Genome-Wide Identiﬁcation and Expression Analysis of UBiA Family Genes Associated with Abiotic Stress in Sunﬂowers ( Helianthus annuus L.)

: The UBiA genes encode a large class of isopentenyltransferases, which are involved in the synthesis of secondary metabolites such as chlorophyll and vitamin E. They performed important functions in the whole plant’s growth and development. Current studies on UBiA genes were not comprehensive enough, especially for sunﬂower UBiA genes. In this study, 10 HaUBiAs were identiﬁed by domain analysis these HaUBiAs had ﬁve major conserved domains and were unevenly distributed on six chromosomes. By constructing phylogenetic trees, 119 UBiA genes were found in 12 species with different evolutionary levels and divided into ﬁve major groups, which contained seven conserved motifs and eight UBiA subsuper family domains. Tissue expression analysis showed that HaUBiAs were highly expressed in the roots, leaves, and seeds. By using promoter analysis, the cis-elements of UBiA genes were mainly in hormone signaling and stress responses. The qRT-PCR results showed that HaUBiA1 and HaUBiA5 responded strongly to abiotic stresses. Under ABA and MeJA treatments, HaUBiA1 signiﬁcantly upregulated, while HaUBiA5 signiﬁcantly decreased. Under cold stress, the expression of UBiA1 was signiﬁcantly upregulated in the roots and stems, while UBiA5 expression was increased only in the leaves. Under anaerobic induction, UBiA1 and UBiA5 were both upregulated in the roots, stems and leaves. In summary, this study systematically classiﬁed the UBiA family and identiﬁed two abiotic stress candidate genes in the sunﬂower. It expands the understanding of the UBiA family and provides a theoretical basis for future abiotic stress studies in sunﬂowers.


