The CDSs of the ADH genes were amplified from the cDNA samples of wild-type Arabidopsis (Col-0) and maize inbred line B73, as well as a genomic DNA sample of E. coli with homologous recombination primers ( Supplementary Table S1 ), separated by 1% agarose gel electrophoresis, purified using the Universal DNA Purification Kit (Tiangen, Beijing, China), and constructed into dicotyledonous expression vector pBI121-cMyc-NY ( Supplementary Figure S1 ) using homologous recombination kit ClonExpress II (Vazyme, Nanjing, China), respectively. The recombined vectors were transformed into competent cells, Trans-T1 of E. coli (TransGen, Beijing, China), incubated in LB liquid medium, and screened on LB plates containing 50 mg/l kanamycin. The vector plasmids were purified using the TIANprep Mini Plasmid Kit (Tiangen, Beijing, China) and then used to transform Agrobacterium tumefaciens competent cells Gv3101-pSoup-19 (Angyubio, Shanghai, China). After screening on LB plates containing 50 mg/l kanamycin and rifampicin and identification by bacterial PCR and sequencing at Kangyou Biotech (Hangzhou, China), the positive monoclonals were incubated in LB liquid medium, recovered, and used to transform wild-type Arabidopsis (Col-0) by floral dipping. Homozygous T₃ lines were screened on MS plates containing 50 mg/l kanamycin, identified by PCR amplification using the same primers as shown in Supplementary Table S1 , and calculated for transformation rate.

The genomic DNA of each homozygous T₃ line was extracted using CTAB buffer. After detection for concentration and purity in an NanoDrop 1000 spectrophotometer (Thermo Fisher Scientific Ltd., Paisley, United Kingdom) and identification for integrity by 1% agarose gel electrophoresis, the samples were resequenced (10 × deep) on a DNBSEQ-T7 platform at Gene^plus (Shenzhen, China). After preliminary evaluation of the raw data for quality, clean reads were filtered by the FASTP software (https://github.com/OpenGene/fastp) and aligned against the sequences of the Arabidopsis genome TAIR10 (http://www.arabidopsis.org) and the expression vector pBI121-cMyc-NY to find out the integration sites of T-DNA. As described by Zhang et al. (2011), specific primers ( Supplementary Table S2 ) were designed using online software Primer-BLAST (https://blast.ncbi.nlm.nih.gov) and used to amplify the flanking sequences of the left (from 1000 bp upstream of the integration sites to 1000 bp of the T-DNA) and right (from 1000 bp of the T-DNA to 1000 bp downstream of the integration sites) borders of the T-DNA integration sites from the DNA samples extracted as above from each of the homozygous T₃ lines. The amplified products were separated by 1% agarose gel electrophoresis, recovered, and sequenced at Kangyou Biotech (Hangzhou, China). The sequenced results were aligned against the sequences of the Arabidopsis genome TAIR10 (http://www.arabidopsis.org) and the expression vector pBI121-cMyc-NY again for confirmation of the number and integration sites of T-DNA.

Three biological replates of seedlings of the homozygous T₃ lines and their type wild control were incubated in a phytotron for 30 days. Consistent plants were sampled from each replicate. RNA was extracted from one-third of each sample using RNAiso Plus (TaKaRa, Dalian, China) and reverse transcribed into cDNA using ABScript Neo RT Master Mix for qPCR with gDNA Remover (ABclonal, Wuhan, China). Specific primers ( Supplementary Table S3 ) were designed using online software Primer-Blast (https://blast.ncbi.nlm.nih.gov) and used for real-time quantitative PCR (RT-qPCR) amplification of the transformed genes AtADH, ZmADH, and EcADH, as well as the reference gene AtUBQ5 using the Universal SYBR Green Fast qPCR Mix (Abclonal, Wuhan, China) in a CFX96 MangerTM Real-Time System (Bio-Rad, CA, USA) with four technical replicates. The relative expression levels of the three transformed genes were calculated, normalized, and analyzed for statistical significance using the 2^−ΔΔCT method of the CFX96 Manger^™ software v 2.0.

Total protein was extracted from another one-third of each of the T₃ seedling samples using the Plant Total Protein Extraction Kit (Coolaber, Beijing, China), detected for concentration using the BCA Protein Assay Kit (Coolaber, Beijing, China), and diluted to gradient standard samples with the extraction buffer. The OD₅₆₂ of the gradient standard samples were detected in a microplate reader Varioskan LUX (Thermo Fisher Scientific Ltd., Paisley, United Kingdom) and used to establish the standard curve. The concentration of total protein in the samples was calculated and diluted to 5 μg/μl with the extraction buffer. Half of each of the diluted protein samples were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) using the SDS-PAGE Gel Preparation Kit (Biosharp, Hefei, China) and transferred onto a polyvinylidene fluoride membrane (Millipore, Billerica, USA). After blocking and rinsing, the membrane was incubated in Myc-Tag pAb antibody (Abclonal, Wuhan, China) solution at 4°C for 3 h, incubated in anti-rabbit IgG (H+L) antibody (Jackson, MS, USA) solution at room temperature for 30 min, rinsed with rinsing buffer for 3 × 10 min, colored using Ultra-sensitive ECL Chemiluminescent Substrate Kit (Powerful Biology, Wuhan, China), and imaged in Gel Doc™ EZ System (Bio-Rad, Hercules, CA, USA).

