BIC2, a Cryptochrome Function Inhibitor, Is Involved in the Regulation of ABA Responses in Arabidopsis

The plant hormone ABA (abscisic acid) is able to regulate plant responses to abiotic stresses via regulating the expression of ABA response genes. BIC1 (Blue-light Inhibitor of Cryptochromes 1) and BIC2 have been identified as the inhibitors of plant cryptochrome functions, and are involved in the regulation of plant development and metabolism in Arabidopsis . In this study, we report the identification of BIC2 as a regulator of ABA responses in Arabidopsis . RT-PCR (Reverse Transcription-Polymerase Chain Reaction) results show that the expression level of BIC1 remained largely unchanged, but that of BIC2 increased significantly in response to ABA treatment. Transfection assays in Arabidopsis protoplasts show that both BIC1 and BIC2 were mainly localized in the nucleus, and were able to activate the expression of the co-transfected reporter gene. Results in seed germination and seedling greening assays show that ABA sensitivity was increased in the transgenic plants overexpressing BIC2, but increased slightly, if any, in the transgenic plants overexpressing BIC1. ABA sensitivity was also increased in the bic2 single mutants in seedling greening assays, but no further increase was observed in the bic1 bic2 double mutants. On the other hand, in root elongation assays, ABA sensitivity was decreased in the transgenic plants overexpressing BIC2, as well as the bic2 single mutants, but no further decrease was observed in the bic1 bic2 double mutants. By using qRT-PCR (quantitative RT-PCR), we further examined how BIC2 may regulate ABA responses in Arabidopsis , and found that inhibition of ABA on the expression of the ABA receptor genes PYL4 (PYR1-Like 4) and PYL5 were decreased, but promotion of ABA on the expression of the protein kinase gene SnRK2.6 (SNF1-Related Protein Kinases 2.6) was enhanced in both the bic1 bic2 double mutants and 35S:BIC2 overexpression transgenic plants. Taken together, our results suggest that BIC2 regulates ABA responses in Arabidopsis possibly by affecting the expression of ABA signaling key regulator genes.


Introduction
CRYs (Cryptochromes) are photolyase-like flavoproteins found in all evolutionary lineages, and plant CRYs are blue light receptors that mediate light regulated plant growth and development and metabolism [1][2][3]. CRYs present as inactive monomers in darkness, but as active homodimers after being photoexcited by blue light to regulate gene expression, and eventually plant growth and development and metabolism in plants [1][2][3][4][5]; whereas, BICs (Blue-light Inhibitor of Cryptochromes) are inhibitors of CRYs. They can bind to CRYs to suppress their blue light-dependent dimerization, photobody formation, phosphory- ing overexpression transgenic plants and mutants generated for BICs, we found that ABA sensitivity is altered in both the BIC2 overexpression transgenic plants and the bic2 mutants in seed germination, seedling greening, and root elongation assays. Quantitative RT-PCR analysis show that ABA regulated expression of some ABA signaling component genes was affected in the overexpression transgenic plants and the mutants.

Expression of BIC2 Is Induced by ABA Treatment
In the process of identifying new regulators of ABA and/or abiotic stress responses by using the strategy described previously [34], we found that BIC2, a cryptochrome function inhibitor gene [7], is an ABA response gene, with a FPKM (Reads Per Kilobase per Million mapped reads) of 2.41 in ABA-treated seedlings, compared to 0.95 in control samples.
To examine if expression of BIC2 is indeed induced by ABA, we examined the expression of BIC2 in response to ABA treatment. The Col (Columbia-0) wild type seedlings were treated with ABA or solvent alone as control for a few hours. RNA was then isolated and the expression level of BIC2 was examined by using RT-PCR. As shown in Figure 1, the expression level of BIC2 increased dramatically in response to ABA treatment. Considering that BIC1 is closely related to BIC2 [7], we also examined the expression level of BIC1 in ABA-treated and control seedlings. We found that the expression level of BIC1 remained largely unchanged in response to ABA treatment ( Figure 1). some ABA signaling component genes was affected in the overexp plants and the mutants.

Expression of BIC2 Is Induced by ABA Treatment
In the process of identifying new regulators of ABA and/or abiot by using the strategy described previously [34], we found that BIC function inhibitor gene [7], is an ABA response gene, with a FPKM (R per Million mapped reads) of 2.41 in ABA-treated seedlings, compare samples.
To examine if expression of BIC2 is indeed induced by ABA, expression of BIC2 in response to ABA treatment. The Col (Colum seedlings were treated with ABA or solvent alone as control for a few then isolated and the expression level of BIC2 was examined by u shown in Figure 1, the expression level of BIC2 increased dramatica ABA treatment. Considering that BIC1 is closely related to BIC2 [7], the expression level of BIC1 in ABA-treated and control seedlings. W expression level of BIC1 remained largely unchanged in response ( Figure 1).