Introduction
Many biologically active substances must be prenylated to become soluble in the membrane to become functional [1][2][3]. UBiA superfamily is a class of proteins that fuse isoprenoid or phytyl to aromatic compound molecules from the aqueous phase of the cell. Proteins with UBiA structural domains are widely found in bacteria, fungi, and plants [4]. In plants, these PTases (prenyltransferases) are integral membrane proteins that contain at least one aspartate-rich motif (e.g., NDxxDxxxD) for the binding of prenyl diphosphate via Mg 2+ ions or similar cations, such as Mn 2+ , Co 2+ or Ni 2+ [5]. The substrates of the prenylation reaction play essential roles in all species. The prenylation reaction is generally an evolutionarily conserved and rate-limiting step of the secondary metabolite biosynthetic pathway. The compounds that can be prenylated by PTase include 4-hydroxybenzoic acid (PHB), homogeneous acid (HGA), 1,4-dihydroxy-2-naphthalene acid (DHNA), flavonoids chlorophyll or proheme, which play important roles in electron 2 of 20 transportation, anti-oxidation, and formation of structural lipids for microbial cell walls and membranes.
PHB Polyprenyltransferase (PPT) has been the most comprehensively studied isopentenyl transferase, which transfers varying lengths of isopentenes to PHB [3]. PPT is the rate-limiting step of UQ (Ubiquinone) biosynthesis [6]. In Arabidopsis thaliana, embryonic retardation is a prominent trait of the UQ-deficient mutant caused by PPT absent in the early stage of zygotic embryogenesis [7]. OsPPT1 exhibits PPT enzymatic activity, enabling the restoration of respiratory ability and UQ synthesis, respectively [8]. The tobacco UQ overexpressed plants showed increased tolerance to oxidative stress induced by methyl violet and salt stress [9].
Based on different isopentenyl donors, homogentisate polyprenytransferase can be divided into three categories, such as homogentisate phytyltransferases (HPT), homogentisate geranylgeranyl transferase (HGGT) and homogentisate solanesyl transferase (HST). HST is involved in the biosynthesis of plastoquinone-9. When α-tocopherol is consumed, plastoquinone-9 can replace α-tocopherol, providing protection against singlet oxygen toxicity [10,11]. HPT and HGGT are key rate-limiting enzymes of Vitamin E synthesis that catalyze the binding of homogentisate acid to different isopentenyl donors. HGGT mostly exists in monocotyledons, and its product has a side chain with three unsaturated bonds [12]. HGGT mutants have wilting phenotypes, and a significant reduction in seed total starch content [13] and rice HPT mutants are sensitive to cold stress [14]. The overexpression of homogentisate phytyltransferase genes in tobacco enhances drought tolerance [15]. Pentenyl flavonoids and isoflavones are mainly accumulated in legumes, which can resist the invasion of pathogens by plants and play an important role in the biosynthesis of the secondary metabolites [16]. ChlorophyII synthase is the final step in chlorophyII biosynthesis, which is the phosphorylation of chlorophyII lactone with phytyl or geranyl [17]. Protoheme IX farnesyltransferase (heme O synthase), connecting the tail of farnesyl to protoheme IX, finally turns heme O to heme A in participating plant respiration. DHNA-phytyltransferases, another member of the UBiA superfamily, are involved in phylloquinone biosynthesis by using DHNA as substrate [18].
Sunflower is the fourth largest oilseed crop in the world and has a high economic value. It is also a pioneer plant in saline areas with strong tolerance to various stresses [19,20]. UBiA transferase were involved in the accumulation of secondary metabolism and were the key rate-limiting enzyme for many important reactions, such as the synthesis of vitamin E chlorophyll and proheme. It plays an important role in plant growth and development. Studying how a sunflower senses and responds to various stresses from nature, can provide new insights and the mechanisms of plant tolerance to stress. However, few studies have been done on the functional analysis of the UBiA family under abiotic stresses. Therefore, this study systematically analyzed the UBiA family in sunflower and other 11 species on different evolutionary nodes. Using a more macroscopic perspective for in-depth analysis of the UBiA genes.
In this study, phylogenetic relationship, conserved domain analysis and other bioinformatics methods were carried out in sunflowers. The UBiA genes of species were highly conserved and were divided into five subgroups. The motifs of the encoded proteins performing the same function were clustered in a similar branch. Promoter analysis revealed that the UBiA genes in all species contained at least one ABA, MeJA, cold stress and anaerobic stress-induced response element. In the sunflower, the UBiA genes were expressed in different tissues. HaUBiAs were responsive to ABA, MeJA, cold stress and anaerobic induction. UBiA1 and UBiA5 were identified as highly responsive genes to both hormones and stress treatments. Our study revealed the important role of the UBiA family in sunflowers and screened out two candidate genes responding to abiotic stress.

Identification and Chromosomal Location of UBiA Genes and in Helianthus annuus
To identify the UBiA family members, genome-wide and full-length transcriptome analyses were performed using HMM software. After removing duplicate transcripts and pseudogenes, 10 UBiA genes were finally obtained in Helianthus annuus. To understand the basic characteristics of HaUBiA genes, the gene structures were analyzed ( Figure 1A). The phylogenetic relationship and conserved domains of these 10 HaUBiAs were analyzed, and the results showed that the sunflower UBiA had six structural domains, including UBiA-HPT, UBiA-HST, UBiA-COX10, UBiA-PPT, UBiA-ATG4, UBiA-ABC4. HPT had the highest number of four sequences ( Figure 1B). Ten HaUBiA genes were unevenly distributed on six chromosomes ( Figure 1C). Collinearity analyses of burdock lettuce and sunflower were performed ( Figure S1). There was a linear relationship in seven pairs of genes between the lettuce and sunflower and four pairs between the burdock and sunflower. The effective length of the coding region sequence is 921~1308 bp. The linearly related gene pairs had a ka/ks ratio between 0.15 to 0.3; it suggested that those genes tend to be selected for purification (Table S1). This result indicated that the UBiA genes of Asteraceae were highly conserved in evolutionary terms.
Via a sequences blast, a total of 119 UBiA genes were extracted and screened from these 12 selected species. Among them, Glycine max had a maximum of 24 UBiA genes, and Synechocystis had a minimum of five UBiA genes ( Table 1). The basic information of these genes was analyzed and summarized (Table 2), including amino acid number, molecular weight, theoretical PI, instability index, aliphatic index, and the grand average of hydropathicity. Among them, PpUBiA6 encoded the largest protein, containing 596 amino acids, while OsUBiA9 encodes the smallest protein, consisting of 205 amino acids. The molecular weight (MW) of the protein was between 21.9-64.2 kDa. The range of isoelectric point (PI) was 6.05-10.63. The range of the instability index was 19.93-58.59. The aliphatic index was 86. 33-134.87. The grand average of hydropathicity was −0.025-0.897. Hydrophilic results showed that UBiA usually had hydrophilic properties.

