Identification of YABBY Transcription Factors and Their Function in ABA and Salinity Response in Nelumbo nucifera

The plant-specific transcription factor family YABBY plays important roles in plant responses to biotic and abiotic stresses. Although the function of YABBY has been identified in many species, systematic analysis in lotus (Nelumbo nucifera) is still relatively lacking. The present study aimed to characterize all of the YABBY genes in lotus and obtain better insights into NnYABBYs in response to salt stress by depending on ABA signaling. Here, we identified nine YABBY genes by searching the whole lotus genome based on the conserved YABBY domain. Further analysis showed that these members were distributed on six different chromosomes and named from YABBY1 to YABBY9, which were divided into five subgroups, including YAB1, YAB2, YAB5, INO, and CRC. The analysis of cis-elements in promotors revealed that NnYABBYs could be involved in plant hormone signaling and plant responses to abiotic stresses. Quantitative real-time PCR (qRT-PCR) showed that NnYABBYs could be up-regulated or down-regulated by ABA, fluridone, and salt treatment. Subcellular localization indicated that NnYABBY4, NnYABBY5, and NnYABBY6 were mainly localized in the cell membrane and cytoplasm. In addition, the intrinsic trans-activity of NnYABBY was tested by a Y2H assay, which revealed that NnYABBY4, NnYABBY5, and NnYABBY6 are deprived of such a property. This study provided a theoretical basis and reference for the functional research of YABBY for the molecular breeding of lotus.


Introduction
Nelumbo nucifera Gaertn. is a perennial aquatic herbaceous plant that is the main aquatic vegetable in China and has horticultural, ornamental, medicinal, and food values [1][2][3][4][5]. The cultivated areas of lotus have reached more than 600,000 hectares, and lotus cultivation forms a unique high-quality production area with unique freshwater lake natural resources in China [6]. Its products, as a tonic food welcomed by consumers, are rich in starch, protein, a variety of vitamins, and mineral nutrition, with good nutrition and health functions. 'Shennong Materia Medica', a Chinese pharmacopeia book, listed lotus as a top-quality tonic that could nourish the blood, muscle, and spleen. Nelumbo nucifera, as a sessile plant that grows in a complex and changing environment, has evolved a sophisticated defense mechanism to cope with stresses, such as abiotic and biotic stresses [7]. The transcription factor families, such as NAC, bZIP, MYB, YABBY, NF-YB, and AP2, are important regulatory elements for plants to respond to external environmental stresses [8,9].
Among the above transcription factors, YABBY, as a small family, receives a lot of attention in several aspects of plant research [10][11][12]. The structural domains of YABBY members, N-terminal C2C2 domain mediating protein interactions, and C-terminal YABBY domain mediating protein-DNA interactions, are highly conserved in different species.
(https://www.arabidopsis.org/, accessed on 10 September 2022). Both protein sequences were inserted into the online tool pfam to confirm the conservative domain of YABBY. Using the conservative domain as a query, nine YABBY gene family members were identified from the lotus genome. Nine NnYABBY genes were named from NnYABBY1 to NnYABBY9 according to the order of their chromosomal locations. The lengths of nine NnYABBY gene-encoded proteins varied from 146 to 211 amino acids (Table 1). Each NnYABBY family member has a different protein molecular weight. The smallest protein is NnYABBY5, and it has a weight of 16481.96 kDa. The longest protein is NnYABBY9, and it has a weight of 23365.56 kDa. The NnYABBY protein sequences had large variations in the theoretical pI (ranging from 5.20 to 9.61). For nine NnYABBY proteins, it was predicted that all proteins did not contain signal peptides.

Phylogenetic Analysis
We selected species such as potato, rice, wheat, soybean, and Arabidopsis for phylogenetic tree analysis, along with lotus ( Figure 1). According to previous studies, YABBY was classified into five categories, including YAB1, YAB2, YAB5, INO, and CRC. NnYABBY3 and NnYABBY7 belong to the YAB2 group. The YAB5 group includes two members, NnYABBY2 and NnYABBY8. NnYABBY1 and NnYABBY9 belong to the YAB1 group. NnYABBY5 belongs to the INO group. NnYABBY4 and NnYABBY6 belong to the CRC group.

