Alfalfa (Medicago sativa cv. Zhao dong) seeds, provided by the Livestock and Veterinary Research Institute of Heilongjiang Academy of Agricultural Sciences, were germinated in distilled water for 3 days. Seedlings were cultivated in Hoagland’s solution (10 μM MnSO₄, 100 μM H₃BO₃, 0.1 μM CuSO₄, 0.1 μM Na₂MoO₄, 30 μM ZnSO₄, 5 μM KI, 0.1 μM CoCl₂, 4 mM CaCl₂, 1 mM MgSO₄, 1 mM KH₂PO₄, 100 μM Fe(III)-EDTA and 5 mM KNO₃, pH 5.8) at 25°C, 16 h light/8 h darkness (Cui et al., 2013). The culture solution was changed every 3 days. Four-week-old alfalfa seedlings were selected for treatment. Alfalfa seedlings were treated with +Cd stress for 3 days to analyze the expression of iron-deficiency response genes. The stress concentration of hydroponics in Hoagland’s solution was −Fe (0 µM Fe-EDTA+0 μM CdCl₂), +Cd (100 µM Fe-EDTA+90 μM CdCl₂), −Fe+Cd (0 µM Fe-EDTA+90 μM CdCl₂), and the control (CK, 100 µM Fe-EDTA+0 μM CdCl₂) (Dai et al., 2017; Yang et al., 2021). Alfalfa seedlings were treated with −Fe, +Cd, −Fe+Cd, and CK for 24 h, and the roots, stems, and leaves were collected, respectively, to analyze the tissue-specific expression of MsbHLH115. Alfalfa seedlings were treated with 50 μM, 70 μM, 90 μM, 100 μM, and 200 μM Cd for 24 h to detect the response of MsbHLH115. Alfalfa seedlings were treated with −Fe, +Cd, −Fe+Cd, and CK for 6, 12, 24, 48, and 72 h to detect the temporal properties of MsbHLH115 expression. The above samples were collected, frozen in liquid nitrogen, and stored at −80°C to extract total RNA for Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR).

The cultivation method of Arabidopsis seeds (ecotype Columbia, Col-0) is as follows: surface-sterilized with 10% NaClO for 15 min and then washed six times with distilled water, seeds were plated on 1/2 MS medium (Murashige and Skoog, 1962) with 1% sucrose, 0.8% agar, and 100 µM Fe-EDTA at pH 5.8, cultivated at 22°C, 16 h light/8 h darkness. The Arabidopsis seedlings were treated with −Fe, +Cd, −Fe+Cd, and CK for phenotype observation and physiological detection.

qRT-PCR was performed by the Bio-Rad CFX96 detection system (Bio-Rad, Hercules, CA, USA) using the Top Green qPCR Super Mix (Transgenic Biotechnology Company, Beijing, China). The relative expression levels of genes were calculated using the 2^-ΔΔCT comparative method as described by (Livak and Schmittgen, 2001). MsACTIN2 (Wang et al., 2015) and AtACTIN2 (AT3G18780) (Czechowski et al., 2005) were used as internal reference genes. The Arabidopsis genes bHLH115, bHLH104, bHLH121, FIT, bHLH39, FRO2, IRT1, VTL, HMA3, MTP3, NAS4, IREG2, SOD1, POD1, RbohB, RbohD, RbohF, BGLU14, GSTL1/2/4/6, AtP5CS, and AtPRODH were detected in the transgenic Arabidopsis and WT under 90 μM CdCl₂ for 24 h. Primers used in this study were designed using Primer 5.0 software (http://www.premierbiosoft.com/primerdesign/) and online NCBI Primer-BLAST (http://www.ncbi.nlm.nih.gov/tools/primer-blast/), and listed in Supplementary Table S1 .

Total RNA was extracted using the Total RNA Extraction Kit (Omega BioTek, Norcross, GA, USA). Then, RNA was reverse transcribed to cDNA using ReverTra Ace^®qPCR RT Master Mix with gDNA Remover (TOYOBO, Japan). Total DNA was extracted using the Total DNA Extraction Kit (OMGAE, USA). Primers are shown in Supplementary Table S1 . The PCR products were cloned into the pMD18-T vector (Takara) and sequenced (Sangon, Shanghai, China). Sequence alignment was performed using DANMAN software. The structured domain was obtained through the SMART (HTTP://smart.embl-heidelberg.de/ accessed on 9 June 2022). Phylogenetic analysis was performed using the MEGA 7.0 with neighbor-joining and a bootstrap repeat value of 1000 times. The cis-elements were analyzed using PlantCare (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/ accessed on 9 June 2022). Finally, TBtools was used to splice.