The ADH standard sample of the ADH ELISA Kit (MLBio, Shanghai, China) was diluted to gradient standard samples and added into the wells of a 96-well plate with three technical replicates. The other half of each diluted protein sample was added into the other wells of the same plate with three replicates. One hundred microliter of HRP antibody was added into each of these wells. The plate was sealed with film, incubated at 37°C for 1 h, and dried by removing the solution in the wells. The wells were washed 5 times by being filled with the diluted washing buffer, incubated at room temperature for 1 min, and dried by removing the solution, added with 50 μl of 0.01 H₂O₂ and 0.1% tetramethylbenzidine, shaken well, incubated at 37°C in the dark for 15 min, added with 50 μl termination solution, shaken well, and read for OD₄₅₀ in a microplate reader Varioskan LUX (Thermo Fisher Scientific Ltd., Paisley, United Kingdom). A standard curve was established based on the concentration of the standard samples and their OD₄₅₀ and used to calculate the concentrations of the enzyme proteins. Due to the different molecular weights of the AtADH, EcADH, and ZmADH proteins, the concentrations were divided by their molecular weight to convert to the accumulation of each enzyme in ng/ml/kDa.

The other one-third of each sample was ground in liquid nitrogen, transferred into an Enppendoff tube, weighed (W), added with 1 ml of the extraction solution of the ADH reaction assay kit CAS:9031-72-5 (Solarbio, Beijing, China), incubated on ice for 20 min, and centrifuged at 4°C and 16000 r/min for 20 min. The supernatant was used to be detected for ADH activity (U/g) using the same assay kit in an ultraviolet spectrophotometer UV8000 (Shimadzu, Japan). In the reversible reaction between ethanol and acetaldehyde, ADH catalyzes the reduction of acetaldehyde by NADH to produce ethanol and nicotinamide adenine dinucleotide (NAD⁺). NADH has an absorption peak at 340 nm, while NAD⁺ does not. According to the manual of the assay kit, the ADH activity was calculated as

Where, ΔA₃₄₀ was the difference of absorbance at 340 nm before and after reaction, ϵ (molar extinction coefficient of NADH) = 6.22 × 10³ l/mol/cm, d (optical diameter of colorimetric dish) = 1 cm, V_{Reaction total} (total volume of reaction) = 1 ml, V_{Sample total} (the added volume of the ADH extraction solution) = 1 ml, T (reaction time) = 1 min, V_Sample (the added volume of the supernatant) = 100 μl, and W was the weight (g) of the ground sample.

The results of the PCR amplification showed that the CDSs of the AtADH, ZmADH, and EcADH genes were successfully cloned ( Supplementary Figure S2 ), conducted into dicotyledonous expression vector pBI121-cMyc-NY, and transformed into Agrobacterium tumefaciens ( Supplementary Figure S3 ). After transformation, 1.2%, 1.4%, and 1.3% T₀ plants were screened on the resistant plates ( Supplementary Figure S4A ). Each of these T₁ lines is segregated in a 3:1 ratio ( Supplementary Figure S4B ). Untill T₃ generation, five, three, and six homozygous lines transformed by the AtADH, ZmADH, and EcADH genes, respectively, were screened on the resistant plates ( Supplementary Figure S4C ) and identified by the PCR amplification ( Figure 1 ).

By resequencing the genomic DNA of these 14 T₃ lines and alignment against the sequences of the Arabidopsis genome and T-DNA, a single T-DNA integrated site was found on chromosomes 5, 5, 1, 3, 4, 1, 3, 5, and 5 of lines AtADH-11, AtADH-17, AtADH-18, ZmADH-1, ZmADH-2, ZmADH-4, EcADH-4, EcADH-5, and EcADH-9, respectively. Each of the integrated sequences contained a complete expression structure of 4404, 4024, 2778, 4500, 13131, 4279, 13124, 11887, and 13114 bp, respectively ( Supplementary Table S4 ). By using the primers ( Supplementary Table S2 ), the sequences flanking the left or right borders, or both, of these integration sites were successfully amplified, sequenced, and aligned against the T-DNA sequences of the expression vector and the Arabidopsis genome with high confidence (E value ≤ 2.90E-40 and similarity ≥ 93.60%) ( Supplementary Table S5 ). Single integrated sites of the T-DNA containing each of the transformed genes AtADH, ZmADH, and EcADH were confirmed on chromosome 5 of line AtADH-11, chromosome 5 of line AtADH-17, chromosome 1 of line AtADH-18, chromosome 3 of line ZmADH-1, chromosome 4 of line ZmADH-2, chromosome 1 of line ZmADH-4, chromosome 3 of line EcADH-4, chromosome 5 of line EcADH-5, and chromosome 5 of line EcADH-9, respectively ( Figure 2 ). These nine homozygous T₃ lines were used for the following detections.

