for Auger transition energies at the FSMRCC level of theory. The ionization potentials resulted from this method with no extra cost are also found to be in agreement with experiment, particularly the outer-valence ones. Interestingly, the FSMRCC and the multiconfiguration Dirac-Fock calculations exhibit an inversion in the 3P energy levels of the xenon atom for N4,5-O23O23 Auger transitions, where the 3Pj state energies appear in the order J = 1, 0, 2, a feature which can be verified experimentally.

GBF1 differentially regulates CAT2 and PAD4 transcription to promote pathogen defense in Arabidopsis thaliana.

G-BOX BINDING FACTOR 1 (GBF1) influences light-regulated seedling development in Arabidopsis, and inhibits CATALASE 2 (CAT2) expression during senescence. CAT2 functions as a scavenger of hydrogen peroxide. The role of GBF1 in the defense response is not known. We report here that GBF1 positively influences the defense against virulent and avirulent strains of Pseudomonas syringae. The gbf1 mutants are susceptible, whereas GBF1 over-expresser transgenic plants are resistant to bacterial pathogens. GBF1 negatively regulates pathogen-induced CAT2 expression and thereby positively regulates the hypersensitive response. In addition to CAT2 promoter, GBF1 binds to the G-box-like element present in the intron of PHYTOALEXIN DEFICIENT 4 (PAD4). This association of GBF1 with PAD4 intron is enhanced upon pathogenesis. GBF1 positively regulates PAD4 transcription in an intron-dependent manner. GBF1-mediated positive regulation of PAD4 expression is also evident in gbf1 mutant and GBF1 over-expression lines. Similar to pad4 mutants, pathogen-induced camalexin and salicylic acid (SA) accumulation, and expression of SA-inducible PATHOGENESIS RELATED1 (PR1) gene are compromised in the gbf1 mutant. Exogenous application of SA rescues the loss-of-defense phenotypes of gbf1 mutant. Thus, altogether, our results demonstrate that GBF1 is an important component of the plant defense response that functions upstream of SA accumulation and, by oppositely regulating CAT2 and PAD4, promotes disease resistance in Arabidopsis.

Interaction of Arabidopsis TGA3 and WRKY53 transcription factors on Cestrum yellow leaf curling virus (CmYLCV) promoter mediates salicylic acid-dependent gene expression in planta.

MAIN CONCLUSION: This paper highlighted a salicylic acid-inducible Caulimoviral promoter fragment from Cestrum yellow leaf curling virus (CmYLCV). Interaction of Arabidopsis transcription factors TGA3 and WRKY53 on CmYLCV promoter resulted in the enhancement of the promoter activity via NPR1-dependent salicylic acid signaling. Several transcriptional promoters isolated from plant-infecting Caulimoviruses are being presently used worldwide as efficient tools for plant gene expression. The CmYLCV promoter has been isolated from the Cestrum yellow leaf curling virus (Caulimoviruses) and characterized more than 12 years ago; also we have earlier reported a near-constitutive, pathogen-inducible CmYLCV promoter fragment (-329 to +137 from transcription start site; TSS) that enhances stronger (3×) expression than the previously reported fragments; all these fragments are highly efficient in monocot and dicot plants (Sahoo et al. Planta 240: 855-875, 2014). Here, we have shown that the full-length CmYLCV promoter fragment (-729 to +137 from TSS) is salicylic acid (SA) inducible. In this context, we have performed an in-depth study to elucidate the factors responsible for SA-inducibility of the CmYLCV promoter. We found that the as-1 1 and W-box1 elements (located at -649 and -640 from the TSS) of the CmYLCV promoter are required for SA-induced activation by recruiting Arabidopsis TGA3 and WRKY53 transcription factors. Consequently, as a nascent observation, we established the physical interaction between TGA3 and WYKY53; also demonstrated that the N-terminal domain of TGA3 is sufficient for the interaction with the full-length WRKY53. Such interaction synergistically activates the CmYLCV promoter activity in planta. Further, we found that activation of the CmYLCV promoter by SA through TGA3 and WRKY53 interaction depends on NPR1. Finally, the findings presented here provide strong support for the direct regulatory roles of TGA3 and WRKY53 in the SA and NPR1-dependent activation of a Caulimoviral promoter (CmYLCV).

A mitogen-activated protein kinase cascade module, MKK3-MPK6 and MYC2, is involved in blue light-mediated seedling development in Arabidopsis.

