Overexpression of a Pak Choi Gene, BcAS2, Causes Leaf Curvature in Arabidopsis thaliana

The LBD (Lateral Organ Boundaries Domain) family are a new group of plant-specific genes, which encode a class of transcription factors containing conserved Lateral Organization Boundary (LOB) domains, and play an important role in regulating the adaxial–abaxial polarity of plant leaves. In Arabidopsis thaliana, ASYMMETRIC LEAVES 2 (AS2) has a typical LOB domain and is involved in determining the adaxial cell fate. In this study, we isolated the BcAS2 gene from the pak choi cultivar “NHCC001”, and analyzed its expression pattern. The results showed that the BcAS2 encoded a protein made up of 202 amino acid residues which were located in the nucleus and cytomembrane. The Yeast two-hybrid system (Y2H) assay indicated that BcAS2 interacts with BcAS1-1 and BcAS1-2 (the homologous genes of AS1 gene in pak choi). In the transgenic Arabidopsis thaliana that overexpressed BcAS2 gene, it presented an abnormal phenotype with a curly shape. Taken together, our findings not only validate the function of BcAS2 in leaf development in Arabidopsis thaliana, but also contribute in unravelling the molecular regulatory mechanism of BcAS2, which fulfills a special role by forming complexes with BcAS1-1/2 in the establishment of the adaxial–abaxial polarity of the lateral organs in pak choi.


Introduction
The flat symmetric leaves are essential for plant development as they provide the place for photosynthesis, respiration and other important physiological activities. It is reported that the leaves originate from the shoot apical meristem (SAM) along proximal-disal, adaxial-abaxial and medial-lateral axis [1,2]. Therefore, the successful establishment of SAM is crucial for the leave polarity [3,4]. The initiation and maintenance of SAM requires the normal expression of a highly conserved class of homeodomain transcription factors encoded by class-I KNOX genes, which include four members: SHOOT MERISTEMLESS (STM); BREVIPEDICELLUS (BP); KNAT2 and KNAT6 [5][6][7][8]. The abnormal embryos lacking SAM appear in the loss-of-function mutation in STM, which also fail to develop any postembryonic vegetative tissue [9,10]. However, the development of leaves will be disrupted if the KNOX genes continue expressing. Hareven's study demonstrates that there would be super-compound leaves if the KNOX genes overexpressed in tomato [11]. The ectopic expression of KNOX genes in maize presents an abnormal phenotype with distal displacement of sheath and auricle tissue into the blade, and produces deeply serrated or segmented leaves in Arabidopsis thaliana [12,13]. Therefore, it is a critical event to down regulate KNOX genes in lateral organ primordia for normal leaf development [14].
ASYMMETRIC LEAVES 2 (AS2), containing a Lateral Organization Boundary (LOB) domain, is a key determinant of adaxial cell fate. It is involved in the leaf morphogenesis and downregulates the expression of KNOX genes via forming a complex with an MYB family transcription factor (TF) AS1 [15,16]. From a previous study, some other components also cooperate with the AS2-AS1 complex in order to maintain the normal development of leaves, such as several TCP family proteins, namely TEOSINTE BRANCHED1, CY-CLOIDEA, and PCF (TCP) [17]. In addition, some epigenetic factors also participate in the pathway of KNOX gene silencing as a partner with AS2-AS1, for instance, HISTONE REPRESSION A FACTOR (HIRA) [18], HISTON DEACETYLASE HDA6 [19]. The AS2-AS1 complex also restrict the YABBY, ARF, and KANAND family genes, which play important roles in deciding adaxial-abaxial polarity during leaf development [20,21]. These results indicate that AS2-AS1 is essential in the establishment of plant leaf polarity.
The interaction mechanism between AS2 and AS1 has been studied by many researchers in Arabidopsis thaliana. We know that AS2 can interact with AS1 in vitro using a yeast two-hybrid system assay [22], and AS2-RS2(the homologous gene of AS1 in maize) are co-located in the nucleus [23,24]. The latest research demonstrates that the AS2, with the zinc-finger DNA-binding motif, is indispensable for the establishment of perinuclear bodies which influences the normal development of leaves [25]. The transgenic lines that overexpress the AS2 gene using the 35S promoter in cockscomb present significantly curly leaves [26].
Pak choi (Brassica rapa ssp. chinensis), belongs to the Brassica genus of Cruciferae and has a close relationship with Arabidopsis thaliana. As a relatively important leafy vegetable, it is popular and widely cultivated in Asia. The leaf morphology is a vital agronomic trait with a great impact on pak choi architecture and yield, while the establishment of leaf polarity is a key event in its leaf development. From a previous study, we know that the AS2 gene is indispensable for adaxial patterning of lateral organs. However, the function of AS2 in pak choi is still unknown. In the present study, we firstly isolated BcAS2 from the cultivar of the pak choi (NHCC001) in order to discover its regulatory mechanism in lamina development. We demonstrated that BcAS2 physically interact with the BcAS1-1/2 in vitro and in vivo via a Y2H assay and colocalization assay. Furthermore, we successfully obtain the transgenic lines overexpressed BcAS2 with an obvious leaf curly phenotype. These findings extend our understanding of AS2 in Cruciferae plants, suggesting that BcAS2 and BcAS1-1/2 were involved in the establishment of leaf polarity in the form of complex in pak choi.