The result of RT-qPCR showed that the transformed endogenous gene AtADH was overexpressed under the drive of the CaMV35S promoter in all three T₃ lines (AtADH-11, AtADH-17, and AtADH-18) transformed by the endogenous gene AtADH, and the relative expression levels were more than 15 times higher than the wild control with only the endogenous AtADH gene ( Figure 3A ). In the three T₃ lines ZmADH-1, ZmADH-2, and ZmADH-4, and the three T₃ lines EcADH-4, EcADH-5, and EcADH-9, the transformed exogenous genes ZmADH and EcADH were overexpressed under the drive of the CaMV35S promoter, and their relative expression levels were several or tens of times higher than those in the three T₃ lines transformed by the endogenous gene AtADH ( Figure 3B ). Taken together, the averages of the relative expression levels of the three T₃ lines transformed by the ZmADH gene and the three T₃ lines transformed by the EcADH gene were both tens of times higher than the average of the relative expression levels of the three T₃ lines transformed by the endogenous AtADH gene ( Figure 3C ). All the above differences were greatly significant ( Figure 3 ).

By Western blotting, the ADH proteins encoded by each of the AtADH, ZmADH, and EcADH genes were detected in the corresponding T₃ lines with a single integration site of the T-DNA ( Figure 4 ). The result of ELISA showed that the accumulation of the ADH protein in the wild-type control was 0.1621 ng/ml/kDa. In the three T₃ lines (AtADH-11, AtADH-17, and AtADH-18) transformed by the endogenous AtADH gene, the accumulation was 0.2237, 0.2462, and 0.2416 ng/ml/kDa, respectively, with no significant difference among them but greatly significantly higher than that in the wild-type control ( Figure 5A ). The accumulation of the ADH protein in the three T₃ lines (ZmADH-1, ZmADH-2, and ZmADH-4) transformed by the exogenous ZmADH gene was 0.2180, 0.2489, and 0.4984 ng/ml/kDa, respectively. The difference between lines ZmADH-1 and ZmADH-2 was not significant, while the difference between these two lines and line ZmADH-4 was significant with a p-value less than 0.01. However, all three lines were greatly significantly or significantly higher than that of the wild-type control ( Figure 5A ). The accumulation of the ADH protein in the three T₃ lines (EcADH-4, EcADH-5, and EcADH-9) transformed by the exogenous EcADH genes was 0.5201, 0.3481, and 0.2660 ng/ml/kDa, respectively, with significant or greatly significant differences among them, but all were significantly higher than that of the wild-type control ( Figure 5A ). However, the accumulation of the ADH proteins in each of the six T₃ lines transformed by the exogenous ZmADH and EcADH genes, respectively, as well as the average accumulations of the ADH protein in the three T₃ lines transformed by the exogenous ZmADH gene and average accumulation of the ADH protein in the three T₃ lines transformed by the exogenous EcADH genes, were both significantly or greatly higher than the average accumulation of the three T₃ lines transformed by the endogenous AtADH gene ( Figure 5B ).

In the wild-type control, the activity of the ADH enzyme was as low as 0.0306 U/g. In the three T₃ lines (AtADH-11, AtADH-17, and AtADH-18) transformed by the endogenous AtADH gene, the activities were 0.0784, 0.0436, and 0.1502 U/g, respectively, with only line AtADH-18 significantly higher than that of the wild-type control. However, the activities of the ADH enzyme in the three T₃ lines (ZmADH-1, ZmADH-2, and ZmADH-4) transformed with the exogenous ZmADH gene were 1.2860, 4.3368, and 5.3720 U/g, respectively, with significant differences among them, but all were significantly higher than that of the wild-type control. The activities of the ADH enzyme in the three T₃ lines (EcADH-4, EcADH-5, and EcADH-9) transformed by the exogenous EcADH gene were 0.0750, 0.4458, and 1.1046 U/g, respectively, with greatly significant or significant differences among them. The latter two lines (EcADH-5 and EcADH-9) were significantly higher than the wild-type control. However, the average activity of the ADH enzyme in the three T₃ lines transformed by the exogenous ZmADH gene and the three T₃ lines transformed by the exogenous EcADH gene were significantly higher than the average activity of the ADH enzyme in the three T₃ lines transformed by the endogenous AtADH gene ( Figure 6 ).