Gene Structures and Motif Analysis of NnYABBYs
We analyzed nine NnYABBY gene structures by using the GFF annotation file of lotus ( Figure 2). We found that most NnYABBY gene members had seven exons. NnYABBY3 and NnYABBY7 had six exons. NnYABBY4 had the fewest exons, only two. NnYABBY1 and NnYABBY9 belonged to the same subgroup in the phylogenetic tree, and they had extremely similar gene structures. The situation was the same for other NnYABBY gene family members. NnYABBY gene family members from the same subfamily had more similar gene structures, while the different subfamily members had more different gene

Gene Structures and Motif Analysis of NnYABBYs
We analyzed nine NnYABBY gene structures by using the GFF annotation file of lotus ( Figure 2). We found that most NnYABBY gene members had seven exons. NnYABBY3 and NnYABBY7 had six exons. NnYABBY4 had the fewest exons, only two. NnYABBY1 and NnYABBY9 belonged to the same subgroup in the phylogenetic tree, and they had extremely similar gene structures. The situation was the same for other NnYABBY gene family members. NnYABBY gene family members from the same subfamily had more similar gene structures, while the different subfamily members had more different gene structures.

Chromosome Localization and Collinearity Analysis
We used the GFF file downloaded from the Nelumbo Genome Database to extract the position of each NnYABBY family member on the chromosome and visualized it using TBtools ( Figure 3). NnYABBY1 was located on an unknown chromosomal segment. Other NnYABBYs were distributed on each chromosome. Chromosome 2 and chromosome 4 contained two YABBY gene family members.
To investigate whether there was tandem replication or fragment replication in the chromosomes of lotus, we performed intra-species covariance analysis [37]. The collinearity analysis was visualized by using MCScanX in TBtools ( Figure 4). Five pairs of geneduplication genes were found in nine NnYABBY gene family members, including NnYABBY1/NnYABBY7, NnYABBY2/NnYABBY8, NnYABBY2/NnYABBY7, NnYABBY2/NnYABBY3, and NnYABBY5/NnYABBY6. Gene duplication occurs more frequently in chromosome 2 than in other chromosomes. There were no fragment duplication gene pairs in chromosome 5 for lotus and Arabidopsis thaliana. During the long evolution of lotus, the emergence of gene duplication events facilitated the creation of new genes and the creation of genes' new functions.
To understand the types of genes and the relative sequences of conservation among different species that diverged from the same ancestral type, we conducted collinearity analysis between lotus and Arabidopsis thaliana. Two pairs of gene duplication genes occurred between lotus and Arabidopsis thaliana. These gene pairs played an important role in driving the evolution of plants. The prediction of the motif showed that all proteins encoded by NnYABBY gene family members had motif 2 near the N terminus, which consisted of the C2C2 zinc-finger domain, which was consistent with the specific structure of the YABBY gene family. We also found that the gene members from the same subfamily, such as NnYABBY1 and NnYABBY9, had similar motifs. NnYABBY1 and NnYABBY9 were from the YAB1 subfamily, and both had motif 12, motif 15, and motif 16, which can distinguish them from other family members. NnYABBY4 and NnYABBY6 were from the CRC subfamily, and both had motif 19, which may be unique to the CRC subfamily. NnYABBY2 and NnYABBY8 from the YAB5 group had the specific motif 18 and the YAB2 group, including NnYABBY3 and NnYABBY7, had the specific motif 17. The phylogenetic tree was analyzed with the gene structure and motifs together, and we hypothesized that the gene structure and characteristics of the motifs had an important influence on distinguishing the different subfamilies. We also hypothesized that they had significant impacts on the function of NnYABBY genes from different subfamilies.

Chromosome Localization and Collinearity Analysis
We used the GFF file downloaded from the Nelumbo Genome Database to extract the position of each NnYABBY family member on the chromosome and visualized it using TBtools ( Figure 3). NnYABBY1 was located on an unknown chromosomal segment. Other NnYABBYs were distributed on each chromosome. Chromosome 2 and chromosome 4 contained two YABBY gene family members.

Protein Structure Analysis and Protein Interaction Network
The secondary structures of the NnYABBY protein family in lotus consist of an alpha helix, extended strand, and random coil. Random coils make up the largest part of the secondary structures, followed by extended strands and alpha helices. The tertiary structures of the proteins were mapped by SWISS-MODEL ( Figure 5). The structures of gene members from the same subfamily were more similar than those of other members. For example, NnYABBY1/NnYABBY9, NnYABBY2/NnYABBY8, and NnYABBY3/NnYABBY7 were from the same subfamilies and their protein structures To investigate whether there was tandem replication or fragment replication in the chromosomes of lotus, we performed intra-species covariance analysis [37]. The collinearity analysis was visualized by using MCScanX in TBtools ( Figure 4). Five pairs of gene-duplication genes were found in nine NnYABBY gene family members, including NnYABBY1/NnYABBY7, NnYABBY2/NnYABBY8, NnYABBY2/NnYABBY7, NnYABBY2/NnYABBY3, and NnYABBY5/NnYABBY6. Gene duplication occurs more frequently in chromosome 2 than in other chromosomes. There were no fragment duplication gene pairs in chromosome 5 for lotus and Arabidopsis thaliana. During the long evolution of lotus, the emergence of gene duplication events facilitated the creation of new genes and the creation of genes' new functions.