Plant and Growth Conditions
The seeds of pak choi cultivar "NHCC001" were provided by Professor Xilin Hou (Nanjing Agricultural University) and grown in illumination incubators under the conditions of light 16 h/24 • C and dark 8 h/24 • C, subsequently. The different tissue (leaf, lobus cardiacus, stem, root, hypocotyl) of 1 month old seedings were sampled and frozen immediately in liquid nitrogen and stored at −70 • C. Nicotiana benthamiana and Arabidopsis thaliana wild type (WT) used in this study were grown in illumination incubators under the same conditions.

Sequence and Analysis of BcAS2
We designed primers based on the sequence of Bra039733 (http://brassicadb.org/ brad/) to clone the full-length CDS (coding sequence) of BcAS2 from a pak choi template by homology cloning according to our previous report (Table S1) [27,28]. The physicochemical characteristics of BcAS2 protein were analyzed by the Expasy website (https://web.expasy. org/protparam/), hereinafter referred to as Protparam. The homologous genes of BcAS2 were searched by the online BLAST sever (https://blast.ncbi.nlm.nih.gov/Blast.cgi). The multiple sequence alignments of homologous proteins were performed through DNAman (download from https://www.lynnon.com/dnaman.html). The conserved motifs were analyzed using the MEME website (http://meme-suite.org/tools/meme). The potential proteins interacting with BcAS2 were predicted by online STRING software (https:// version11.string-db.org/cgi/network.pl?taskId=xScSvWWALyMg). We downloaded the protein sequences of predicted results from the Brad website (http://brassicadb.org/brad/ searchGene.php) and traced the homologous genes in Arabidopsis thaliana through the blast tool on the Tair website (https://www.arabidopsis.org/Blast/index.jsp).

Subcellular Localization
The protein-coding region of BcAS2 was cloned into pENTRTM D-TOPO (Invitrogen, Carlsbad, CA, USA) firstly, then the 35S: BcAS2-GFP was generated by LR reaction between pENTRTM D-TOPO-BcAS2 and pEarlyGate103 vector for subcellular localization. The BcAS2, BcAS1-1/2 were cloned into pCAMBIA1302-mcherry and pCAMBIA1302 respectively to generate the BcAS2-mcherry and BcAS1-1/2-GFP fusions for co-localization. The main primers used in our study are listed in Table S1. These plasmids were transformed into Agrobacterium strain GV3101 for infiltrating into tobacco leaves. Then the leaves were co-injected with 35S:BcAS2-GFP/35S: GFP and DAPI (nucleus specific dye). About 70 h after infiltration, we collected the images by confocal laser scanning microscopy (Zeiss, LSM 500, Oberkochen, Germany).