Figure 1.
Expression of BICs in response to ABA treatment. Fourteen-day-old wild type Arabidopsis were mock-treated or treated with 50 μM ABA on a sha in darkness. RNA was then isolated and used for RT-PCR analysis with 30 c expression of BIC1 and BIC2. The expression ACT2 was used as a control.

BIC1 and BIC2 Activate Reporter Gene Expression in Transfected Protop
It has been shown that BICs are involved in CRYs mediated gen and that SmBICs are able to bind directly to the promoter re anthocyanin biosynthesis genes in yeast one-hybrid analysis [15]. Arabidopsis BICs appear to be nuclear proteins as indicated by subc assays in seedlings of the 35S:BIC1-GFP 35S:BIC2-GFP transgenic plan examined if BICs may have transcription activities in transfe protoplasts.
We first examined the subcellular localization of BIC1 and BI Arabidopsis protoplast transient transfection assays, to confirm if nuclear proteins. Plasmids of GFP-BIC1 and GFP-BIC2 effec co-transfected, respectively, with the nucleus indicator gene NLS-RFP protoplasts. The GFP and RFP fluorescence were observed und microscope after the transfected protoplasts were incubated overnig shown in Figure 2A, GFP and RFP fluorescence were mainly observ indicating that BIC1 and BIC2 were mainly localized in the nucleus. Expression of BICs in response to ABA treatment. Fourteen-day-old seedlings of the Col wild type Arabidopsis were mock-treated or treated with 50 µM ABA on a shaker at 40 rpm for 4 h in darkness. RNA was then isolated and used for RT-PCR analysis with 30 cycles to examine the expression of BIC1 and BIC2. The expression ACT2 was used as a control.

BIC1 and BIC2 Activate Reporter Gene Expression in Transfected Protoplasts
It has been shown that BICs are involved in CRYs mediated gene expression [6][7][8][9], and that SmBICs are able to bind directly to the promoter region of eggplant anthocyanin biosynthesis genes in yeast one-hybrid analysis [15]. Considering that Arabidopsis BICs appear to be nuclear proteins as indicated by subcellular localization assays in seedlings of the 35S:BIC1-GFP 35S:BIC2-GFP transgenic plants [7], we therefore examined if BICs may have transcription activities in transfected Arabidopsis protoplasts.
We first examined the subcellular localization of BIC1 and BIC2 proteins using Arabidopsis protoplast transient transfection assays, to confirm if they are indeed nuclear proteins. Plasmids of GFP-BIC1 and GFP-BIC2 effector genes were co-transfected, respectively, with the nucleus indicator gene NLS-RFP into Arabidopsis protoplasts. The GFP and RFP fluorescence were observed under a fluorescence microscope after the transfected protoplasts were incubated overnight in darkness. As shown in Figure 2A, GFP and RFP fluorescence were mainly observed in the nucleus, indicating that BIC1 and BIC2 were mainly localized in the nucleus.
We then examined the transcriptional activities of BIC1 and BIC2 by using protoplast transfection. Plasmids of the effect gene GD-BIC1, GD-BIC2, and the control gene GD were co-transfected, respectively, with the reporter gene Gal4:GUS into Arabidopsis protoplasts. The transfected protoplasts were incubated overnight in darkness, and GUS activities were measured by using a microplate reader. As shown in Figure 2B, expression of the reporter gene was activated by co-transfection of the effector gene GD-BIC1 or GD-BIC2, gene GD were co-transfected, respectively, with the reporter ge Arabidopsis protoplasts. The transfected protoplasts were incub darkness, and GUS activities were measured by using a microplate re Figure 2B, expression of the reporter gene was activated by co-transfec gene GD-BIC1 or GD-BIC2, when compared with the co-transfe indicating that BIC1 and BIC2 have transcription activation activities. Figure 2. Subcellular localization and transcriptional activities of BIC1 and B localization of BIC1 and BIC2. Plasmids of the GFP-BIC1 and GFP-BIC2 genes respectively, with the nuclear indicator gene NLS-RFP into Arabidopsis prot the Col wild type plants. The transfected protoplasts were incubated in dark temperature, and then GFP and RFP fluorescence were examined un microscope. (B) Transcriptional activities of BIC1 and BIC2. Plasmids of th GD-BIC1, and GD-BIC2 were co-transfected, respectively with the reporter Arabidopsis protoplasts isolated from the Col wild type plants. The transfec incubated in dark for 20-22 h at room temperature, and then GUS activity wa microplate reader. Data represent the mean ± SD of three replicates.