MsbHLH115 without the stop codon was inserted into the pBWA(V)HS vector (provided by BioRun Biotechnology Co., Ltd, Wuhan, China), fused with the green fluorescent protein (GFP) driven by the 35S promoter. The recombinational pBWA(V)HS-35S::MsbHLH115-Glosgfp plasmid was transformed into Arabidopsis protoplasts through polyethylene glycol treatment (Yoo et al., 2007). nuclear localization of the MsbHLH115-GFP fusion protein. Red fluorescent protein (RFP) is used as a nuclear localization marker. Fluorescence signals were visualized after 12-16 h using a confocal laser-scanning microscope (Olympus FluoView FV1000, Olympus, Tokyo, Japan).

The MsbHLH115 promoter fragment was digested with Bgl II and BamH I to construct a pBI121-MsbHLH115pro::GUS and transformed into Agrobacterium rhizogenes K599 by the freeze-thaw method. Soybean (Glycine max cv. Williams 82) hairy roots were induced by needle insertion of K599 (Kereszt et al., 2007). The soybean complex was cultivated in 1/2 Hoagland’s solution for 7 days. The hairy roots were divided into two parts and treated with −Fe, +Cd, −Fe+Cd, and CK for 24 h, respectively (Li et al., 2023). Histochemical staining for GUS was conducted following the method described previously (Li et al., 2023). The photograph was taken using a stereomicroscope (EZ4-HD LEICA, Germany) coupled with a color charge-coupled device (CCD) camera (Zeiss, Germany). The GUS enzyme activity was detected using a plant β-glucuronidase GUS ELISA kit (Coolaber, SL7160) and enzyme-labeler machine (Ferdi Bio, FlexA-200). The absorbance (OD value) was measured at 450 nm wavelength. The GUS enzyme activity was calculated according to the standard curve previously drawn.

The MsbHLH115 gene was digested with Xba I and BamH I and inserted into pBI121. The recombinant vector pBI121-35S:: MsbHLH115 was introduced into Agrobacterium tumefacient GV3101 by freeze-thaw method and then transformed into Arabidopsis by the filter-dipping process (Clough and Bent, 1998). PCR and qRT-PCR confirmed the MsbHLH115 transgenic Arabidopsis. The primers used are listed in Supplementary Table S1 . The MsbHLH115 transgenic Arabidopsis produced offspring by self-breeding. T₃ seedlings of MsbHLH115 transgenic Arabidopsis and WT were cultivated in 1/2 MS medium for 5 days. The growth of MsbHLH115ox lines and WT plants were treated with different combinations of Fe and Cd treatments for 7 days. The growth of #12-2 plants was compared when different root parts were in the split medium with combinations of Fe and Cd treatments for 7 days. A root-tip elongation experiment was applied on MsbHLH115ox lines and WT. The plants were planted on a split medium and were treated with different combinations of Fe and Cd treatments. The part near the root tip (RT) and the part distal the root tip (RS) were exposed to different treatments for 7 days. Phenotypic photographs were taken, and root length and fresh weight were recorded.