Agrobacterium-Mediated Transformation
To obtain transgenic plants overexpressing BcAS2, Arabidopsis thalina (Columbia) was transformed with 35S:pEarleyGate103-BcAS2-GFP using the floral-dip method [29]. The T0 seeds of the transgenic plants were screened on MS solid medium containing 50 mg/L Basta and 16 mg/L Timetin. After about 10 days, only the positive seedlings could grow normally. The seeds obtained from the positive seedlings were transgenic seeds of the T1 generation, which were further selected on MS solid medium with the same resistance until T3 generation. Then the T3 lines were selected for the qPCR analysis and phenotypic observation.

Real-Time Quantitative PCR (qPCR) Analysis
The total RNA was extracted by RNA simple total RNA extraction kit (TIANGEN Beijing, China). The cDNA was synthesized using the PrimeScript TM II 1st Strand cDNA Synthesis Kit (TaKaRa, Dalian, China). The AtActin of Arabidopsis thaliana and BcActin of pak choi were used as house-keeping genes, respectively. Gene-specific primers used in this study are given in Table S1. The qPCR was performed using SYBR Green Master Mix (Yeasen, Shanghai, China). The data were calculated using the 2 −∆∆CT method [30].

Sequence and Expression Analysis of BcAS2
The online software ProtParam showed that the full length coding sequence (CDS) of BcAS2 encoded 202 amino acid residues with molecular weight of 22.09 kDa and theoretical isoelectric point (pI) of 7.1. Multiple sequence alignment of BcAS2 and AS2-like proteins from different crops (Brassica rapa, Raphanus sativus, Arabidopsis thaliana, Pistacia vera, Ricinus communis) showed that they had a highly conserved LOB domain at the N-temial(9-107 amino acid sites) ( Figure 1A). In addition, the results of motif analysis indicated that there were identical motifs among BcAS2, BrAS2 and RsLOB6 proteins which were similar to motifs in AtAS2. Interestingly, they all belong to Cruciferae, suggesting that the function of BcAS2 gene in pak choi may be similar to that in Arabidopsis thaliana ( Figure 1B).

Sequence and Expression Analysis of BcAS2
The online software ProtParam showed that the full length coding sequence (CDS) of BcAS2 encoded 202 amino acid residues with molecular weight of 22.09 kDa and theoretical isoelectric point (pI) of 7.1. Multiple sequence alignment of BcAS2 and AS2like proteins from different crops (Brassica rapa, Raphanus sativus, Arabidopsis thaliana, Pistacia vera, Ricinus communis) showed that they had a highly conserved LOB domain at the N-temial(9-107 amino acid sites) ( Figure 1A). In addition, the results of motif analysis indicated that there were identical motifs among BcAS2, BrAS2 and RsLOB6 proteins which were similar to motifs in AtAS2. Interestingly, they all belong to Cruciferae, suggesting that the function of BcAS2 gene in pak choi may be similar to that in Arabidopsis thaliana ( Figure 1B). To verify the expression pattern of BcAS2 in different tissues of pak choi, the qPCR assays were performed to identify the expression level using the samples of 1-month old "NHCC001". The results showed that BcAS2 was highly expressed in the lobus cardiacus and the leaves, followed by stems, but lower in roots and hypocotyls ( Figure 2). This result was consistent with the previous studies on the role of AS2 gene in leaf development [16,23]. To verify the expression pattern of BcAS2 in different tissues of pak choi, the qPCR assays were performed to identify the expression level using the samples of 1-month old "NHCC001". The results showed that BcAS2 was highly expressed in the lobus cardiacus and the leaves, followed by stems, but lower in roots and hypocotyls ( Figure 2). This result was consistent with the previous studies on the role of AS2 gene in leaf development [16,23].