Identification and Phylogenetic Analysis of UBiA Families in 12 Species
To investigate the genetic phylogenetic relationship of the UBiA gene family, 12 different species representing evolutionary nodes were selected. According to the evolutionary level from low to high, a temporal tree of these species was constructed. Figure 2A shows the origin time, evolution degree and genetic relationship between 12 different species.
Through phylogenetic tree analysis, the number of UBiA members in the first four groups was similar, between 14-16, while the number in Group 5 was as high as 56. This suggested that these UBiA members were conserved during the plant evolution, but still, some differences between species had different numbers in each branch. The number of UBiA members in Group 5 increased in more evolved species and in dicotyledonous plants; the UBiA members expanded to different degrees in this group, especially in soybeans ( Figure 2).

Structure Analysis of the UBiA Family Members
To explore the potential functions of the UBiA family members, the conserved motifs and protein domains of 119 UBiA proteins were analyzed (Figure 3). The members of the UBiA family can be divided into five major groups. The classification of UBiA family genes is shown in Table 1. The members of each class of the UBiA family are present in each species. Group 1 corresponds to PHB polyprenyltransferase (PPT), Group 2 corresponds to protoheme IX farnesyltransferases (COX10), Group 3 corresponds to chlorophyll synthases (ATG4), Group 4 corresponds to DHNA-phytyltransferases (ABC4) in phylloquinone biosynthesis, Group 5 corresponds to Homogentisate polyprenytransferase (HPT) ( Figure S2). To further understand the structural features of UBiA family genes, an in-depth analysis of their conserved motifs and structural domains was performed.  Groups 1 to 4, each group corresponds to one conserved domain. Group 1 corresponds to PPT, Group 2 corresponds to COX10, Group 3 corresponds to ATG4, and Group 4 corresponds to ABC4. Group 5 contains four domains, including HPT, HST, PT, HGGT. Each species had one or more UBiA genes in each of the five groups, except for HGGT, which occurred only in Oryza sativa. ATG4 was missing in Group 2 of Arctium lappa. These results indicate that the UBiA genes were also highly conserved across species, with clear distinctions between the different structural domains. For motif analysis, seven different motifs were predicted based on UBiA protein sequences ( Figures 3B and S3). The length of the motifs varied from 21 to 50 bp. Conserved motifs of genes that perform the same function are arranged in a fixed order. In the case of PHB polyprenyltransferase, the two motifs are motif 6 and motif 2. Chlorophyll synthase contains three motifs in the order of: motif 1, motif 7 and motif 2. DHNAphytyltransferases when only motif 2 was present, suggesting that motif 2 is the key motif for DHNA-phytyltransferases. The Protoheme IX farnesyltransferase contains only motif 6. This indicates that motif 6 is the core motif of this class of enzymes. Homogentisate polyprenytransferase consisted of six motifs arranged in the order of motif number 5-1-3-7-2-4.
For domain analysis, all selected genes contained the UBiA domain ( Figure 3C). In Groups 1 to 4, each group corresponds to one conserved domain. Group 1 corresponds to PPT, Group 2 corresponds to COX10, Group 3 corresponds to ATG4, and Group 4 corresponds to ABC4. Group 5 contains four domains, including HPT, HST, PT, HGGT. Each species had one or more UBiA genes in each of the five groups, except for HGGT, which occurred only in Oryza sativa. ATG4 was missing in Group 2 of Arctium lappa. These results indicate that the UBiA genes were also highly conserved across species, with clear distinctions between the different structural domains.