Mitogen-activated protein kinase (MAPK) pathways are involved in several signal transduction processes in eukaryotes. Light signal transduction pathways have been extensively studied in plants; however, the connection between MAPK and light signaling pathways is currently unknown. Here, we show that MKK3-MPK6 is activated by blue light in a MYC2-dependent manner. MPK6 physically interacts with and phosphorylates a basic helix-loop-helix transcription factor, MYC2, and is phosphorylated by a MAPK kinase, MKK3. Furthermore, MYC2 binds to the MPK6 promoter and regulates its expression in a feedback regulatory mechanism in blue light signaling. We present mutational and physiological studies that illustrate the function of the MKK3-MPK6-MYC2 module in Arabidopsis thaliana seedling development and provide a revised mechanistic view of photomorphogenesis.

Arabidopsis CAM7 and HY5 physically interact and directly bind to the HY5 promoter to regulate its expression and thereby promote photomorphogenesis.

Arabidopsis thaliana CALMODULIN7 (CAM7), a unique member of the calmodulin gene family, plays a crucial role as a transcriptional regulator in seedling development. The elongated HYPOCOTYL5 (HY5) bZIP protein, an integrator of multiple signaling pathways, also plays an important role in photomorphogenic growth and light-regulated gene expression. CAM7 acts synergistically with HY5 to promote photomorphogenesis at various wavelengths of light. Although the genetic relationships between CAM7 and HY5 in light-mediated seedling development have been demonstrated, the molecular connectivity between CAM7 and HY5 is unknown. Furthermore, whereas HY5-mediated gene regulation has been fairly well investigated, the transcriptional regulation of HY5 is largely unknown. Here, we report that HY5 expression is regulated by HY5 and CAM7 at various wavelengths of light and also at various stages of development. In vitro and in vivo DNA-protein interaction studies suggest that HY5 and CAM7 bind to closely located T/G- and E-box cis-acting elements present in the HY5 promoter, respectively. Furthermore, CAM7 and HY5 physically interact and regulate the expression of HY5 in a concerted manner. Taken together, these results demonstrate that CAM7 and HY5 directly interact with the HY5 promoter to mediate the transcriptional activity of HY5 during Arabidopsis seedling development.

A simplified ab initio treatment of diradicaloid structures produced from stretching and breaking chemical bonds.

The present investigation reports on the prospect of using state specific multireference perturbation theory (SSMRPT) with an improved virtual orbital complete active space configuration interaction (IVO-CASCI) reference function (IVO-SSMRPT) to generate potential energy surfaces (PESs) for molecular systems [such as CH4, C2H6, C2H4, H2O2, LiH, and KN] by stretching and breaking of suitable bonds with modest basis sets. We have also revisited the dissociation energy profile of triplet ketene which exhibits a step-like structure in the observed rate. The application of the method has also been made to the ionization energies of H2O. Although the perturbative corrections are obtained by the diagonalization of the effective Hamiltonian, in IVO-SSMRPT, only one physically relevant solution is achievable. It is parameter free and does not require any threshold to avoid the intruder problem. It is strictly size-extensive and size-consistent provided that local orbitals are used. The PESs obtained with our approach are smooth all along the reaction path. Our estimates are in close agreement with the available reference data indicating that IVO-SSMRPT is a robust paradigm for the accurate computation of ground, excited and ionized states as it captures the mutual inter-play of different flavors of correlation effects in a balanced and accurate way.

Four-Component Relativistic State-Specific Multireference Perturbation Theory with a Simplified Treatment of Static Correlation.

The relativistic multireference (MR) perturbative approach is one of the most successful tools for the description of computationally demanding molecular systems of heavy elements. We present here the ground state dissociation energy surfaces, equilibrium bond lengths, harmonic frequencies, and dissociation energies of Ag2, Cu2, Au2, and I2 computed using the four-component (4c) relativistic spinors based state-specific MR perturbation theory (SSMRPT) with improved virtual orbital complete active space configuration interaction (IVO-CASCI) functions. The IVO-CASCI method is a simple, robust, useful and lower cost alternative to the complete active space self-consistent field approach for treating quasidegenerate situations. The redeeming features of the resulting method, termed as 4c-IVO-SSMRPT, lies in (i) manifestly size-extensivity, (ii) exemption from intruder problems, (iii) the freedom of convenient multipartitionings of the Hamiltonian, (iv) flexibility of the relaxed and unrelaxed descriptions of the reference coefficients, and (v) manageable cost/accuracy ratio. The present method delivers accurate descriptions of dissociation processes of heavy element systems. Close agreement with reference values has been found for the calculated molecular constants indicating that our 4c-IVOSSMRPT provides a robust and economic protocol for determining the structural properties for the ground state of heavy element molecules with eloquent MR character as it treats correlation and relativity on equal footing.