Light-Grown Seedlings of the BICs Overexpression Plants Produced Lon bic1 bic2 Double Mutants Produced Shorter Hypocotyls
After showing the BIC2 is an ABA response gene ( Figure 1), and nuclear proteins and function as transcription activators (Figure examine the roles of BICs in regulating ABA responses in Arabid overexpression transgenic plants and mutants were generated. T transgenic plants were generated by transforming the Col wild typ 35S:BIC1 and 35S:BIC2 construct, respectively, and selecting homo plants in the T3 generation. Two homozygous of overexpression tra for BIC1 and BIC2, respectively, i.e., 35S:BIC1 #2 and #10, and 35S ( Figure 3A), were used for the experiments. RT-PCR results show t level of BIC1/BIC2 was increased in the transgenic lines. Quantitati show that the expression level of BIC1 increased ~5 and ~20 folds in transgenic lines, respectively, whereas that of BIC2 increased ~40 35S:BIC2 transgenic lines, respectively.
Single mutants were isolated from T-DNA insertion lines, and g CRISPR/Cas9 to edit BIC1 and BIC2, respectively, in the Col wild ty insertion lines GK-014D08 and SM-3-21731 were obtained from A isolate the bic1-1 and the bic2-1 single mutants, respectively, which are in a previously study [7]. In addition, we generated gene-edited singl and BIC2, respectively, by using CRISPR/Cas9 gene editing in the Co (B) Transcriptional activities of BIC1 and BIC2. Plasmids of the effector genes GD, GD-BIC1, and GD-BIC2 were co-transfected, respectively with the reporter gene Gal4:GUS into Arabidopsis protoplasts isolated from the Col wild type plants. The transfected protoplasts were incubated in dark for 20-22 h at room temperature, and then GUS activity was assayed by using a microplate reader. Data represent the mean ± SD of three replicates.