Expression Profile of UBiA Genes in Helianthus annuus
To analyze the biological function of 10 UBiA genes in Helianthus annuus, we performed RT-PCR analysis ( Figure 4A). The UBiA genes were expressed in the highest amount in the leaves, followed by roots and stems, which were hardly expressed in the floral organs. We wanted to understand the pattern of the UBiA genes during seed maturation. Therefore, we selected six time points during the ripening process to analyze the expression of UBiA genes in seeds ( Figure S4). We found three different expression patterns in seeds. In the first few days after flowering, the expression level of HaUBiA1 and HaUBiA2 was high and decreased during the subsequent seed ripening process. The expression of HaUBiA genes mostly increased as the seeds matured in an upward trend. The high expression level at the beginning of the seed setting may be related to pollination. The second expression pattern was that these HaUBiAs, including HaUBiA3, HaUBiA4, HaUBiA7 and HaUBiA9, increased gradually when seed maturing was at a maximum at late maturity. The third expression pattern was the genes, such as UBiA5 and UBiA6 were upregulated only at one period. In short, we can infer that HaUBiAs may play an important role in the process from pollination to seed maturity ( Figure 4B).

Cis-Regulatory Elements Analysis of HaUBiA Promoters
To further explore the putative functions of UBiA family genes in Helianthus annuus, 2000 bp upstream sequences of 119 genes were extracted and analyzed potential cis-elements using PlantCARE (http://bioinformatics.psb. ugent.be/webtools/plantcare/html, accessed on 1 November 2022). The predicted transcription factor (TF) binding patterns were classified into three categories: hormone-related elements, stress-related elements and growth and development-related elements.
We first analyzed the promoter regions of UBiA genes in sunflowers ( Figure 5A). They all had responsive elements, including MeJA, ABA, cold stress, and anaerobic induction. The same results were seen in the promoter regions of 119 UBiA genes ( Figure  5B). MeJA responsiveness contained two different motifs named TGACG-motif and CGTCA-motif, respectively. The ABA response contained 10 different ABRE motifs and the cold stress responsive element had only one LTR motif. The anaerobic stress response

Cis-Regulatory Elements Analysis of HaUBiA Promoters
To further explore the putative functions of UBiA family genes in Helianthus annuus, 2000 bp upstream sequences of 119 genes were extracted and analyzed potential ciselements using PlantCARE (http://bioinformatics.psb.ugent.be/webtools/plantcare/html, accessed on 1 November 2022). The predicted transcription factor (TF) binding patterns were classified into three categories: hormone-related elements, stress-related elements and growth and development-related elements.
We first analyzed the promoter regions of UBiA genes in sunflowers ( Figure 5A). They all had responsive elements, including MeJA, ABA, cold stress, and anaerobic induction. The same results were seen in the promoter regions of 119 UBiA genes ( Figure 5B). MeJA responsiveness contained two different motifs named TGACG-motif and CGTCA-motif, respectively. The ABA response contained 10 different ABRE motifs and the cold stress responsive element had only one LTR motif. The anaerobic stress response contains only one unique ARE motif. These results suggest that UBiA is widely involved in plant growth and development and plays an important role in plant response to stress and hormone response and is highly conserved across species.