Subcellular Localization of BcAS2 Protein
To investigate where BcAS2 functions in the leaf cell, we observed the localization of BcAS2 using the Agrobacterium injection method. From the images captured, we found that the protein of 35S:BcAS2-GFP fusions were expressed in the nucleus and cytomembrane mainly (Figure 3), indicating that BcAS2 may not only function as a transcription factor but also as some cytomembrane proteins. The expression level of BcAS2 in leaf, stem, root, hypocotyl, lobus cardiacus of 1-month-old pak choi cultivar "NHCC001". The data represent the average of the three replicates, and the error bars represent the standard deviation between the replicates. ** and * mean significant differences between different tissues, *, p < 0.05, **, p < 0.01.

Subcellular Localization of BcAS2 Protein
To investigate where BcAS2 functions in the leaf cell, we observed the localization of BcAS2 using the Agrobacterium injection method. From the images captured, we found that the protein of 35S:BcAS2-GFP fusions were expressed in the nucleus and cytomembrane mainly (Figure 3), indicating that BcAS2 may not only function as a transcription factor but also as some cytomembrane proteins.   The expression level of BcAS2 in leaf, stem, root, hypocotyl, lobus cardiacus of 1-month-old pak choi cultivar "NHCC001". The data represent the average of the three replicates, and the error bars represent the standard deviation between the replicates. ** and * mean significant differences between different tissues, *, p < 0.05, **, p < 0.01.

Subcellular Localization of BcAS2 Protein
To investigate where BcAS2 functions in the leaf cell, we observed the localization of BcAS2 using the Agrobacterium injection method. From the images captured, we found that the protein of 35S:BcAS2-GFP fusions were expressed in the nucleus and cytomembrane mainly (Figure 3), indicating that BcAS2 may not only function as a transcription factor but also as some cytomembrane proteins.