Light-Grown Seedlings of the BICs Overexpression Plants Produced Longer, Whereas the bic1 bic2 Double Mutants Produced Shorter Hypocotyls
After showing the BIC2 is an ABA response gene (Figure 1), and BIC1 and BIC2 are nuclear proteins and function as transcription activators (Figure 2), we wanted to examine the roles of BICs in regulating ABA responses in Arabidopsis. To do that, overexpression transgenic plants and mutants were generated. The overexpression transgenic plants were generated by transforming the Col wild type plants with the 35S:BIC1 and 35S:BIC2 construct, respectively, and selecting homozygous transgenic plants in the T3 generation. Two homozygous of overexpression transgenic plant lines for BIC1 and BIC2, respectively, i.e., 35S:BIC1 #2 and #10, and 35S:BIC2 #31 and #36 ( Figure 3A), were used for the experiments. RT-PCR results show that the expression level of BIC1/BIC2 was increased in the transgenic lines. Quantitative RT-PCR results show that the expression level of BIC1 increased~5 and~20 folds in the two 35S:BIC1 transgenic lines, respectively, whereas that of BIC2 increased~40 folds in the two 35S:BIC2 transgenic lines, respectively.
Single mutants were isolated from T-DNA insertion lines, and generated by using CRISPR/Cas9 to edit BIC1 and BIC2, respectively, in the Col wild type plants. T-DNA insertion lines GK-014D08 and SM-3-21731 were obtained from ABRC, and used to isolate the bic1-1 and the bic2-1 single mutants, respectively, which are the mutants used in a previously study [7]. In addition, we generated gene-edited single mutants for BIC1 and BIC2, respectively, by using CRISPR/Cas9 gene editing in the Col wild type plants. Two target sequences for each of the two genes were selected and used to generate CRISPR/Cas9 constructs for plant transformation. The transgene-free mutants were isolated in T2 or T3 generations by isolated Cas9-free plants and sequencing the BIC1 and BIC2 genome sequences. Two gene edited single mutants, i.e., bic1-c1 and bic2-c1, were obtained and used for the experiments. Double mutants were generated by using CRISPR/Cas9 to edit BIC2 in the bic1-1 mutant plants, and two double mutants, i.e., bic1 bic2-c1 and -c2, were obtained and used for the experiments. the target sequences in BIC2 were edited. An 88 bp deletion occurred between the two target sequences in the bic1 bic2-c1 mutant, and a 97 bp deletion together with 4 nucleotide substitutions occurred in the bic1 bic2-c2 mutant ( Figure 3B). All of the mutation resulted in amino acid substitutions and premature stops for BIC1 and BIC2, respectively, in the mutants obtained ( Figure 3C). Expression level of BICs in the overexpression transgenic plants. RNA was isolated from 14-day-old seedlings and used for RT-PCR and q-RT-PCR analysis. The expression ACT2 was used as a control for RT-PCR analysis and an inner control for qRT-PCR analysis. In the qRT-PCR analysis, the expression level of BIC1/BIC2 in the Col was set as 1. Lanes without numbers indicated homozygous lines did not used in the experiments. (B) Alignment of the nucleotide sequences of BIC1 and BIC2 in Col wild type, the bic1 and bic2 single, and the bic1 bic2 double mutants. The bic1 and bic2 single mutants were obtained by transforming the Col wild type plants with pHEE-BIC1 and pHEE-BIC2 constructs, respectively, and the bic1 bic2 double mutants were obtained by transforming the bic1-1 single mutant plants with the pHEE-BIC2 construct. DNA was isolated from leaves of T2 or T3 plants and used for PCR amplification of Cas9 to identify transgene-free mutants and for amplification of BIC1 and BIC2 for sequencing to identify homozygous mutants. The sequencing results of the homozygous mutants were aligned with wild type genome sequence of BIC1 and BIC2. Underlines indicate the PAM sites. (C) Alignment of the amino acid sequences of BIC1 and BIC2 in the Col wild type, the bic1 and bic2 single, and the bic1 bic2 double mutants. Coding sequences of BIC1 and BIC2 in the mutants were subjected to ORFfinder (https://www.ncbi.nlm.nih.gov/orffinder/ (accessed on 1 June 2019)) for ORF analysis, and predicted amino acid sequences were used for alignment with the amino acid sequences of wild type BIC1 and BIC2, respectively. Identical amino acids are shaded in black and similar in gray.
By growing all the overexpression transgenic plants and the mutants generated together with the Col wild type in ½ MS plates in light and dark conditions, we RNA was isolated from 14-day-old seedlings and used for RT-PCR and q-RT-PCR analysis. The expression ACT2 was used as a control for RT-PCR analysis and an inner control for qRT-PCR analysis. In the qRT-PCR analysis, the expression level of BIC1/BIC2 in the Col was set as 1. Lanes without numbers indicated homozygous lines did not used in the experiments. (B) Alignment of the nucleotide sequences of BIC1 and BIC2 in Col wild type, the bic1 and bic2 single, and the bic1 bic2 double mutants. The bic1 and bic2 single mutants were obtained by transforming the Col wild type plants with pHEE-BIC1 and pHEE-BIC2 constructs, respectively, and the bic1 bic2 double mutants were obtained by transforming the bic1-1 single mutant plants with the pHEE-BIC2 construct. DNA was isolated from leaves of T2 or T3 plants and used for PCR amplification of Cas9 to identify transgene-free mutants and for amplification of BIC1 and BIC2 for sequencing to identify homozygous mutants. The sequencing results of the homozygous mutants were aligned with wild type genome sequence of BIC1 and BIC2. Underlines indicate the PAM sites. (C) Alignment of the amino acid sequences of BIC1 and BIC2 in the Col wild type, the bic1 and bic2 single, and the bic1 bic2 double mutants. Coding sequences of BIC1 and BIC2 in the mutants were subjected to ORFfinder (https://www.ncbi.nlm.nih.gov/orffinder/ (accessed on 1 June 2019)) for ORF analysis, and predicted amino acid sequences were used for alignment with the amino acid sequences of wild type BIC1 and BIC2, respectively. Identical amino acids are shaded in black and similar in gray.
In the bic1-c1 mutant, both of the two target sequences in BIC1 were edited, and a 175 bp deletion occurred between the two target sequences. In the bic2-c1 mutant, however, only one target sequence in BIC2 was edited, and a single nucleotide was inserted in the first target sequence ( Figure 3B). In the bic1 bic2 double mutants, both of the target sequences in BIC2 were edited. An 88 bp deletion occurred between the two target sequences in the bic1 bic2-c1 mutant, and a 97 bp deletion together with 4 nucleotide substitutions occurred in the bic1 bic2-c2 mutant ( Figure 3B). All of the mutation resulted in amino acid substitutions and premature stops for BIC1 and BIC2, respectively, in the mutants obtained ( Figure 3C).
By growing all the overexpression transgenic plants and the mutants generated together with the Col wild type in 1 /2 MS plates in light and dark conditions, we observed hypocotyl elongation. We found that under the dark-grown conditions, all the seedlings including the Col wild type plants, the 35S:BIC1 and 35S:BIC2 transgenic plants, the bic1 and bic2 single, and the bic1 bic2 double mutants produced hypocotyls with similar length ( Figure 4A). However, under light-grown condition, seedlings of the 35S:BIC1 and 35S:BIC2 transgenic plant produced longer hypocotyls, whereas seedlings of the bic1 bic2 double mutants produced shorter hypocotyls compared with the Col wild type seedlings ( Figure 4A).
OR PEER REVIEW 6 of 16 observed hypocotyl elongation. We found that under the dark-grown conditions, all the seedlings including the Col wild type plants, the 35S:BIC1 and 35S:BIC2 transgenic plants, the bic1 and bic2 single, and the bic1 bic2 double mutants produced hypocotyls with similar length ( Figure 4A). However, under light-grown condition, seedlings of the 35S:BIC1 and 35S:BIC2 transgenic plant produced longer hypocotyls, whereas seedlings of the bic1 bic2 double mutants produced shorter hypocotyls compared with the Col wild type seedlings ( Figure 4A). Quantitative results show that the hypocotyl length of the light-grown seedlings of the Col wild type was ~3 mm, that of the 35S:BIC1 and 35S:BIC2 transgenic plant seedlings was ~6 mm, that of the bic1 and bic2 single mutant seedlings was similar to that of the Col wild type seedlings, but that of the bic1 bic2 double mutant seedlings was only ~2 mm. On the other hand, dark-grown seedlings of the all the plants produced hypocotyls at ~21 mm ( Figure 4B). These results are comparable to the results obtained under blue light-grown and dark-grown conditions [7].