Expression Patterns of HaUBiA Genes under the Treatments of Different Hormones
Many responsive elements on the upstream HaUBiA genes were associated with MeJA and ABA by promoter analysis software, and then qRT-PCR analysis was used to verify whether these genes were responding to the above hormone signals (Figure 6). Except for HaUBiA8, the other nine genes showed three different expression patterns after ABA treatment. The first category of genes (seven genes), whose expression increased at the beginning of the treatment and decreased dramatically in the end. The second category of genes (two genes) showed less change during the whole treatment. The third category presented increased expression with the increase in treatment time.
Three expression patterns of HaUBiA existed under MeJA treatment. The first category, whose expression increased at the beginning and gradually decreased with treat-

Expression Patterns of HaUBiA Genes under the Treatments of Different Hormones
Many responsive elements on the upstream HaUBiA genes were associated with MeJA and ABA by promoter analysis software, and then qRT-PCR analysis was used to verify whether these genes were responding to the above hormone signals (Figure 6). Except for HaUBiA8, the other nine genes showed three different expression patterns after ABA treatment. The first category of genes (seven genes), whose expression increased at the beginning of the treatment and decreased dramatically in the end. The second category of genes (two genes) showed less change during the whole treatment. The third category presented increased expression with the increase in treatment time. was like the first category but eventually remained higher than before treatment, included four genes. The expression of genes in the third category decreased with increasing treatment time, including only one gene. These results above showed that most HaUBiA responded to ABA and MeJA treatment. The results indicate that HaUBiA genes were able to respond to ABA and MeJA signaling. The different response patterns suggest that ABA and MeJA may involve in the synthesis of secondary sunflower metabolites and plays a regulatory role to some extent. All these results correspond to the results of promoter analysis and indicate that HaUBiA plays an important role in sunflowers.

Expression Analysis of HaUBiA under Different Stresses
The expression pattern of the HaUBiA gene in cold stress and anaerobic induction. Three tissue sites were chosen (roots, stems and leaves) to analyze the expression under stress treatment. During the cold stress treatment, all genes, except for HaUBiA4 and HaU-BiA9, were expressed in different tissue, and the expression was significantly changed in at least one tissue during the cold stress treatment ( Figure 7A). Among these genes, UBiA2 was expressed in various tissues, and it was significantly downregulated under cold Three expression patterns of HaUBiA existed under MeJA treatment. The first category, whose expression increased at the beginning and gradually decreased with treatment time, included five genes. The second category of genes, in which the expression was like the first category but eventually remained higher than before treatment, included four genes. The expression of genes in the third category decreased with increasing treatment time, including only one gene. These results above showed that most HaUBiA responded to ABA and MeJA treatment. The results indicate that HaUBiA genes were able to respond to ABA and MeJA signaling. The different response patterns suggest that ABA and MeJA may involve in the synthesis of secondary sunflower metabolites and plays a regulatory role to some extent. All these results correspond to the results of promoter analysis and indicate that HaUBiA plays an important role in sunflowers.

Expression Analysis of HaUBiA under Different Stresses
The expression pattern of the HaUBiA gene in cold stress and anaerobic induction. Three tissue sites were chosen (roots, stems and leaves) to analyze the expression under stress treatment. During the cold stress treatment, all genes, except for HaUBiA4 and HaUBiA9, were expressed in different tissue, and the expression was significantly changed in at least one tissue during the cold stress treatment ( Figure 7A). Among these genes, UBiA2 was expressed in various tissues, and it was significantly downregulated under cold stress. UBiA2 may be an important gene widely involved in sunflower cryoprotection. So, to speak, the HaUBiA genes had a strong response to the cold stress signal. Under anaerobic induction, the expression of all the HaUBiA genes was changed at least in one tissue ( Figure 7B). In contrast, HaUBiA1 and HaUBiA5 were significantly upregulated in all tissue, and it was speculated that they may serve as important regulatory genes for maintaining organism survival under anaerobic conditions. The results suggested that HaUBiA genes were strongly induced under cold stress and anaerobic induction. It maybe plays an important function in sunflower resistance to adversity stress. stress. UBiA2 may be an important gene widely involved in sunflower cryoprotection. So, to speak, the HaUBiA genes had a strong response to the cold stress signal. Under anaerobic induction, the expression of all the HaUBiA genes was changed at least in one tissue ( Figure 7B). In contrast, HaUBiA1 and HaUBiA5 were significantly upregulated in all tissue, and it was speculated that they may serve as important regulatory genes for maintaining organism survival under anaerobic conditions. The results suggested that HaUBiA genes were strongly induced under cold stress and anaerobic induction. It maybe plays an important function in sunflower resistance to adversity stress.