Interaction Between BcAS2 and BsAS1-1/2 In Vivo and In Vitro
In total, 10 potential interacting proteins of BcAS2 were predicted by online software STRING. Based on the blast result of the Tair database, we found that these proteins mainly belonged to four gene families, including KANADI family (Bra023570, Bra008613, Bra033844, Bra023254), YABBY family (Bra037320, Bra00538), MYB family (Bra000011, Bra005177), and KNOX family (Bra000638, STM) ( Figure S1). The result of multiple sequence analysis showed that the proteins identified in Figure S1 were highly homologous to AtKAN1, AtKAN2, AtYAB2, and AtYAB3 ( Figure S2). Among these results, we were surprised to find that AS1 is one of the proteins that might interact with BcAS2, for many researches in Arabidopsis thaliana have demonstrated that AS2 and AS1 usually function as a complex [22,24].
From a previous study, we realized that the AS2 of Arabidopsis thaliana had no transcript activation ability [12], but it still needed further verification in pak choi. Therefore, the yeast two-hybrid screen method was performed in our study to validate our hypothesis. The result showed that the yeast Y2H gold strains co-transformed with pGBTK7-BcAS2/BcAS2-2 and pGADT7 (empty vector) could grow well on SD/-Trp/-Leu/-His/-Ade medium as well as the positive control (pGBTK7-53 + pGADT7-T). While the BcAS2-1 and pGADT7 failed to grow on SD/-Trp/-Leu/-His/-Ade medium, indicating that the full length of BcAS2 had transcript activation ability while BcAS2-1 had not ( Figure 4A). In order to further verify the interaction between BcAS2 and BcAS1-1/2, we generated the yeast Y2H gold strains co-transformed with pGBTK7-BcAS2-1 and pGADT7-BcAS1-1/2. The result emerged that all the experimental groups can grow on selective medium (SD/-Trp/-Leu/-His/-Ade), while the negative control cannot grow, illustrating that the BcAS2 protein interacted with BcAS1-1 and BcAS1-2 proteins ( Figure 4B).
In total, 10 potential interacting proteins of BcAS2 were predicted by online software STRING. Based on the blast result of the Tair database, we found that these proteins mainly belonged to four gene families, including KANADI family (Bra023570, Bra008613, Bra033844, Bra023254), YABBY family (Bra037320, Bra00538), MYB family (Bra000011, Bra005177), and KNOX family (Bra000638, STM) ( Figure S1). The result of multiple sequence analysis showed that the proteins identified in figure S1 were highly homologous to AtKAN1, AtKAN2, AtYAB2, and AtYAB3 ( Figure S2). Among these results, we were surprised to find that AS1 is one of the proteins that might interact with BcAS2, for many researches in Arabidopsis thaliana have demonstrated that AS2 and AS1 usually function as a complex [22,24].
From a previous study, we realized that the AS2 of Arabidopsis thaliana had no transcript activation ability [12], but it still needed further verification in pak choi. Therefore, the yeast two-hybrid screen method was performed in our study to validate our hypothesis. The result showed that the yeast Y2H gold strains co-transformed with pGBTK7-BcAS2/BcAS2-2 and pGADT7 (empty vector) could grow well on SD/-Trp/-Leu/-His/-Ade medium as well as the positive control (pGBTK7-53 + pGADT7-T). While the BcAS2-1 and pGADT7 failed to grow on SD/-Trp/-Leu/-His/-Ade medium, indicating that the full length of BcAS2 had transcript activation ability while BcAS2-1 had not ( Figure 4A). In order to further verify the interaction between BcAS2 and BcAS1-1/2, we generated the yeast Y2H gold strains co-transformed with pGBTK7-BcAS2-1 and pGADT7-BcAS1-1/2. The result emerged that all the experimental groups can grow on selective medium (SD/-Trp/-Leu/-His/-Ade), while the negative control cannot grow, illustrating that the BcAS2 protein interacted with BcAS1-1 and BcAS1-2 proteins ( Figure 4B). Based on the interaction between the BcAS2 and BcAS1-1/2 in the Y2H system, we further confirmed their relationship by a co-localization assay in tobacco leaves transiently co-infiltrated with the BcAS2-mcherry and BcAS1-1/2-GFP. From the images we captured, we can observe that the red fluorescence of BcAS2-mcherry fusion protein was mainly Based on the interaction between the BcAS2 and BcAS1-1/2 in the Y2H system, we further confirmed their relationship by a co-localization assay in tobacco leaves transiently co-infiltrated with the BcAS2-mcherry and BcAS1-1/2-GFP. From the images we captured, we can observe that the red fluorescence of BcAS2-mcherry fusion protein was mainly expressed in the cell membrane and nucleus, which is also consistent with the subcellular localization result of BcAS2 in this study. The green fluorescence of BcAS1-1/2-GFP was mainly expressed in the nucleus. The co-expression of BcAS2 and BcAS1-1/2 in the nucleus resulted in fusion yellow fluorescence ( Figure 5). The interaction between BcAS2 and BcAS1-1/2 in the yeast two-hybrid system and the co-localization between them allowed us to propose a mechanism that they might also form a complex to function as a leaf polarity regulator in pak choi. expressed in the cell membrane and nucleus, which is also consistent with the subcellular localization result of BcAS2 in this study. The green fluorescence of BcAS1-1/2 -GFP was mainly expressed in the nucleus. The co-expression of BcAS2 and BcAS1-1/2 in the nucleus resulted in fusion yellow fluorescence ( Figure 5). The interaction between BcAS2 and BcAS1-1/2 in the yeast two-hybrid system and the co-localization between them allowed us to propose a mechanism that they might also form a complex to function as a leaf polarity regulator in pak choi. Figure 5. The BcAS2-mCherry and BcAS1-1/2-GFP were co-localized in the nucleus. From left to right, the fluorescence signals in the nucleus, nucleus, and cytomembrane, nucleus due to BcAS1-1/2-GFP, BcAS2-mChery and the co-expression between them, respectively. Scale bars = 50 μM.