ABA Sensitivity Is Altered in Both the 35S:BIC2 Transgenic Plants and the bic2 Mutants
The expression level of BIC2 was increased in response to ABA treatment (Figure 1), suggesting that BIC2 may be involved in the regulation of ABA responses. We therefore compared ABA responses of the overexpression transgenic plants and the mutants of BICs obtained with the Col wild type plants, by using ABA inhibited seed germination, seedling greening, and root elongation assays.
In the seed germination assays, all the seeds in the control plates, including seeds of Col wild type, the 35S:BIC1 and 35S:BIC2 transgenic plants, the bic1 and bic2 single, and and bic2 single, and the bic1 bic2 double mutants were germinated and grown on vertically placed 1 /2 MS plates solidified with 1.5% agar in a growth room under light for 5 days or dark condition for 7 days, then photographed using a digital camera. (B) Hypocotyl length of light-and dark-grown seedlings. Hypocotyl length of 5-day-old light-grown seedlings or 7-day-old dark-grown seedlings on vertically placed 1 /2 MS plates was measured. Data represent the mean ± SD of 15-18 seedlings.
Quantitative results show that the hypocotyl length of the light-grown seedlings of the Col wild type was~3 mm, that of the 35S:BIC1 and 35S:BIC2 transgenic plant seedlings was~6 mm, that of the bic1 and bic2 single mutant seedlings was similar to that of the Col wild type seedlings, but that of the bic1 bic2 double mutant seedlings was only~2 mm. On the other hand, dark-grown seedlings of the all the plants produced hypocotyls at~21 mm ( Figure 4B). These results are comparable to the results obtained under blue light-grown and dark-grown conditions [7].

ABA Sensitivity Is Altered in Both the 35S:BIC2 Transgenic Plants and the bic2 Mutants
The expression level of BIC2 was increased in response to ABA treatment (Figure 1), suggesting that BIC2 may be involved in the regulation of ABA responses. We therefore compared ABA responses of the overexpression transgenic plants and the mutants of BICs obtained with the Col wild type plants, by using ABA inhibited seed germination, seedling greening, and root elongation assays.
In the seed germination assays, all the seeds in the control plates, including seeds of Col wild type, the 35S:BIC1 and 35S:BIC2 transgenic plants, the bic1 and bic2 single, and However, on the ABA-containing plates, lower germination rate was observed for seeds of the 35S:BIC1 and 35S:BIC2 transgenic plants at most of the time points examined, as compared to seeds of the Col wild type plants. We also noted that seeds of the 35S:BIC2 transgenic plant have a relative lower germination rate compared with seeds of the 35S:BIC1 transgenic plants. On the other hand, no difference was observed for between seeds of the Col wild type and seeds of the bic1 and bic2 single and the bic1 bic2 double mutants ( Figure 5). Nevertheless, these results show that ABA sensitivity is increased in the 35S:BIC1 and 35S:BIC2 transgenic plants.
In the seedling greening assays, clearly increased ABA sensitivity was also observed for the 35S:BIC2 transgenic plants, but only a slight increase was observed for the 35S:BIC1 transgenic plants ( Figure 6A). To our surprise, we found that ABA sensitivity also increased in the bic2 single mutants ( Figure 6A), but did not further increase in the bic1 bic2 double mutants ( Figure 6B). However, different ABA responses were observed for the two bic1 single mutants, increased ABA sensitivity was observed in the bic1-1 mutant, but that of the bic1-c1 mutant was largely similar to the Col wild type plants ( Figure 6B). However, on the ABA-containing plates, lower germination rate was observed for seeds of the 35S:BIC1 and 35S:BIC2 transgenic plants at most of the time points examined, as compared to seeds of the Col wild type plants. We also noted that seeds of the 35S:BIC2 transgenic plant have a relative lower germination rate compared with seeds of the 35S:BIC1 transgenic plants. On the other hand, no difference was observed for between seeds of the Col wild type and seeds of the bic1 and bic2 single and the bic1 bic2 double mutants ( Figure 5). Nevertheless, these results show that ABA sensitivity is increased in the 35S:BIC1 and 35S:BIC2 transgenic plants.
In the seedling greening assays, clearly increased ABA sensitivity was also observed for the 35S:BIC2 transgenic plants, but only a slight increase was observed for the 35S:BIC1 transgenic plants ( Figure 6A). To our surprise, we found that ABA sensitivity also increased in the bic2 single mutants ( Figure 6A), but did not further increase in the bic1 bic2 double mutants ( Figure 6B). However, different ABA responses were observed for the two bic1 single mutants, increased ABA sensitivity was observed in the bic1-1 mutant, but that of the bic1-c1 mutant was largely similar to the Col wild type plants ( Figure 6B).
Quantitative results show that the percentage of greening seedlings reduced greatly in the 35S:BIC2 transgenic plants, the bic2 and the bic1-1 single, and the bic1 bic2 double mutants, but only reduced slightly in the 35S:BIC1 transgenic plants. No difference was observed between the bic1-c1 single mutant and the Col wild type plants ( Figure 6C).
In the root elongation assays, ABA inhibited root elongation of all the plants. However, decreased ABA sensitivity was observed for the 35S:BIC2 and the 35S:BIC1 transgenic plants, the bic1-1 and bic2 single mutants, and the bic1 bic2 double mutants ( Figure 7A). Quantitative results show that ABA sensitivity in the bic1-c1 single mutant was largely similar to the Col wild type plants, and no further changes were observed in the bic1 bic2 double mutants when compared to the bic1-1 and bic2 single mutants ( Figure 7B). Quantitative results show that the percentage of greening seedlings reduced greatly in the 35S:BIC2 transgenic plants, the bic2 and the bic1-1 single, and the bic1 bic2 double mutants, but only reduced slightly in the 35S:BIC1 transgenic plants. No difference was observed between the bic1-c1 single mutant and the Col wild type plants ( Figure 6C).
In the root elongation assays, ABA inhibited root elongation of all the plants. However, decreased ABA sensitivity was observed for the 35S:BIC2 and the 35S:BIC1 transgenic plants, the bic1-1 and bic2 single mutants, and the bic1 bic2 double mutants ( Figure 7A). Quantitative results show that ABA sensitivity in the bic1-c1 single mutant was largely similar to the Col wild type plants, and no further changes were observed in the bic1 bic2 double mutants when compared to the bic1-1 and bic2 single mutants ( Figure 7B).  seedlings. Sterilized seeds of the Col wild type, the 35S:BIC1 and 35S:BIC2 transgenic plants, the bic1 and bic2 single, and the bic1 bic2 double mutants were germinated and grown on vertically placed 1 /2 MS plates solidified with 1.5% agar in a growth room for 5 days. The seedlings were then transferred to control plates and plates containing 10 µM ABA and grown for 8 more days, then pictures were taken by using a digital camera. (B) Percentage of root inhibition by ABA. Length of new elongated roots was measured, and percentage of inhibition was calculated. Data represent the mean ± SD of 20-22 seedlings.