Protein Structure Analysis and Protein Interaction Network
The secondary structures of the NnYABBY protein family in lotus consist of an alpha helix, extended strand, and random coil. Random coils make up the largest part of the secondary structures, followed by extended strands and alpha helices. The tertiary structures of the proteins were mapped by SWISS-MODEL ( Figure 5). The structures of gene members from the same subfamily were more similar than those of other members. For example, NnYABBY1/NnYABBY9, NnYABBY2/NnYABBY8, and NnYABBY3/NnYABBY7 were from the same subfamilies and their protein structures To understand the types of genes and the relative sequences of conservation among different species that diverged from the same ancestral type, we conducted collinearity analysis between lotus and Arabidopsis thaliana. Two pairs of gene duplication genes occurred between lotus and Arabidopsis thaliana. These gene pairs played an important role in driving the evolution of plants.

Protein Structure Analysis and Protein Interaction Network
The secondary structures of the NnYABBY protein family in lotus consist of an alpha helix, extended strand, and random coil. Random coils make up the largest part of the secondary structures, followed by extended strands and alpha helices. The tertiary structures of the proteins were mapped by SWISS-MODEL ( Figure 5). The structures of gene members from the same subfamily were more similar than those of other members. For example, NnYABBY1/NnYABBY9, NnYABBY2/NnYABBY8, and NnYABBY3/NnYABBY7 were from the same subfamilies and their protein structures were more similar than those of others. The differences in protein structures corresponded to the classification of NnYABBY family members and may contribute to their different functions in lotus roots.  We entered nine NnYABBY protein sequences into the STRING website and used the default parameters to visualize the network of NnYABBY protein interactions ( Figure 6). Interaction networks of single YAB proteins were also constructed ( Figure S1). In the network, nine YAB proteins needed to interact with other proteins to be able to connect to a large network of protein interactions, according to the predictions of the STRING system. Interestingly, NnYABBY proteins from the same subfamily were more similarly positioned on the interaction network and the predicted proteins that interacted with them were also identical. BEL2, KAN4, and CRR could interact with multiple NnYABBY family members. KAN4, a member of the GARP family, is extensively involved in the flavonoid synthesis pathway and is able to regulate the flavonoid content of seeds in Arabidopsis [38]. The protein interactions of NnYABBY with KAN4 suggest that NnYABBY may be involved in flavonoid synthesis and regulates the expression of related genes in the flavonoid synthesis pathway. We entered nine NnYABBY protein sequences into the STRING website and used the default parameters to visualize the network of NnYABBY protein interactions ( Figure 6). Interaction networks of single YAB proteins were also constructed ( Figure S1). In the network, nine YAB proteins needed to interact with other proteins to be able to connect to a large network of protein interactions, according to the predictions of the STRING system. Interestingly, NnYABBY proteins from the same subfamily were more similarly positioned on the interaction network and the predicted proteins that interacted with them were also identical. BEL2, KAN4, and CRR could interact with multiple NnYABBY family members. KAN4, a member of the GARP family, is extensively involved in the flavonoid synthesis pathway and is able to regulate the flavonoid content of seeds in Arabidopsis [38]. The protein interactions of NnYABBY with KAN4 suggest that NnYABBY may be involved in flavonoid synthesis and regulates the expression of related genes in the flavonoid synthesis pathway.

Analysis of cis-Acting Elements in Promoter Regions of NnYABBY Genes
To understand the response of NnYABBY to biotic and abiotic stresses, we analyzed the promoter sequence of NnYABBY for cis-acting elements (Figure 7). A total of eighteen different cis-acting elements were identified in nine NnYABBYs. First, a variety of phytohormone response elements were found, including elements for abscisic acid (ACGTG motif), auxin (AACGAC motif), salicylic acid (CCATCTTTTT motif), and gibberellin (TCTGTTG motif). Second, we also found stress-responsive elements in promotors, such as elements for drought (CAACTG motif), low-temperature (CCGAAA motif), anaerobic (AAACCA motif), and anoxic conditions. Third, several light-responsive elements were widely distributed among the nine NnYABBYs which were also the most numerous cisacting elements. Therefore, NnYABBY genes may be involved in a variety of physiological and biochemical reactions. Different NnYABBYs have different cis-acting elements, so they may have different functions during plant growth and development.