Overexpression of BcAS2 Resulted in Upward-Curling Leaves and Downregulated Some Polarity-Related Genes
To understand better the regulation of BcAS2 during leaf polarity formation in Pak choi, we generated the transgenic Arabidopsis thaliana with the ectopic expression in BcAS2. The result of qPCR reveled a much higher transcript level of BcAS2 in overexpression lines (named as OE3, OE8, OE9, OE12) compared with WT ( Figure 6A). Further, it was obvious that the leaves of overexpression lines presented the upward curling phenotype ( Figure 6B), which is in line with the transgenic AS2 lines in Celosia cristata [26]. We further examined the transcription levels of eight genes (AtYAB3, AtYAB2, AtYAB3, At-KAN1, AtKAN2, AtKAN3, AtARF3, AtARF4) which have been reported that they were related to leaf abaxialization in a previous study [31,32]. Our result showed that the transcript levels of the eight genes were extremely reduced in transgenic BcAS2 lines compared with WT ( Figure 6C). Therefore, we speculated that the curly leaves of BcAS2 transgenic plants may be attributed to the interaction between abaxial and abaxial polarity regulators, but the specific regulatory pathway remains to be studied. Figure 5. The BcAS2-mCherry and BcAS1-1/2-GFP were co-localized in the nucleus. From left to right, the fluorescence signals in the nucleus, nucleus, and cytomembrane, nucleus due to BcAS1-1/2-GFP, BcAS2-mChery and the co-expression between them, respectively. Scale bars = 50 µM.

Overexpression of BcAS2 Resulted in Upward-Curling Leaves and Downregulated Some Polarity-Related Genes
To understand better the regulation of BcAS2 during leaf polarity formation in Pak choi, we generated the transgenic Arabidopsis thaliana with the ectopic expression in BcAS2. The result of qPCR reveled a much higher transcript level of BcAS2 in overexpression lines (named as OE3, OE8, OE9, OE12) compared with WT ( Figure 6A). Further, it was obvious that the leaves of overexpression lines presented the upward curling phenotype ( Figure 6B), which is in line with the transgenic AS2 lines in Celosia cristata [26]. We further examined the transcription levels of eight genes (AtYAB3, AtYAB2, AtYAB3, AtKAN1, AtKAN2, AtKAN3, AtARF3, AtARF4) which have been reported that they were related to leaf abaxialization in a previous study [31,32]. Our result showed that the transcript levels of the eight genes were extremely reduced in transgenic BcAS2 lines compared with WT ( Figure 6C). Therefore, we speculated that the curly leaves of BcAS2 transgenic plants may be attributed to the interaction between abaxial and abaxial polarity regulators, but the specific regulatory pathway remains to be studied.

Discussion
The leaves play an important role in the success of plants which are regulated by a complex genetic network [33]. The polarity of the plant's lateral organ is established and young leaves are formed with the continuous division of leaf cells among the three asymmetrical axes. Bowman found that only when the adaxial-abaxial polarity was established can the leaf dysplasia begin [34]. Among these leaf polarity regulators, the LBD gene fam-