The Expression of Some ABA Signaling Core Regulator Genes Were Affected in the bic1 bic2 Double Mutants and the 35S:BIC2 Transgenic Plants
Even though ABA response was not consistent in the two bic1 mutants, our above results show that that ABA sensitivity was altered in 35S:BIC2 transgenic plants, and the bic2 single and bic1 bic2 double mutants compared to the Col wild type (Figures 5-7). These results indicate that BIC2 is involved in the regulation of ABA responses in Arabidopsis. To examine how BIC2 may regulate ABA responses in Arabidopsis , we wanted to examine if the expression of the ABA signaling core regulator genes was affected by BIC2. Considering that similar ABA sensitivity was observed in the bic2 single and bic1 bic2 double mutants (Figure 6), and the bic1 bic2 mutants showed light-mediated growth phenotypes (Figure 4), we used the bic1 bic2 double mutants rather than the bic2 single mutants for the experiment. As shown in Figure 8A, inhibition of ABA on the expression of PYL genes PYL4 and PYL5 were decreased, whereas promotion of ABA on the expression of the SnRK2 gene SnRK2.6 was enhanced in the bic1 bic2 mutants. We then examined the expression of the ABA signaling core regulator genes in the 35S:BIC2 transgenic plants, and the results were similar to that obtained in the bic1 bic2 mutants; i.e., inhibition of ABA on the expression of PYL4 and PYL5 were decreased, whereas promotion of ABA on the expression of the SnRK2.6 was enhanced in the 35S:BIC2 transgenic plants ( Figure 8B). These results suggest that BIC2 may regulate ABA responses in Arabidopsis by regulating the expression of ABA signaling core regulator genes.   analysis. Expression of ACT2 was used as an inner reference gene, and fold changes were calculated by comparing the transcript level of the corresponding genes in ABA treated and control seedlings. Data represent the mean ± SD of three replicates.