Several previous studies have reported that bHLH115 plays a role in iron homeostasis. In this study, we performed experiments focused on Cd treatment rather than iron deficiency treatment to better explain its role in Cd tolerance, The Arabidopsis leaves were collected and frozen at −80°C for physiological index determination. The total chlorophyll content was measured according to the method described by (Yavari et al., 2021). Leaves from seedlings grown on a medium were collected and ground to powder in liquid nitrogen. The powder was resuspended in 80% (v/v) acetone on ice and centrifuged at 10,000 g at 4°C for 5 min. A664 and A648 were inspected by spectroscopy absorbance measurements, and the chlorophyll a and b contents were calculated according to those described previously. NBT and DAB staining methods refer to (Zhang et al., 2011). For NBT staining, the leaves were soaked in darkness with 1 mg/mL nitro blue tetrazole (pH 7.8) for 40-60 min. For DAB staining, the leaves were washed with 1 mg/mL diaminobenzidine (pH 7.0) in the dark for 8 h. The leaves were then boiled in an ethanol glycerin (3:1) solution for 20 min and photographed. Evans Blue staining was performed as described by (Baker and Mock, 1994). Root damage was evaluated by staining with Evans blue solution (0.25%, w/v), The leaves were then boiled in an ethanol glycerin (3:1) solution for 20 min and photographed. H₂O₂ content was determined according to Elstner, 1976 and the specific procedures were carried out as per the manufacturer’s instructions. The tissues were homogenized in an ice bath with 0.1% trichloroacetic acid (TCA) and centrifuged at 12,000 ×g for 15 min. The reaction solution contained 0.5 mL of potassium dihydrogen phosphate buffer (pH 7.0), 1 mL of 1 M potassium iodide (KI), and 0.5 mL of supernatant. Following that, the mixture was kept at 25°C for 1 h and its absorbance was measured at the wavelength of 390 nm. The H₂O₂ content was calculated according to the standard curve obtained using different concentrations of H₂O₂. O₂ ⁻ content was determined according to Elstner, 1976 and the specific procedures were carried out as per the manufacturer’s instructions. The O₂ ⁻ content was determined by monitoring the absorbance of azo compounds at 530 nm (Willekens et al., 1997). The malondialdehyde (MDA) content of the detailed experimental method refers to (Puckette et al., 2007). Approximately 0.1 g plant tissue was homogenized in 1.5 mL 5% TCA that contained 0.25% TBA, incubated at 100°C for 30 min, cooled to room temperature, and then centrifuged at 15000 ×g for 10 min. The concentration of TBA-reactive substance was then determined by measuring the absorbance of the resulting supernatant at 440, 532, and 600 nm. SOD activity was determined refer to (Giannopolitis et al., 1977) by monitoring the absorbance of blue formazan generated by the reaction of the remaining O₂ ⁻ with nitro-blue tetrazolium at 560 nm. The Catalase (CAT) activity was determined refer to (Soydam Aydin et al., 2013), tested in potassium phosphate buffer (pH 7.8) containing 3 mM H₂O₂ at 240 nm. The glutathione-s-transferase (GST) activity was determined using a previously described method (Nagalakshmi and Prasad, 2001). The reaction mixture contained 10 μL of 0.1 M 1-chloro-2,4-dinitrobenzene (CDNB), 100 μL of 10 mM GSH, 500 μL of 0.2 M KPO4 buffer, and 390 μL of distilled water. and the increase in absorbance was measured at the wavelength of 340 nm. One unit of GST was defined as the amount of enzyme that increased the absorbance by a unit of 1 per min, at the wavelength of 340 nm. Ferric-chelate reductase activity was determined using a previously described method (Li et al., 2023). The roots were immerged into Fe (III) reductase detection solution (0.1 mM Fe (III)-EDTA and 0.3 mM ferrozine), and 10 intact plants were used in each independent experiment. The reaction solution was placed in darkness at 22°C, and then the supernatant was analyzed spectrophotometrically at 562 nm. Lastly, the FCR activity was measured with the use of a molecular extinction coefficient of 28.6 mM^-1cm^-1.

The 13-day-old Arabidopsis seedlings were treated with or without 90 μM CdCl₂ for 3 days. The roots and shoots were counted and harvested separately, and then washed with ddH₂O 3~4 times. After being dried in a conventional oven at 70°C, the samples were digested completely in 65%~68% HNO₃ at 120°C. The Fe, Cd, Zn, Mn, and Cu concentrations were determined as described by (Takahashi et al., 2019). Using inductively coupled plasma emission spectrometry ICP-OES8000 (Perkin Elmer, USA). The Fe, Cd, Zn, Mn, and Cu concentrations were calculated according to the method previously described (Wu et al., 2012).

The MsbHLH115 ORF sequence was cloned into the pGADT7 vector to construct the recombinant vector pGADT7-MsbHLH115. The E-box element sequence (CAAATG) and mutated E-box element sequence (ACAATG) were cloned into the pAbAi vector to form pE-box-AbAi and pmE-box-AbAi. The p53-AbAi+pGADT7-p53, pE-box-AbAi+pGADT7-MsbHLH115, and pmE-box-AbAi+pGADT7-MsbHLH115 were transformed into yeast strain Y1H. the yeast was grown in SD/-Leu medium (Takara, Shanghai, China) containing 300 ng/mL AbA. The interaction was studied using a yeast single hybridization system (Clontech, Palo Alto, CA).