Discussion
The leaves play an important role in the success of plants which are regulated by a complex genetic network [33]. The polarity of the plant's lateral organ is established and young leaves are formed with the continuous division of leaf cells among the three asymmetrical axes. Bowman found that only when the adaxial-abaxial polarity was established can the leaf dysplasia begin [34]. Among these leaf polarity regulators, the LBD gene family encoded a class of protein with an LOB domain only found in plants, which regulates the plant-specific processes in many species. While the biological role of LBD genes in pak choi is poorly understood. Here, we isolated the BcAS2 gene from pak choi cultivar "NHCC001", which had a typical LOB domain and was homologous to AS2 in Arabidopsis. The multiple sequence and motif analysis revealed a high similarity between BcAS2 and AS2, implying that BcAS2 may also participate in specifying the latter's organ shape in pak choi. We profiled the expression level of BcAS2 in different tissues at the 1-month-old stage. From the results, we found that the expression of BcAS2 was higher in leaves and the lobus cardiacus compared with other tissues, which allowed us to speculate that BcAS2 mainly played a role in the leaves of pak choi.
Previous reports showed that AS2 mediated the adaxial polarity with a MYB TF AS1 as a complex [33]. Therefore, we want to verify whether there is a similar mechanism in the regulation of BcAS2 on leaf development in pak choi. We predicted the potential proteins that might interact with BcAS2 using the online server STRING ( Figure S1). From the results, we obtained 10 potential proteins, including BcAS1-1/2. To gain further insight into their relationship, we performed a yeast two-hybrid screen to verify our conjecture. As expected, the result revealed that BcAS2 could bind with BcAS1-1/2 in yeast strains. In order to explain the interaction between them in more depth, we utilized a co-localization assay to analyze the localization under the Agrobacterium tumefaciens-mediated transient transformation system. According to the picture we captured, it was evident that they co-expressed in the nucleus, which was in line with a previous study [35].
We first overexpressed BcAS2 in Arabidopsis thaliana to investigate the function of BcAS2 more intuitively. Through phenotypic observation, we found that the over-expressed BcAS2 phenotypes of transgenic lines reflected dramatically curved leaves, which were similar to kan1 and kan2, and arf3 and arf4 double mutants but different from the transgenic Arabidopsis thaliana with a high expression in a wheat YAABY gene, TaYAB1 [36][37][38]. Considering that the homologous genes of BcAS2 potential interaction genes in Arbidopsis thaliana and the main function of KANADI ARF and YABBY genes are promoting the development of leaf abaxial axis [39][40][41][42], we speculated that the high transcript level of BcAS2 in the plant results in the decreased expression of some abaxial polarity relative genes to improve the adaxial development. With this in mind, we examined eight genes that are vital in the determination of abaxial cell fate, namely AtKAN1, AtKAN2, AtKAN3, AtYAB1, AtYAB2, AtYAB3, AtARF3, and AtARF4 [34][35][36]. The result indicated that they were evidently down-regulated in transgenic lines compared with WT, especially the AtKAN1, AtKAN2, AtARF3, and AtYAB3, which was positive for our conjecture. Significantly, the elevated BcAS2 expression level also downregulated the AtARF3 and AtARF4, which responded to auxin, an important endogenous hormone participating in the regulation of plant development [42][43][44]. There is a lot of evidence that has proved that auxin signal transduction, polar transport, and regulation are closely related to leaf morphology and development. The pin1 mutant, with inactivated PIN1 (encoding a polar auxin transport protein) in Arabidopsis, showed wider leaves, fused cotyledon, and abnormal phyllotaxis, and the rosette leaves of axr mutant showed irregularity in shape and tended to curl downward [35,45]. Hay found that the accumulation of auxin can inhibit KNOX genes with AS1 in the process of leaf primordium formation [31,45], and the auxin response factor ARF3/4 was indirectly repressed by the AS1-AS2 complex through activating the miR390-tasirR-ARF pathway [46]. This evidence allowed us to assumed that the BcAS2-BcAS1-1/2 complex was involved in the process of auxin affecting leaf morphological development Genes 2021, 12, 102 9 of 11 in pak choi. Nevertheless, the correctness of our conjecture remains to be further studied in future.

Conclusions
In summary, we firstly cloned BcAS2 from pak choi, which has a highly conserved LOB domain, and a high expression level in leaves. This study provided preliminary proof about the function of the BcAS2-BcAS1-1/2 complex in leaf polarity according to the yeast two-hybrid, co-localization, and transgenic assays. This finding contributed to a better understanding about the regulatory mechanism of leaf shape in pak choi or other Brassica species. In addition, the special leaf phenotype caused by overexpression of BcAS2 can provide an insight for the morphological improvement of pak choi in order to increase its economic value.
Supplementary Materials: The following are available online at https://www.mdpi.com/2073-442 5/12/1/102/s1, Figure S1: The prediction of interaction protein with BcAS2, Figure S2: The multiple sequence analysis between the potential interaction genes of BcAS2 and the leaf polarity genes in Arbidopsis thaliana Table S1: Primers were used in this study, Table S2: Protein sequences of AS2 like genes in various plants.