The diversity and complexity of KFB structures make their functions diversified.
Although both the F-box proteins and the Kelch containing proteins can bind to other proteins to mediate the substrates degradation via ubiquitylation pathway in all organisms, some studies have found that proteins that co-exist with the F-box domain and Kelch motifs were only observed in eukaryotes [35, 39, 40]. Compared to KFB in human and other animals, a large number of KFB members were identified in plants . More than 103, 68 and 31 KFB members were identified in Arabidopsis thaliana, Populus trichocarpa and Salvia miltiorrhiza, respectively . To date, multiple KFB genes have been isolated from chickpea, Arabidopsis, wheat and so on [14–16], but the potato KFB members have not been systematically identified and investigated. In this study, 44 KFB genes from potato (Solanum tuberosum) were identified and analyzed in phylogenetic relationship, extron-intro organization, motif composition, chromosomal location, syntenic relationship and expression patterns. However, these 44 members may not represent all the KFB genes in the potato genome. The main reason is the lack of strictly conserved sequences in the F-box domains and Kelch motifs [3, 9], in which only a few amino acid residues are relatively invariant (Fig. 1, Additional file 1: Table S2 and S3). Therefore, it is possible that there exist other StKFB members that have not been detected.
By analyzing the protein sequences of F-box domains of StKFBs, we found that L at the 8th and 20th positions, P at the 9th position, I at the 16th position, and C or S at the 32nd position were highly conserved residues, which is consistent with the results of existing research . Besides, D (aspartic acid), L, P, V (valine) at the 11th, 17th, 21st and 31st positions, respectively, were also conserved in F-box domains of StKFBs. However, these relatively conserved amino acids were discontinuous and thus showed low identity, which makes identification of KFB members difficult. Kelch motif is the secondary domain of KFB proteins , and characterized by 8 highly conserved amino acids: 4 hydrophobic amino acid residues, 2 glycine (G) and 2 aromatic amino acid residues (Y or W) (Additional file 1: Table S3). Multiple Kelch motifs would be folded to form a β-propeller with a pocket that coordinates ions required for enzyme activity and is the most likely site for KFB substrate binding . The motif distribution of StKFB members were further analyzed. Based on the sequence and location information of these motifs, it is speculated that Motif 1, 8 and 12 were part of F-box domain, while Motif 2, 3, 6, 9 and 17 were Kelch domains. These different motifs belong to the same domain, showing the variability of this domain. On the other hand, some motifs like Motifs 8 and 12 were found only in the members of Group I and Group II, indicating the specificity and relative conservatism of these motifs.
F-box domains and Kelch domains have been identified as essential components for degradation of regulatory proteins via UPS . The F-box domain recognizes and binds with SKP1 to form the SCF E3 ubiquitin ligase complex; while Kelch domain is responsible for selectively interacting with target proteins . Therefore, the variability of the Kelch domain is important for the recognition of different substrates, which has been demonstrated in both animals and plants. For example, α-Scruin, a Kelch repeat protein in Limulus spermatozoa, has been demonstrated to bind with F-actin and participate in actin stabilization and crosslinking. While β-scruin, having 67% sequence identity with α-Scruin, was located in the actin-free acrosomal vesicle and had different binding partners from α-scruin . In Arabidopsis, AtKFB50 (At3g59940) and AtKFBCHS (At1g23390) respectively recognized and bind to PAL and CHS, mediating their proteolysis [14, 29]. Besides, the number of Kelch repeats varies in different KFB family members, which may also be a vital factor that causes the difference in KFB functions . In this study, most potato KFB members (30/44) contain 1-2 Kelch motifs, followed by those containing 3 Kelch motifs (8 members). StKFB members containing 4-6 Kelch motifs are the fewest, with only 6 members in total. Although it is known that β-propellers structure formed by multiple Kelch repeats can produce different contact sites and interact with different partners, the most key residues associated with substrate proteins remain unknown. Moreover, due to the low sequence similarity of the Kelch motifs, it is almost impossible to infer its function from the primary sequence of KFB. In addition, many of them have degenerated Kelch motifs, suggesting that they might be pseudogenes or their functions may be divergent . Therefore, the binding substrates of these StKFB members and their functions need further experimental verification.