Discussion
BIC1 and BIC2 can bind to CRYs to suppress their activities, thereby affecting CRYs mediated gene expression and photoresponses, eventually light mediated plant growth and development, and metabolisms [2,[6][7][8][9]. In this study, we provide evidences that BIC2 is also involved in the regulation of ABA responses in Arabidopsis.
First, we identified BIC2 as an ABA response gene, and RT-PCR result show that the expression level of BIC2 increased dramatically in response to ABA treatment ( Figure 1). Second, by generating transgenic overexpression plants and mutants for BIC2 gene, and using them for ABA sensitivity assays, we found that the 35S:BIC2 transgenic plants showed altered sensitivity to ABA treatment in seed germination, seedling greening, and root elongation assays ( Figures 5-7), and the bic2 single mutants showed altered ABA sensitivity too in seedlings greening and root elongation assays. Third, the ABA-regulated expression of some ABA signaling core regulator genes including PYL4, PYL5 and SnRK 2.6 was changed in the bic1 bic2 double mutants and the 35S:BIC2 transgenic plants ( Figure 8).
The bic1 bic2 double mutants, but not the bic2 single mutants, produced shorter hypocotyl under light-grown condition (Figure 4), a result similar to the observations under blue light condition [7], suggesting that the bic1 bic2 double mutants we generated should be loss-of-function mutants for both BIC1 and BIC2 genes. In addition, even though no difference in ABA sensitivity was observed for the bic2 single and the bic1 bic2 double mutants (Figures 6 and 7), slight changes in ABA sensitivity were observed for the 35S:BIC1 transgenic plants ( Figures 5 and 7), suggesting that BIC1 may also have a role in regulating ABA responses. Therefore, the bic1 bic2 double mutants, rather than the bic2 single mutants, were used to examine the expression of ABA signaling core regulator genes in our experiments. However, it may be of interest to examine the expression of ABA signaling core regulator genes in the bic2 single mutants to see if BIC1 has a role, and if BIC1 and BIC2 may have redundant functions in regulating ABA responses in Arabidopsis.
On the other hand, previous experiments have shown that the expression of BIC1 was undetectable in the bic1-1 T-DNA insertion mutant, indicating that bic1-1 is a lossof-function mutant [7]. Our bic1 bic2 double mutants generated in the bic1-1 mutant showed shorter hypocotyl phenotype under light-grown condition, a phenotype similar to the T-DNA insertion bic1 bic2 double mutants under blue light-grown condition [7], further confirming that the T-DNA insertion in BIC1 resulted in loss-of-function mutation. However, ABA sensitivity was changed greatly in the bic1-1 mutant, but not the bic1-c1 single mutant in seedling greening assays (Figures 6 and 7). Considering that gene editing of BIC1 in the bic1-c1 mutant resulted in a few amino acid substitutions and premature stops for BIC1, and produced a truncated BIC1 protein with only the first 16 amino acids remained unchanged, it is possible that the N-terminal of BIC1 is critical for its function in regulating ABA responses. However, considering that insertion of the T-DNA in BIC1 in the bic1-1 mutant may also result in a C-terminal truncated BIC1 protein, we could not rule out the possibility that other genes may be also affected in the gene edited bic1-c1 mutant. On the other hand, similar ABA sensitivity was observed in the bic1-1 and bic2 single and the bic1 bic2 double mutants in seedling greening assays ( Figure 6), even though we could not rule out the possibility that BIC1 and BIC2 may have redundant functions in regulating ABA responses as they are closely related proteins. It is possible that BIC1 and BIC2 may function in parallel pathways to regulate ABA response in Arabidopsis. It is worthwhile to generate other bic1 mutants to further examine if that is the case.
The protoplast transfection assays indicated that BIC2 is a nuclear protein and is able to activate the expression of the co-transfected reporter gene ( Figure 2B), suggesting that BIC2 may function as a transcription activator. However, similar changes of some ABA signaling core regulator genes were observed in the bic1 bic2 double mutants and the 35S:BIC2 transgenic plants (Figure 7), suggesting that overexpressing and knockout of BIC2 have similar effects on the expression of the ABA signaling core regulator genes. Thus, it is very unlikely that BIC2 may directly regulate the expression of these ABA signaling core regulator genes. It is possible that protein homeostasis of BIC2 may be important for its functions in regulating ABA responses, and accumulation of more or less BIC2 proteins resulted in similar changes in expression of ABA signaling core regulator genes, therefore, similar ABA responses in Arabidopsis.

Plant Materials and Growth Conditions
All the mutants and transgenic plants are in the Col background, and Col Arabidopsis was used for plant transformation and protoplast isolation. The T-DNA insertion lines GK-014D08 and SM-3-21731 were obtained from the Arabidopsis Biological Resource Center (Ohio State University, Columbus, OH, USA), and used to identify the homozygous bic1-1 and bic2-1 mutants, respectively. The bic1-c1 and bic2-c1 single and the bic1 bic2-c1 and -c2 double mutants were generated by using CRISPR/Cas9 gene editing.
For ABA treatment and gene expression analysis, seeds of the Col, the 35S:BIC1 and 35S:BIC2 transgenic plants, the bic1 and bic2 single, and the bic1 bic2 double mutants were surface sterilized and sown on plates containing 1 /2 MS salts solidified with 1% or 1.5% agar. The plates were kept at 4 • C in darkness for 2 days, and then transferred into a plant growth room. To generate plants for plant transformation and protoplast isolation, seeds of the Col wild type and bic1-1 mutant plants were germinated and grown in soil pots. The conditions of the growth room were described previously [37].

ABA Treatment, RNA Isolation, RT-PCR and Quantitative RT-PCR (qRT-PCR)
To examine the expression of BICs in response to ABA treatment in the Col wild type seedlings, and the expression of ABA signaling component genes in seedlings of the Col wild type, the bic1 bic2 double mutants, and the 35S:BIC2 transgenic plants, 14-day-old seedlings were treated with 50 µM ABA in darkness on a shaker at 40 rpm for 4 h and collected and frozen in liquid N 2 used for RNA isolation. Seedlings treated with the solvent methanol were used as a control.
Total RNA was isolated by using an EasyPure plant RNA kit (Transgen Biotech, Beijing, China), and 2 µg of the total RNA isolated was subjected to first-strand cDNA synthesis by using an EasyScript First-strand DNA Synthesis Super Mix (TransGen Biotech). Synthesized cDNA was used as a template for RT-PCR or qRT-PCR analysis. The expression of ACT2 (ACTIN2) was used as an inner control. The primers used for qRT-PCR analysis of ABA signaling component genes were as described previously [33,34].