Analysis of cis-Acting Elements in Promoter Regions of NnYABBY Genes
To understand the response of NnYABBY to biotic and abiotic stresses, we analyzed the promoter sequence of NnYABBY for cis-acting elements (Figure 7). A total of eighteen different cis-acting elements were identified in nine NnYABBYs. First, a variety of phytohormone response elements were found, including elements for abscisic acid (ACGTG motif), auxin (AACGAC motif), salicylic acid (CCATCTTTTT motif), and gibberellin (TCT-GTTG motif). Second, we also found stress-responsive elements in promotors, such as elements for drought (CAACTG motif), low-temperature (CCGAAA motif), anaerobic (AAACCA motif), and anoxic conditions. Third, several light-responsive elements were widely distributed among the nine NnYABBYs which were also the most numerous cisacting elements. Therefore, NnYABBY genes may be involved in a variety of physiological and biochemical reactions. Different NnYABBYs have different cis-acting elements, so they may have different functions during plant growth and development.

Expression Patterns by Quantitative Real-Time PCR (qRT-PCR)
In order to explore the response of YABBY members to hormone stress, lotus leaves were sprayed with three different hormone treatments, including ABA, ABA+FL, and NaCl. Blades of the same leaf area were selected for qPCR experiments to ensure the accuracy of the results. After spraying the lotus leaves with 100 μmol/L abscisic acid (ABA), all nine YABBY members responded to the ABA treatment to varying degrees ( Figure 8A). Except for NnYABBY2, NnYABBY3, and NnYABBY7, the expression levels of the other members were up-regulated, with NnYABBY4, NnYABBY5, and NnYABBY6 episodes occupying peaks. ABA treatment resulted in the down-regulation of NnYABBY2 and NnYABBY3 expression after spraying. ABA treatment had no obvious effect on NnYABBY7, and its expression did not seem to change. Fluridone, an inhibitor of ABA, was sprayed with ABA together, and the concentration of the FL solution was the same as that of ABA [27]. We hope to prove whether these NnYABBY genes are actually regulated by ABA from the other side. We expected that the expression of some genes positively regulated by ABA would return to normal if we sprayed ABA and Fluridone on the lotus leaves at the same time. The results of the experiment ( Figure 8B) agreed with our expectation. The expression levels of NnYABBY4, NnYABBY5, and NnYABBY6, which were up-regulated by ABA, became normal when we sprayed ABA and Fluridone. The expression levels of NnYABBY2 and NnYABBY3 were down-regulated within eight hours when sprayed with ABA and FL and up-regulated after 8 h when sprayed with ABA and FL. The expression levels of other members were up-regulated after spraying with ABA and FL. Spraying with NaCI could up-regulate the expression levels of NnYABBY1, NnAYBBY6, and NnYABBY9. NnYABBY4, NnYABBY5, and NnYABBY6 had the most obvious responses to hormones ( Figure 8C). They may play important roles in hormone responses and salt stress responses.

Expression Patterns by Quantitative Real-Time PCR (qRT-PCR)
In order to explore the response of YABBY members to hormone stress, lotus leaves were sprayed with three different hormone treatments, including ABA, ABA+FL, and NaCl. Blades of the same leaf area were selected for qPCR experiments to ensure the accuracy of the results. After spraying the lotus leaves with 100 µmol/L abscisic acid (ABA), all nine YABBY members responded to the ABA treatment to varying degrees ( Figure 8A). Except for NnYABBY2, NnYABBY3, and NnYABBY7, the expression levels of the other members were up-regulated, with NnYABBY4, NnYABBY5, and NnYABBY6 episodes occupying peaks. ABA treatment resulted in the down-regulation of NnYABBY2 and NnYABBY3 expression after spraying. ABA treatment had no obvious effect on NnYABBY7, and its expression did not seem to change. Fluridone, an inhibitor of ABA, was sprayed with ABA together, and the concentration of the FL solution was the same as that of ABA [27]. We hope to prove whether these NnYABBY genes are actually regulated by ABA from the other side. We expected that the expression of some genes positively regulated by ABA would return to normal if we sprayed ABA and Fluridone on the lotus leaves at the same time. The results of the experiment ( Figure 8B) agreed with our expectation. The expression levels of NnYABBY4, NnYABBY5, and NnYABBY6, which were up-regulated by ABA, became normal when we sprayed ABA and Fluridone. The expression levels of NnYABBY2 and NnYABBY3 were down-regulated within eight hours when sprayed with ABA and FL and up-regulated after 8 h when sprayed with ABA and FL. The expression levels of other members were up-regulated after spraying with ABA and FL. Spraying with NaCI could up-regulate the expression levels of NnYABBY1, NnAYBBY6, and NnYABBY9. NnYABBY4, NnYABBY5, and NnYABBY6 had the most obvious responses to hormones ( Figure 8C). They may play important roles in hormone responses and salt stress responses.