The oligonucleotide probe E-box (CAAATG) synthesized the gene promoter and was labeled biotin at the 3’ end by Sangon (Shanghai, China). The MsbHLH115 coding sequence was cloned into the pGEX4T-1 vector (provided by BioRun Biotechnology Co., Ltd, Wuhan, China), and EMSA analysis was performed using the photolytic chemiluminescence EMSA kit (Pierce, Rockford, IL, USA).

The MsbHLH121 promoter was inserted into the pGreenII 0800-LUC (provided by BioRun Biotechnology Co., Ltd, Wuhan, China) to produce a report generator. The MsbHLH115 ORF was inserted into pGreenII 62-SK to produce effectors. The mixture of the above plasmid fusion was injected into the lower epidermis of Nicotiana benthamiana leaves with a 1mL syringe without the needle. The infiltrated tobacco plants were cultured in low light for 2 days. Added 100 μL of freshly prepared Revilla substrate working liquid into the reaction solution, quickly mixed, and detected the Revilla luciferase activity immediately in the fluorescence detector.

pBI121-MsbHLH121pro::GUS and pBI121-3×E-Box::GUS recombinant vectors were constructed and transformed into Agrobacterium GV3101 by freeze-thaw method with pBI121-35S::GUS vector as the control. These vectors infiltrated into 3-week-old tobacco leaves, respectively. GUS activity was detected.

All of the data presented here were mean values for each treatment. There are at least three independent biological replicates in every experiment. SPSS 19.0 software and Origin 2018 software were utilized to perform the statistical analysis and produce the graphs, respectively. Student’s t-test was carried out between WT and transgenic plants in the same treatment, and one-way analysis of variance (ANOVA) followed by Duncan’s multiple range test was carried out between the control and treatments.

To explore the effect of Cd stress on Fe homeostasis, the expression levels of the genes involved in Fe-deficient response under Cd exposure in wild-type alfalfa. Compared with wild-type plants grown on standard medium, the results showed that MsbHLH115, MsbHLH121, MsbHLH25, MsbHLH68, MsFIT, MsWRKY33, MsWRKY40, MsYSL6, and MsNAS were significantly up-regulated; meanwhile, MsFRO2, MsIRT1, and MsIRO3 were considerably down-regulated by Cd stress (p<0.05) ( Figure 1A ). Interestingly, MsbHLH115 was most strongly induced by Cd at the transcription level, suggesting that MsbHLH115 might be an essential gene involved in the network of Fe and Cd regulation in plants.

MsbHLH115 was expressed in roots, leaves, and stems of the alfalfa, mainly in roots. MsbHLH115 was significantly up-regulated by −Fe and +Cd stress and considerably up-regulated by −Fe+Cd stress in roots, while down-regulated by −Fe+Cd stress in the stems and leaves of alfalfa ( Figure 2A ). The MsbHLH115 expression was significantly increased by −Fe and +Cd at 6 h, 12 h, and 24 h, and was significantly down-regulated at 72 h. The MsbHLH115 expression was significantly up-regulated by −Fe+Cd at 12 h and 24 h and was significantly down-regulated at 48 h and 72 h ( Figure 2B ). Following the increase of Cd concentration, MsbHLH115 expression was significantly increased, reaching maximum expression at 90 µM, and then decreased ( Figure 2C ).

In the CK, the MsbHLH115pro transformed soybean hairy root showed β-D-glucuronidase (GUS) signals, indicating that the MsbHLH115pro had expression activity. The hairy root tip showed dark GUS staining in all treatments. Under −Fe and +Cd stress, the hairy root was darker blue. Under −Fe+Cd stress, the hairy root staining was darker than that in the CK but shallower than that in −Fe or +Cd treatment ( Figure 2D ). The value of GUS activity in soybean hairy roots is −Fe>+Cd>−Fe+Cd>CK (p<0.05) ( Figure 2E ). This result was consistent with the quantification of gene expression, indicating that MsbHLH115 responds to −Fe, +Cd, and −Fe+Cd stress in plants.

A total of 26 bHLHs containing typical bHLH domains were assigned to bHLHIVc, bHLHIVb, and bHLHIb subfamilies. Evolutionary analysis showed that MsbHLH115 belonged to the bHLHIVc subfamily. MsbHLH115 is most closely related to MtbHLH115, GmbHLH115, AtbHLH115, and AtbHLH105 ( Figure 1B ). There are some cis-regulatory elements such as G-box and E-box in the promoters of bHLH family genes. Several cis-elements involved in stress response and hormone regulation were found in the MsbHLH115 promoter ( Figure 1C ).