A confinement induced spectroscopic study of noble gas atoms using equation of motion architecture: Encapsulation within fullerene's voids.

A relativistic study of spectroscopic properties of the endohedral fullerenes Ng@C60q (where Ng = He, Ne and q=0,±1,±2 are the charges) associated with the C60 molecule has been done using the equation of motion coupled cluster (EOM-CC) methodology. Specific properties estimated are the transition energies, dipole oscillator strengths, and transition probabilities for the low-lying excitations 1s2(1S0) → 1snp (1P1) (n = 2, 3, 4) for He@C60q and 1s22s22p6 (1S0) → 1s22s22p5ns∕nd (1P1) (n = 3, 4) for Ne@C60q, which have been compared with those for the isolated atom to depict the confinement effect of the host molecule on the encapsulated atom. This is accomplished by introducing an effective potential to the atomic Hamiltonian induced by the fullerene moiety and its charge. The EOM-CC results have been compared with those estimated with the random phase approximation (and configuration interaction singles) to understand the effect of electron correlation under such confinement. The systematic and interesting behavior of the properties is highlighted indicating the effect of fullerene cage potential on the redistribution of electron density of the guest atom.

Improved virtual orbitals in state specific multireference perturbation theory for prototypes of quasidegenerate electronic structure.

The state-specific multireference perturbation theory (SSMRPT) with an improved virtual orbital complete active space configuration interaction (IVO-CASCI) reference function [called as IVO-SSMRPT] is used to investigate the energy surface, geometrical parameters, molecular properties of spectroscopic interest for the systems/situations [such as BeH2, BeCH2, MgCH2, Si2H4, unimolecular dissociation of H2CO, and intramolecular reaction pathways of 1,3-butadiene] where the effect of quasidegeneracy cannot be neglected. The merit of using the IVO-CASCI rather than complete active space self-consistent field (CASSCF) is that it is free from iterations beyond those in the initial SCF calculation and the convergence difficulties that plague CASSCF calculations with increasing size of the CAS. While IVO-CASCI describes the non-dynamical correlation, the SSMRPT scheme is a good second-order perturbative approximation to account for the rest of the correlation energy. Our IVO-SSMRPT method is instrumental in avoiding intruder states in an size-extensive manner and allows the revision of the content of wave function in the model space. It can treat model as well as real systems with predictive accuracy, as is evident from the fairly nice accordance between our estimates, and high-level theoretical results. Our estimates also corroborate well with some experimental findings.

Communication: Viewing the ground and excited electronic structures of platinum and its ion through the window of relativistic coupled cluster method.

Highly accurate electronic structure calculations are often needed to supplement scant experimental data. We report the ground 3D3 and some selected low lying excited/ionized states of Pt and its ions obtained using the Fock space multireference coupled cluster method with four-component relativistic spinors. The present work establishes the stability of the 2S1/2 state of its negative ion and reproduces the binding energy of this state within 10 cm-1. The first ionization potential (cm-1) is estimated to be 72 005, deviating from the experiment by just 200 (0.3%). We also report the magnetic hyperfine coupling constants (A) of Pt and its ions. The present calculation provides the A value (GHz) of the 3D3 state of Pt to be 5.78 exhibiting very good agreement with the experimental data of 5.70. To our knowledge, this is the first relativistic ab initio calculation of the ionization potential and magnetic hyperfine coupling constant for the neutral and ionic states of Pt at a high level of correlation treatment.

Interaction of MYC2 and GBF1 results in functional antagonism in blue light-mediated Arabidopsis seedling development.

Regulations of Arabidopsis seedling growth by two proteins, which belong to different classes of transcription factors, are poorly understood. MYC2 and GBF1 belong to bHLH and bZIP classes of transcription factors, respectively, and function in cryptochrome-mediated blue light signaling. Here, we have investigated the molecular and functional interrelation of MYC2 and GBF1 in blue light-mediated photomorphogenesis. Our study reveals that MYC2 and GBF1 colocalize and physically interact in the nucleus. This interaction requires the N-terminal domain of each protein. The atmyc2 gbf1 double mutant analyses and transgenic studies have revealed that MYC2 and GBF1 act antagonistically and inhibit the activity of each other to regulate hypocotyl growth and several other biological processes. This study further reveals that MYC2 and GBF1 bind to HYH promoter and inhibit each other through non-DNA binding bHLH-bZIP heterodimers. These results, taken together, provide insights into the mechanistic view on the concerted regulatory role of MYC2 and GBF1 in Arabidopsis seedling development.