Discussion
UBiA family genes are widely involved in the synthesis of various secondary metabolites in plants. However, the evolution of UBiA family genes in the plant is not clear. In this study, 119 UBiA genes from 12 species with different degrees of evolution were selected to analyze phylogenetic relationships, structural domain composition, gene structure and cis-acting elements. In addition, the responses and the potential functions of HaUBiA family genes to cold stress, anaerobic induction, MeJA, and ABA were detected and explored. By constructing phylogenetic trees, a clear classification among UBiAs with five major clades with 119 UBiA family members of 12 species. Each branch corresponds to different functions, and these members were distributed among species at different levels of evolution. The number of UBiA members increased in plants with higher levels of evolution. In soybean, new functions emerged in UBiA, but the number of branches did not change. This suggested that the UBiA family of members had evolved to some extent during evolution, but natural selection is still relatively conserved. A comprehensive analysis of structural domains and motifs showed that most UBiA members contained seven conserved motifs, the most conserved of which was NDxxDxxxD. Interestingly, the conserved motifs of the same functional members between species were arranged in a certain order. This rule applied to most of the UBiA members. The tissue expression results showed that the UBiA genes were expressed in various tissues, with higher expression in the roots, stems and leaves, and accumulated during seed maturation.
In soybean, secondary metabolic genes acquire new functions from primary metabolic genes through positive selection driving gene replication and new functionalization [21]. Therefore, the expansion of the UBiA gene in Compositae also has the possibility to drive new functions. Transcriptional regulation plays a dominant role in the regulation of gene expression and is mainly controlled by its cis-acting elements at the gene promoters [22]. To explore the potential functions of UBiA family genes, we analyzed the cis-acting elements of all UBiA family promoter regions. The results showed that the largest proportion of cisacting elements responded to MeJA, ABA and anaerobic induction, and low temperature and this result was consistent in all selected species, suggesting that the promoters of the UBiA genes are conserved and highly responsive to hormone treatment. Our study detected many ABA-and MeJA-responsive elements in the Helianthus annuus UBiA family. We selected all 10 UBiA genes in sunflower and tested their expression by RT-qPCR analysis under cold, anaerobic induction MeJA and ABA treatments. In hormone treatment, most genes in all 10 HaUBiA genes undergo expression changes in the leaves. Sunflower responds to treatments with both hormones. ABA and MeJA signaling can promote stomatal closure and regulate the expression of related genes [23,24]. Therefore, we hypothesized that sunflower drought resistance might be due to the hormones involved in regulating stomatal closure. In addition, several genes were significantly induced in the leaves, stems, and roots during anaerobic and cold stress, indicating that these genes are involved in hormone response and play important functions in resistance to adversity stress.
Mutations in rice HPT, a key gene for vitamin E synthesis, showed overall growth retardation throughout the growth period, which caused most agronomic traits to be damaged and cold tolerance reduced [14]. In lettuce, HPT was preferentially expressed in mature leaves. The expression of LsHPT was significantly increased under drought and high light stress [25]. Reactive oxygen species (S), a singlet oxygen that affects lipids, proteins and nucleic acids [26,27]. Using HGA as a substrate, plants can synthesize αtocopherol (HPT catalyze) and plastochromanol-8 (HST catalyze). Tocopherol can be used as a singlet oxygen scavenger in photosystem II [18]. The unsaturated side chains of plastochromanol-8 also have quenching activity for singlet oxygen in the hydrophobic environment [28]. Chlorophyll synthase (UBiA3) can also indirectly affect the synthesis of vitamin E. In Arabidopsis, 80% of PDP (prenoid phytyl diphosphate) synthesis must be catalyzed by Chlorophyll synthase [29]. NAC transcription factors regulate cold stress in Arabidopsis [30]. In Arabidopsis, the homologs gene of HaUBiA5 were able to bind to NAC transcription factors [31]. HaUBiA5 might bind to NAC proteins ( Figure S5). We can speculate that UBiA5 may be regulated by NAC transcription factors in leaves.
UBiAs not only play an important role in plants but are also necessary to maintain the growth of animals. COX10 mutations in humans trigger mitochondrial causes such as Leigh syndrome [31,32], a factor that may increase the risk of Alzheimer's disease [33]. PBH isopentenyl transferase mutation is the cause of multisystem disease and nephropathy in infants [34,35]. In summary, the UBiA gene plays an important role in the growth and development of both animals and plants and is an indispensable key gene. In this study, we systematically analyzed the UBiA genes among different species using sunflowers as the entry point at the species evolutionary level and provided a molecular basis for an in-depth study of HaUBiA genes.  [36]. A hidden Markov model was used to evaluate the UBiA domain, and the UBiA family genes of 12 different species were identified with an E value < 10 −5 . CDS was extracted from 12 genomes and translated into proteins. After duplicate transcripts were removed, 119 protein sequences were obtained [37]. The protein parameter CalC of TBtools software (https://github.com/CJ-Chen/TBtools, accessed on 2 November 2022) was used to analyze the number of amino acids, molecular weight, theoretical pI, instability index, aliphatic index, and grand average of hydropathicity.