During the transient expression of the fusion protein in Arabidopsis protoplasts, green fluorescence was visible throughout the cytoplasm in protoplasts with the 35S:: GFP control. In protoplasts transiently expressing 35S::MsbHLH115-GFP, nuclear localization of the MsbHLH115-GFP fusion protein was revealed by a nuclear localization marker RFP. The alignment of the predominant red fluorescence in the nucleus with the GFP fluorescence signals confirmed the localization of MsbHLH115 in the nucleus ( Figure 3 ).

To investigate the biological function of MsbHLH115, we generated Arabidopsis overexpressed MsbHLH115 lines (MsbHLH115ox). Three transgenic lines with high MsbHLH115 expression levels (ox#2-1, ox#5-1, and ox#12-2) were selected for further analysis ( Figure 4B ). Under normal conditions(CK), −Fe, +Cd, and −Fe+Cd stress, the MsbHLH115ox lines showed significantly longer roots, higher biomass, and higher chlorophyll content than WT. The degree of impact on plant growth was −Fe+Cd >−Fe>+Cd(p<0.05) ( Figures 4A, C–E ). The severe growth inhibition caused by −Fe+Cd stress indicated that iron deficiency exacerbated the Cd toxicity in plants.

Under +Cd stress, the Nitro-Blue tetrazolium chloride (NBT), 3,3-diaminobenzidine (DAB), and Evans blue staining in the MsbHLH115ox leaves were lighter ( Figure 5A ) than WT; the O₂ ^-, H₂O₂, and MDA content in the three MsbHLH115ox lines was lower than WT ( Figures 5B–D ); the CAT, SOD, and GST activity of the three MsbHLH115ox lines were significantly higher than WT ( Figures 5E–G ). The result indicated that overexpression of MsbHLH115 enhanced the tolerance of Arabidopsis to Cd stress and reduced the production of reactive oxygen species (ROS) in Arabidopsis under Cd stress.

Considering that Fe deprivation visibly elevated the susceptibility of transgenic plants to Cd stress, especially root growth, a split-root experiment was employed to further explore the impact of Fe status on Cd toxicity to plants. We first examined the effects of different Fe and Cd supplies on the growth of MsbHLH115ox line #12-2. In the split medium, the root growth of #12-2 was inhibited by Cd under iron supply conditions ( Figure 6A ); the Cd inhibition of root growth was more obvious under iron deficiency ( Figure 6B ); and the Cd inhibition of plant root growth under iron deficiency was more obvious than that under Fe supply ( Figure 6C ). The result showed that the root elongation of MsbHLH115ox plants was inhibited by Cd, which was exacerbated by iron deficiency ( Figures 6A–C , Supplementary Figure S1 ). We further compared the growth of #12-2 and WT plants when the part near the root tip (RT) and the part distal the root tip (RS) was grown in different conditions. The #12-2 root was longer than WT in the following cases, i. RT or RS was grown in Cd stress and the other part was stressed by iron deficiency ( Figures 6D, G ); ii. RT was grown in the −Fe+Cd stress and RS was grown in the +Fe−Cd medium ( Figures 6E, H ); and iii. RT was grown in Cd stress and RS was grown in the +Fe+Cd medium ( Figures 6F, I ). In other cases, the difference in growth of #12-2 and WT plants was not obvious. The root elongation was affected by the total dose of Cd. In the presence of iron, MsbHLH115 can rescue the root growth inhibition caused by Cd, whether the iron is in RT or RS; but in the absence of iron in the medium, especially in RT, the rescue function of MsbHLH115 was negatively affected. The results showed that the iron in the rhizosphere played a leading role in maintaining root elongation under Cd stress.

The Fe concentration in the shoots and roots of three MsbHLH115ox lines was significantly higher than that in WT under both CK and Cd stress. The Fe concentration in shoots and roots of MsbHLH115ox lines and WT significantly decreased by Cd stress ( Figures 7A, B ). The Cd concentration showed no significant difference in he shoots and roots of MsbHLH115ox lines and WT. The results indicated that overexpression of MsbHLH115 does not affect Cd absorption and translocation ( Figure 7C ). The Ferric-chelate reductase (FCR) activity in MsbHLH115ox lines was significantly higher than that in WT under CK and Cd stress ( Figure 7D ). The results indicated that MsbHLH115 positively affects Fe accumulation and translocation in plants. Cd exposure reduced the plant’s ability to absorb Fe and decreased Fe accumulation in plants.