Subcellular Localization
According to the quantitative results (Figure 8), the relative expressions of NnYABBY4, NnYABBY5, and NnYABBY6 changed most significantly under various hormone treatments. Subcellular localization can reflect the specific locations of the proteins encoded by YABBYs. To better understand the molecular role of YABBY in response to hormones and stress in lotus leaves, the above three members were selected for subcellular location. The control protein we used was 1300-GFP, and its green fluorescence appeared in the nucleus, cytoplasm, and cell membrane. The green fluorescence of

Subcellular Localization
According to the quantitative results (Figure 8), the relative expressions of NnYABBY4, NnYABBY5, and NnYABBY6 changed most significantly under various hormone treatments. Subcellular localization can reflect the specific locations of the proteins encoded by YABBYs. To better understand the molecular role of YABBY in response to hormones and stress in lotus leaves, the above three members were selected for subcellular location. The control protein we used was 1300-GFP, and its green fluorescence appeared in the nucleus, cytoplasm, and cell membrane. The green fluorescence of NnYABBY4, NnYABBY5, and NnYABBY6 proteins all appeared on the cell membrane and cytoplasm. The cell membrane and cytoplasm are probably the most important places where NnYABBY4, NnYABBY5, and NnYABBY6 perform their functions ( Figure 9). NnYABBY4, NnYABBY5, and NnYABBY6 proteins all appeared on the cell membrane and cytoplasm. The cell membrane and cytoplasm are probably the most important places where NnYABBY4, NnYABBY5, and NnYABBY6 perform their functions (Figure 9). Figure 9. Subcellular localization of the YABBY proteins in lotus. The picture above is the control and that below shows NnYABBY5, NnYABBY6, and NnYABBY7. Scale bar = 20 μm.

Determination of Transcriptional Activation Function
The full-length cDNA of NnYABBY4, NnYABBY5, and NnYABBY6 was cloned into the PGBKT7 vector, and its self-activation ability was verified by a yeast experiment (Figure 10). The negative control pGBKT7, NnYABBY4, NnYABBY5, and NnYABBY6 could grow on SD/−Trp medium, which confirmed that the experimental procedures and practices were correct. Their inability to grow monoclonal colonies on SD/−Trp/−Ade/−His medium indicated that NnYABBY4, NnYABBY5, and NnYABBY6 lacked self-activation ability. They may require the help of other proteins to function and could be used as bait to search for genes that interact with them in cDNA libraries to further analyze the potential functions of NnYABBY proteins. Figure 9. Subcellular localization of the YABBY proteins in lotus. The picture above is the control and that below shows NnYABBY5, NnYABBY6, and NnYABBY7. Scale bar = 20 µm.

Determination of Transcriptional Activation Function
The full-length cDNA of NnYABBY4, NnYABBY5, and NnYABBY6 was cloned into the PGBKT7 vector, and its self-activation ability was verified by a yeast experiment ( Figure 10). The negative control pGBKT7, NnYABBY4, NnYABBY5, and NnYABBY6 could grow on SD/−Trp medium, which confirmed that the experimental procedures and practices were correct. Their inability to grow monoclonal colonies on SD/−Trp/−Ade/−His medium indicated that NnYABBY4, NnYABBY5, and NnYABBY6 lacked self-activation ability. They may require the help of other proteins to function and could be used as bait to search for genes that interact with them in cDNA libraries to further analyze the potential functions of NnYABBY proteins.

Discussion
The YABBY family has a typical zinc-finger structural domain and a YABBY structural domain, which are specific transcription factors. In addition to maintaining organ polarity and meristem activity, YABBY proteins can also promote the growth of lamina by regulating the signal transduction processes between the front and back of the leaf [39]. Six members of the YABBY gene family in Arabidopsis have different expression patterns. The expressions of FIL and YAB3 are located in the abaxial domain of lateral organs [18,21,27]. YAB2 and YAB5 are expressed in abaxial regions of the aboveground lateral organ primordia, whereas the other two (CRC and INO) are expressed in the abaxial domains of the carpels and the outer integument of the ovules, respectively [21,39]. FIL, YAB2, YAB3, and YAB5 have functions in the establishment of leaf polarity, the general genetic program of leaf expansion, and leaf formation [27]. When the genes for FIL, YAB2, and YAB5 were mutated in Arabidopsis and thus became non-functional, we found that the mutant leaves lost their abaxial and adaxial identities in Arabidopsis [40,41]. The mutants of these YABBY genes could bring about radial filamentous floral organs [20,42]. Members of the YABBY family have a role in the outer tepals and leaf development, and when they lose their function, it leads to a reduction in leaf growth [43].
Although articles about the YABBY family have been reported for other species, there are few reports of the YABBY family in lotus. The number of YABBY genes in lotus still needs to be determined. Therefore, we identified nine members of the NnYABBY gene family in lotus by searching the genome database of lotus based on the raw HMM models for the conserved YABBY domain and C2-C2 domain. Based on the taxonomy of the model plants and numerous well-studied species, we divided NnYABBY in lotus into five subfamilies [44,45]. According to the genome sequence information, the gene structure of NnYABBY family members was visualized (Figure 2A). Different NnYABBY family members contain different numbers of exons and introns. The MEME website was used to visualize the motif structure of YABBY members ( Figure 2B). Members of the NnYABBY gene family from the same subfamily had similar motif structures. Gene structure and motif structure are likely to be an important basis for dividing different subfamilies. Different motifs may lead to different functions of YABBY family members, which lead to different roles in plant growth and development. Almost every subfamily has its own unique motif, which is not found in other subfamilies. Motif 18 only appeared in NnYABBY2 and