Phylogenetic Tree Analysis of UBiA Proteins
Protein sequences of UBiA members were compared using the ClustalW program. Phylogenetic trees were constructed by the neighbor-joining method with UBiA members and their homologous sequences in different species using MEGA software (version 7.0). Tree nodes were evaluated through 1000 repeated bootstrap analyses [38]. The final version of phylogenetic tree was showed using ITOL (https://itol.cmbl.del, accessed on 3 November 2022) modification [39].

Chromosomal Location Density, Collinearity, and Ka/Ks Analysis of UBiA Genes
Based on gene annotation information, TBtools was used to analyze the chromosome location and chromosome density of Helianthus annuus L. UBiA family genes. The data source is based on the NCBI database. Helianthus annuus, Lactuca sativa and Arctium lappa were selected to analyze the collinearity of the UBiA family among compositae plants using the multicollinearity scan toolkit. Ka and Ks are calculated using the simple Ka/ Ks calculator in TBtools [37,40].

Plant Materials and Stress Treatments
In this study, AZB, a variety of Helianthus annuus, was used as experimental material to analyze the expression pattern of UBiA family members. Fourteen-day-old seedlings were treated with 15 µmol/L ABA and 200 µmol/L MeJA. Leaves were then collected for RNA extraction and RT-qPCR analysis. For stress treatment, the plant materials were placed in a 4 • C environment and given 16 h of light and 8 h of darkness for 2 days for low-temperature treatment; two-week-old seedlings were completely submerged in water with 16 h of light and 8 h of darkness for one day for anaerobic stress treatment. After treatment, roots, stems, and leaves were collected for RNA extraction and qRT-PCR analysis.

RNA Extraction and Quantitative/Real-Time-PCR (RT-qPCR) Analysis
The total RNA of plant materials was extracted using the OmniPlant RNA Kit to obtain cDNA for subsequent experiments. Use CFX384 real-time system (Biorad, Hercules, CA, USA) to complete qRT-PCR experiment. The reagent used was ChamQ universal SYBR qPCR Master Mix (Vazyme, Nanjing, China). The experiment was repeated three times. The internal reference gene was the tubline of Helianthus annuus L. The list of primers used in the experiment is in Table S2.

Conclusions
In this study, a systematic analysis of the UBiA family was performed. The conservation of UBiA genes across species was verified. Two candidate genes of abiotic stresses were screened in the sunflower. In summary, our study has expanded the understanding of UBiA genes. It provides a reference for abiotic stress studies in sunflowers and offers new research perspectives for crop breeding. However, the functions of HaUBiA1 and HaUBiA5 in abiotic stress will require further study.