Zn concentration in MsbHLH115ox was significantly higher than that in WT under Cd treatment or not. Cd stress significantly reduced Zn concentration in MsbHLH115ox lines and WT ( Figures 8A, B ). The results showed that Cd stress reduced Zn absorption, and led to a decrease in Zn accumulation in plants while MsbHLH115 promotes Zn absorption and transport in plants. Mn and Cu concentration in MsbHLH115ox lines was higher than that in WT under Cd treatment or not, with no significant difference ( Figures 8C–F ). The results showed that Cd stress reduced Mn and Cu absorption and accumulation and MsbHLH115 had little effect on Mn and Cu absorption and transport.

As shown in Figure 9 , the expression level of Fe uptake and translocation genes (IRT1, FRO2, VTL, NAS4, IREG2, HMA3, and MTP3) was higher in the three MsbHLH115ox lines than in the WT, regardless of whether they were under CK or Cd-exposed conditions. Under Cd stress, the expressions of IRT1, FRO2, and VTL expressions decreased, while the expressions of NAS4, HMA3, IREG2, and MTP3 increased in the MsbHLH115ox lines. The terms of these five transcription factors (bHLH115, bHLH39, FIT, bHLH121, and bHLH104) were higher in the MsbHLH115ox lines than in the WT and decreased by Cd stress. The expression of four GSTs was higher in the MsbHLH115ox lines and was enhanced by Cd stress. The expression of RbohB, RbohD, and RbohF was higher in the MsbHLH115ox lines and was enhanced by Cd stress. The expression of SOD1, POD1, and BGLU14, was higher in the MsbHLH115ox lines and was enhanced by Cd stress. The P5CS expression was higher in the MsbHLH115ox lines and was enhanced by Cd stress, while PRODH was higher in the MsbHLH115ox lines but was inhibited by Cd stress ( Figure 9A ). The promoters of bHLH115, FIT, bHLH121, bHLH104, IRT1, FRO2, VTL, and HMA3 contain E-box elements, while NAS4, IREG2, and MTP3 have no E-box element ( Figure 9B ).

On SD/-Leu medium without AbA, p53-AbAi+pGADT7-MsbHLH115, pE-box-AbAi +pGADT7-MsbHLH115, and pmE-box-AbAi+pGADT7-MsbHLH115 transformed yeast strains can grow at diluted concentrations from 10^-1 to 10^-3. The background expression of pE-box-AbAi(linearized) in yeast was severely inhibited by 300 ng/mL AbA. On SD/-Leu medium supplemented with 300 ng/mL AbA, p53-AbAi+pGADT7-MsbHLH115 and pmE-box-AbAi +pGADT7-MsbHLH115 did not grow, while pE-box-AbAi+pGADT7-MsbHLH115 could grow. The yeast one-hybrid (Y1H) assay showed that MsbHLH115 can bind to E-box cis-element ( Figure 10A ). The results of EMSA analysis showed that there was a migration band between the E-box probe and the protein GST- MsbHLH115. The 20× competitive examination can compete with part of the migration band of the protein GST- MsbHLH115, and the 100× competitive examination can compete with most of the migration band of the probe and the protein GST- MsbHLH115. No migration band exists between the mutant probe and GST- MsbHLH115, and the E-box interacts with GST- MsbHLH115, further proving that the MsbHLH115 fusion protein can directly bind to the E-box ( Figure 10B ). LUC test showed that the enzyme activity of double luciferase was significantly higher than that of the control, indicating that MsbHLH115 could directly bind to MsbHLH121 promoter ( Figure 10C ). The tobacco leaves that co-transformed pBI121-MsbHLH115 and pBI121-MsbHLH121pro::GUS showed more blue spots than those that co-transformed pBI121-MsbHLH115 and pBI121-3×E-box::GUS. However, no blue dots appeared in tobacco leaves of co-transformed pBI121-MsbHLH115 and pBI121-GUS, indicating that MsbHLH115 protein can recognize E-box element and interact with MsbHLH121 promoter ( Figure 10D ).