In the three T₃ lines transformed by the endogenous AtADH gene, the relative expression levels of the transformed genes detected by RT-qPCR inevitably included the expression of the endogenous AtADH gene of the recipient wild-type Arabidopsis. However, the transformed exogenous AtADH gene, driven by the constitutive strong promoter CaMV35S, significantly increased their expression levels together with the expression of the endogenous AtADH gene. The relative expression levels were more than 15 times higher than those in the wild-type control ( Figure 3A ). In the six T₃ lines transformed by the exogenous ZmADH and EcADH genes, the expression levels of the exogenous genes were also significantly increased by ten to tens of times compared to the three T₃ lines transformed by the endogenous AtADH genes ( Figure 3B ). This result indicated that the expression of the transformed exogenous genes was less regulated by endogenous mechanisms and more mRNA could be accumulated, although they were under the drive of the same constitutive strong promoter as the transformed endogenous genes. The same results of database and literature research were also reviewed by Khush (2001); Showalter et al. (2009); Chen et al. (2019), and Yu et al. (2022). The significant differences in the relative expression levels of the transformed genes among the different T₃ lines transformed with the same gene could be due to the different integration sites of the transformed genes in the Arabidopsis genome and their impact on the expression of the transformed genes ( Figure 2 ). As we discussed in a previous review (Yu et al., 2022), the transformed exogenous genes overexpress under the control of constitutive promoters and confer recipient crops with novel traits that are usually absent or function in different pathways in the recipient crops themselves. In addition to the influence of the genetic background, growth stage, and environment, the heterologous expression of the transformed exogenous genes in recipient crops is usually less regulated at the transcriptional level (Khush, 2001; Showalter et al., 2009; Chen et al., 2019).

By Western blotting, the ADH proteins encoded by each of the transformed AtADH, ZmADH, and EcADH genes were detected in each of the three corresponding T₃ lines ( Figure 4 ). The result of ELISA showed that the accumulation of the ADH protein in the three T₃ lines transformed by the endogenous AtADH gene was significantly higher than that in the wild-type control ( Figure 5A ). The most important was that the accumulation of the ADH proteins in each of the six T₃ lines transformed by the exogenous ZmADH and EcADH genes, respectively, as well as the average accumulations of the ADH protein in each of the three T₃ lines transformed by the exogenous ZmADH and EcADH genes, respectively, were both significantly higher than the average accumulation of the three T₃ lines transformed by the endogenous AtADH gene ( Figure 5B ). This result indicated that the accumulation of the proteins encoded by exogenous genes was higher than that encoded by endogenous genes. The proteins encoded by the transformed exogenous genes accumulate and function independently of the endogenous metabolic pathways and confer recipient crops with novel or enhanced traits (Adamczyk and Sumerford, 2001; Palma et al., 2014; Melo et al., 2016). In the 13 commercial cotton varieties, the accumulation of the Cry protein was also found to nonlinearly correlate with the heterologous transcription levels of the transformed exogenous Cry1Ac gene (Adamczyk and Sumerford, 2001). This indicates that there are few endogenous factors that affect the accumulation of exogenous proteins, which can function independently of the endogenous metabolic pathways of recipient crops (Palma et al., 2014; Melo et al., 2016). However, endogenous proteins encoded by the overexpressed endogenous genes are usually regulated in complex networks with functional redundance or replaceable pathways and are difficult to confer the desirable phenotypes significantly (Winkel, 2004; Fiehn and Weckwerth, 2010; Wan et al., 2017).

The result of the enzyme reaction assay showed that the activities of the ADH enzyme in the three T₃ lines transformed by the exogenous ZmADH gene and two T₃ lines transformed by the exogenous EcADH gene were greatly significantly higher than that of the wild-type control ( Figure 6A ). Moreover, the average ADH enzyme activity in the three T₃ lines transformed by the exogenous ZmADH gene and in the three T₃ lines transformed by the exogenous EcADH gene were both significantly higher than the average ADH enzyme activity in the three T₃ lines transformed by the endogenous AtADH gene ( Figure 6B ). This result indicated that the enzyme activity of the proteins encoded by exogenous genes was higher than that of the proteins encoded by endogenous genes.

One of the T₃ lines transformed by the exogenous EcADH gene did not show a significant increase in enzyme activity ( Figure 6A ), although its relative expression level of and accumulation of the EcADH protein encoded by the transformed exogenous EcADH gene were significantly increased ( Figures 3B , 5A ). This exception should be investigated further. In addition, more homozygous transgenic lines should be transformed and screened for elimination of potential biases probably caused by variable T-DNA integration sites or operational deviation on specific methodologies like RT-qPCR, ELISA, and enzyme activity assay.