Discussion
The YABBY family has a typical zinc-finger structural domain and a YABBY structural domain, which are specific transcription factors. In addition to maintaining organ polarity and meristem activity, YABBY proteins can also promote the growth of lamina by regulating the signal transduction processes between the front and back of the leaf [39]. Six members of the YABBY gene family in Arabidopsis have different expression patterns. The expressions of FIL and YAB3 are located in the abaxial domain of lateral organs [18,21,27]. YAB2 and YAB5 are expressed in abaxial regions of the aboveground lateral organ primordia, whereas the other two (CRC and INO) are expressed in the abaxial domains of the carpels and the outer integument of the ovules, respectively [21,39]. FIL, YAB2, YAB3, and YAB5 have functions in the establishment of leaf polarity, the general genetic program of leaf expansion, and leaf formation [27]. When the genes for FIL, YAB2, and YAB5 were mutated in Arabidopsis and thus became non-functional, we found that the mutant leaves lost their abaxial and adaxial identities in Arabidopsis [40,41]. The mutants of these YABBY genes could bring about radial filamentous floral organs [20,42]. Members of the YABBY family have a role in the outer tepals and leaf development, and when they lose their function, it leads to a reduction in leaf growth [43].
Although articles about the YABBY family have been reported for other species, there are few reports of the YABBY family in lotus. The number of YABBY genes in lotus still needs to be determined. Therefore, we identified nine members of the NnYABBY gene family in lotus by searching the genome database of lotus based on the raw HMM models for the conserved YABBY domain and C2-C2 domain. Based on the taxonomy of the model plants and numerous well-studied species, we divided NnYABBY in lotus into five subfamilies [44,45]. According to the genome sequence information, the gene structure of NnYABBY family members was visualized (Figure 2A). Different NnYABBY family members contain different numbers of exons and introns. The MEME website was used to visualize the motif structure of YABBY members ( Figure 2B). Members of the NnYABBY gene family from the same subfamily had similar motif structures. Gene structure and motif structure are likely to be an important basis for dividing different subfamilies. Different motifs may lead to different functions of YABBY family members, which lead to different roles in plant growth and development. Almost every subfamily has its own unique motif, which is not found in other subfamilies. Motif 18 only appeared in NnYABBY2 and NnYABBY8 from the YAB5 subgroup. The YAB1 subfamily has the most unique motifs, including motif 12, motif 15, and motif 16. NnYABBY3 and NnYABBY7 in the YAB2 subfamily have motif 17. Each unique motif has value to be further investigated, and they may be closely related to the unique functions of their respective subfamilies. Motif 19 is unique to the CRC subfamily, which is required for carpel development. It is likely that motif 19 is an important functional region of the CRC family, which could promote the longitudinal growth of the developing gynoecium, suggesting that motif 19 has the possibility to be related to a unique function in the carpel development of lotus. Nine NnYABBY gene family members were evenly distributed on the eight chromosomes of lotus ( Figure 3). Collinearity analysis showed that there were five pairs of gene duplication in the eight chromosomes of lotus ( Figure 4). Between the two species of lotus and Arabidopsis, two pairs of gene duplication occurred. The condition of gene pair duplication is possibly related to the division of NnYABBY family members. The secondary protein structures of the YABBY family members were predicted using the Phyre2 website, and the tertiary structures were visualized using the SWISS website. Each NnYABBY family member had multiple genes that interacted with it and were involved in multiple plant growth and development processes together. Valuable genes interacting with YABBY remain to be discovered. The prediction of protein interaction networks for nine NnYABBY members showed that KAN4, BEL1, and CRR1/3 could interact with several NnYABBY gene family members. CRR, as a dihydrodipicolinate reductase, functions in the lysine biosynthetic pathway and is able to expand the ovaries of rice [46,47]. YABBY proteins are likely to interact with CRR1, and thus participate in the cyclic electron transport around photosystem I. The biological processes in plants that YABBY members are involved in by interacting with other genes remain to be discovered, and this is of great significance. CRR3 was found to be a disease-resistance gene in Brassica rapa [48]. Among these stresses, salt stress, as the main environmental stimulus affecting plant growth and development, has been widely investigated in many crops (Shavrukov, 2013 #72;Shavrukov, 2013 #72;Shavrukov, 2013 #72;Shavrukov, 2013 #72;Shavrukov, 2013 #72) [49]. These studies have unearthed and identified many excellent plant salt-tolerance genes (Bian, 2020 #73; Gong, 2020 #74) [50,51]. Among our quantitative results (Figure 8), YABBY4, 5, and 6 showed high changes in relative expression under conditions of hormonal stress and salt stress. Therefore, we hypothesize that YAB is likely to be involved in the responses to biotic and abiotic stresses through binding to certain genes. The results of self-activation verification experiments showed that YABBY4, YABBY5, and YABBY6 had no self-activation activity. They probably need some interacting proteins to bind to them in order to function in lotus. It is necessary to discover the protein that could bind to YABBY members.
Lotus is a persistent perennial herb in the lotus family and is one of the most important aquatic vegetables in China [52]. This aquatic vegetable is rich in nutrients, mainly starch, fat, sugar, protein, alkaloid, riboflavin, flavonoids, carotene, vitamin C, and so on [53,54]. Lotus root has not only edible value and medicinal value, but is also an important commodity for foreign exchange. However, salt stress has an important impact on the yield and quality of lotus root [55]. Therefore, determining the key resistance genes is an important task to breed high-quality and high-yielding lotus root varieties. Our results suggest that YABBY4, YABBY5, and YABBY6 may be key genes for improving stress tolerance in lotus roots, which is likely to improve the lotus root yield under biotic and abiotic stresses.