Constructs
The reporter construct Gal4:GUS, the effector construct GD, and the nuclear indicator construct NLS-RFP used for protoplast transfection were as described previously [40,41].
To generate constructs for protoplast transfection, the full-length open reading frame (ORF) of BIC1 and BIC2 were amplified by RT-PCR using RNA isolated from 12-day-old Col seedlings as template, and cloned in frame with an N-terminal GD and GFP tag into the pUC19 vector under the control of the double CaMV 35S promoter.
To generate 35S:BIC1 and 35S:BIC2 constructs for plant transformation, the full-length ORF of BIC1 and BIC2 were amplified by RT-PCR, cloned in frame with an N-terminal HA tag into the pUC19 vector under the control of the double CaMV 35S promoter, and then digested with proper enzymes and subcloned into the binary vector pPZP211 [42].

Plant Transformation and Over-Expression Transgenic Plants and Mutants Isolation
The Col wild type and the bic1-1 mutant plants about 5 weeks old with several mature flowers were transformed with the 35:BIC1 and 35:BIC2 construct in pPZP211 or CRISPR/Cas9 gene editing constructs by using floral dip method via Agrobacterium tumefaciens strain GV3101 mediated transformation [44]. Homozygous overexpression transgenic plants and Cas9-free homozygous mutants were selected by following the produces as described previously [45,46].

Plasmid DNA Isolation, Protoplasts Isolation and Transfection
Plasmid DNA of the reporter and the effector were isolated by using the GoldHi Endo Free Plasmid Maxi Kit (CWBIO, Taizhou, China) following the manufacture's procedures, and the concentration of the plasmid DNA was measured by using a NanoDrop (ThermoFisher, Waltham, MA, USA).
Protoplasts were isolated from leaves of~4-week-old Col wild type plants and transfected as described previously [34,[47][48][49][50]. In brief, for protein subcellular location assay, plasmids of GFP-BIC1 and GFP-BIC2 effector constructs were co-transfected, respectively, with the NLS-RFP nuclear indicator construct into the protoplasts isolated. For transcriptional activity assays, plasmids of GD, GD-BIC1, and GD-BIC2 effect constructs were co-transfected, respectively, with the Gal4:GUS reporter construct into the protoplasts isolated. The transfected protoplasts were incubated at room temperature for 20-22 h under darkness, and then GFP and RFP florescence were examined under a fluorescence microscope (Olympus, Tokyo, Japan), whereas GUS activities were measured by using a Synergy HT fluorescence microplate reader (BioTEK, Charlotte, VT, USA). The experiments were repeated at least twice.

Hypocotyl Elongation Assays
For hypocotyl elongation assay, sterilized seeds of the Col wild type, the 35S:BIC1 and 35S:BIC2 transgenic plants, the bic1 and bic2 single, and the bic1 bic2 double mutants were germinated and grown on vertically placed 1 /2 MS plates solidified with 1.5% agar in a growth room under light or dark conditions. In all the assays, 15-18 seedlings for each genotype were used, and the experiments were repeated at least twice.

ABA Sensitivity Assays
ABA inhibited seed germination and seedling greening were assayed as described previously [33,34,37,51]. Briefly, surface sterilized seeds of the Col wild type, the 35S:BIC1 and 35S:BIC2 transgenic plants, the bic1 and bic2 single, and the bic1 bic2 double mutants were sown on plates with 1 /2 MS salts solidified with 1.0% agar in the presence or absence of 1 µM ABA, kept at 4 • C in darkness for 2 days, and then transferred to a growth room. Germinated seeds were counted every 12 h after the transfer from 12h and 36h, respectively, for the control and the ABA treated plates. Pictures were taken 13 and 14 days after the transfer, and seedlings with green cotyledons were counted. The experiments were repeated at least twice.
Root elongation assays were performed as described previously [33]. Briefly, sterilized seeds of the Col wild type, the 35S:BIC1 and 35S:BIC2 transgenic plants, the bic1 and bic2 single, and the bic1 bic2 double mutants were sown on 1 /2 MS plates solidified with 1.5% agar, kept at 4 • C in darkness for 2 days, transferred to a growth room and grown vertically for 5 days, and then transferred to control plates and plates containing 10 µM ABA grown vertically for 8 more days. The length of new elongated roots was measured, and percentage of inhibition was calculated.

Conclusions
Our results show that BIC2 is an ABA responsive gene, and BIC1 and BIC2 act as a transcription activator and are involved in the regulating of ABA response in Arabidopsis possible by affecting the expression of some ABA signaling core regulator genes.