Identification of YABBY Family Members in Lotus
Genome data, cDNA sequences, and peptide sequences of lotus (Nelumbo nucifera Gaertn.) were downloaded from the Nelumbo Genome Database (http://nelumbo.biocloud. net/nelumbo/home, accessed on 17 December 2022) [2]. We identified the conserved domain of YABBY by analyzing the Arabidopsis YABBY sequences obtained from the TAIR10 database (https://www.arabidopsis.org/, accessed on 17 December 2022) using the pfam website (http://pfam.xfam.org/search/sequence, accessed on 17 December 2022). Then, we identified all members of the YABBY family in lotus by tBLASTN. These YABBYs' sequence information was used to predict the physicochemical properties of proteins, such as the amino acid length, theoretical isoelectric point, and signal peptides.

Phylogenetic Analysis
The YABBY protein sequence information of wheat, potato, maize, and rice was downloaded from the NCBI database (https://www.ncbi.nlm.nih.gov/, accessed on 17 December 2022). Protein sequences were used for phylogenetic analysis. The MEGA11 software was used to construct a neighbor-joining phylogenetic tree for YABBY family members [4,56]. The pair-wise estimates of the genetic distances were used to calculate the branch lengths in a bootstrap analysis of 1000 replicates.

Gene Structure Analysis and Motif Identification
The exon-intron structures of the NnYABBY family genes were analyzed based on the GFF annotation file obtained from the Nelumbo Genome Database. Motifs of the NnYABBY proteins were identified using MEME's online tools (http://meme-suite.org/tools/meme, accessed on 17 December 2022) [57]. The number of motifs was set to 10. The structure view was drawn by TBtools [58].

Analysis of NnYABBY Protein Structure and the Network of Proteins Interaction
The main forms of the secondary structures of NnYABBY proteins include α-helices, β-folds, and random coils. We predicted the secondary structure of NnYABBY proteins using the Phyre2 website (http://www.sbg.bio.ic.ac.uk/~phyre2/html/page.cgi?id=index, accessed on 17 December 2022). The SWISS website was used to predict the tertiary structures of NnYABBY proteins and export the corresponding model images (https:// swissmodel.expasy.org/, accessed on 17 December 2022).
The STRING database (https://cn.string-db.org/, accessed on 17 December 2022) is one of several online resources dedicated to organism-wide protein association networks [59]. The protein interaction network of NnYABBY members was analyzed using the STRING website.

Chromosome Localization and Collinearity Analysis
We used the annotation GFF files of the lotus genome to visualize the chromosomal localization of NnYABBY genes using the TBtools software. We predicted collinearity between genomes and visualized it by using MCScanX and Circos in TBtools [60].

Analysis of cis-Acting Elements in Promoter Regions of NnYABBY Genes
The upstream 2000 bp of every NnYABBY gene was used as its promoter sequence. The cis-acting elements were analyzed using PlantCARE (http://bioinformatics.psb.ugent.be/ webtools/plantcare/html/, accessed on 17 December 2022). TBtools was used to visualize the number of cis-acting elements in promoters.

Expression Patterns by Quantitative Real-Time PCR (qRT-PCR)
All material plants were grown in the aquatic vegetable experimental field at Yangzhou University, Yangzhou, China. Lotus plants that had grown for two weeks were used in the qRT-PCR experiment. We applied different treatments to the lotus seedlings, including ABA, ABA+FL, and NaCl. The concentration of the ABA and FL solutions was 100 µmol/L. The concentration of the NaCl solution was 200 mmol/L. Untreated lotus was used as the control. We collected samples at 0, 2, 4, 8, 12, and 24 h after various treatments, then dropped them into liquid nitrogen immediately and stored them at −80 • C. The total RNA was extracted using a polysaccharide polyphenol plant total RNA extraction kit (Proteinssci Company). We used 1% agarose gel for electrophoresis, and the results showed three clear and bright bands, especially the 28s band, indicating that the quality of RNA extraction was good. Then, the cDNA was prepared by using reverse transcription kits (Vazyme company, Nanjing, China). This kit contained 5× gDNA Wiper Mix, which can eliminate gDNA contamination in RNA samples effectively. Experiments were conducted according to the system requirements of the reverse transcription kit, and the amount of RNA added to each sample was 1 µL. The cDNA of each sample for qPCR was diluted to 300 ng/µL. We designed the specific qRT-PCR primers (Table S1) based on the coding sequences (CDS) of every NnYABBY gene on NCBI. ChamQ Universal SYBR qPCR Master Mix (Vazyme Biotech, Nanjing, China) was used in the qPCR experiments. The mix solution of qRT-PCR was 2 × ChamQ SYBR qPCR Master Mix, 10 µL; Primer F, 1 µL; Primer R, 1 µL; Template cDNA, 1 µL; and ddH 2 O to 20 µL. The qPCR procedure was set up according to the requirements of the kit above. The data were quantitatively analyzed by the 2 −∆∆CT method [61]. The PCR procedure was run using CFX Connect (BIO-RAD, America). NnTUA was used as a reference gene in the qRT-PCR experiments (Zhao, 2022 #80) [6]. The primers for the TUA gene can be viewed in supplemental data (Table S1).

Subcellular Localization
The CDS sequences of NnYABBYs whose stop codons were removed were cloned using 2×Heiff Canace Plus PCR Master Mix (YEASEN, Shanghai, China). NnYABBYs were respectively built into the pCAMBIA-1300 vectors' fluorescent protein N-terminal GFP using Hieff Clone Plus One Step Cloning Kit (YEASEN, Shanghai, China). Two restriction sites, SacI and XbaI, were used to construct the vector. NnYABBYs with GFP fusion expression vectors and empty vectors were transferred into the leaves of tobacco (Nicotiana benthamiana). The tobacco plants were kept in the dark for 3 days. Then, we observed them under a confocal laser scanning microscope (TCS SP8 STED, Wetzlar, Germany).

Determination of Transcriptional Activation Function
NnYABBYs CDS was cloned into a pGBKT7 vector and these plasmids were transformed into a Y2H yeast strain. Two restriction sites, BamHI and EcoRI, were used to construct the vector. The transformed yeast strain grew on SD/-Trp plates and SD/-Ade/-His/-Trp plates at 30 • C for 3-6 days.

Statistical Analysis
The statistical analysis method we used was Student's t-test in the relevant experiments. p values of <0.05 were considered statistically significant (*), and p values of <0.01 were considered highly statistically significant (**).

Conclusions
In conclusion, a total of nine NnYABBY gene members of the lotus were identified and systematically analyzed. The results showed that NnYABBY genes from the lotus could be classified into five different subgroups, YAB1, YAB2, YAB5, INO, and CRC. The gene duplication phenomenon may contribute to the evolution of lotus and the expansion of NnYABBY family genes. The prediction of the protein interaction network revealed that NnYABBYs may be involved in the regulation of flavonoid biosynthesis. The qRT-PCR analysis indicated that NnYABBY4, NnYABBY5, and NnYABBY6 are sensitive to ABA and salt treatment. They may be involved in the physiological responses of plants to abiotic stresses. NnYABBYs have tremendous research value in modern genetic and biological techniques.
Supplementary Materials: The following supporting information can be downloaded at: https:// www.mdpi.com/article/10.3390/plants12020380/s1, Figure S1: Diagram of the interaction network between YABBY and other proteins; Table S1: Primers in this study; Table S2: Secondary structure prediction of the NnYABBY protein sequence.