The evolution of the StKFB family is relatively stable, and the duplicated genes may result in functional differentiation of StKFB members.
Previous studies implied that KFB family originated before the branching of animals and plants, and may have undergone a rapid evolution in some land plants . Sun et al. have found that one of the KFB subfamilies (G5) included large numbers of KFB genes in Arabidopsis, but had very few members in rice, pine and poplar, suggesting that a rapid gene birth of KFBs has occurred in Arabidopsis . Also, a phylogenetic analysis of KFB proteins from S. miltiorrhiza, Arabidopsis, rice, human, mice and C. reinhardtii showed that 67 of 69 KFB members in Group I were belong to Arabidopsis . Similarly, in our results of KFB family evolutionary relationship among potato, Arabidopsis, rice and upland cotton, we found that 71 of the 76 members of Group I were Arabidopsis KFBs and only 5 KFBs were from other plants that we analyzed (Fig. 4). These results indicated that KFB members may be relatively stable in most plants, and only a few plants, such as Arabidopsis, may have experienced drastic expansion.
One of the main driving forces of gene expansion is the occurrence of gene duplication events . Multiple KFB genes in the G5 subfamily of Arabidopsis were found to be tandemly arrayed on the same chromosome, which probably led to the gene evolution . Potato KFB family did not seem to undergo a rapid gene birth event like Arabidopsis KFBs. Forty-four StKFB genes were unevenly located on 12 potato chromosomes, including 2 pairs of tandem duplications (StKFB15/StKFB16, StKFB40/StKFB41) and 1 pair of segmental duplications (StKFB16/StKFB29) (Fig. 2). The Ka/Ks ratios of three pairs of duplicated StKFB genes were all less than 1, suggesting that the duplicated StKFBs might have undergone great selection constraint during evolution. Also, the Ka/Ks values of the orthologous pairs of KFB genes between potato and other plants were all less than 1, denoting that the corresponding homologous KFBs have not experienced positive selection (Additional file 1: Table S5). Besides, the syntenic analysis of KFB genes in different plants showed that the numbers of syntenic KFB pairs between potato and other dicots (Arabidopsis, pepper, tomato and upland cotton) were more than those between potato and the monocot (rice), indicating that potato KFBs had a closer syntenic relationship with those in dicots. Furthermore, multiple KFB orthologous pairs between potato and other two solanaceae plants (tomato and pepper) were arrayed on corresponding chromosomes and in corresponding orders, speculating that the syntenic relationship of potato KFBs was closer to the KFBs in tomato and pepper. The closely related gene members in the phylogenetic tree may have similar structure and function . Therefore, phylogenetic analysis can be used as a preliminary method to study the potential function of the unknown StKFBs.
The existence of duplicated KFBs may result in redundancy of their function [35, 42]. For instance, two duplicated genes in Arabidopsis, LKP1/ZTL/AtKFB98 and LKP2/FKL2/AtKFB22, were found to share redundant functions in controlling the circadian clock and flowering time . Both AtKFB29 and AtKFB32 were involved in the anther development, indicating that they may participate in the similar biological processes and have redundant functions . However, numerous studies have confirmed that gene evolution caused by gene duplication may also lead to the loss of original functions and the generation of new functions. In tartary buckwheat, several duplicated FtARFs (like FtARF7 and FtARF13) were highly expressed in different organs . Similarly, many tandemly duplicated AtKFB members of G5 showed preferential expression in certain organs . In this study, potato duplicated KFBs showed the different expression patterns in various potato organs and under diversified stresses (Fig. 7a and 7b). StKFB41 was highly expressed in mannitol-treated potato plants, but StKFB40 did not show obvious expression. Besides, StKFB16 was mainly expressed in shoots and immature fruits, while its tandemly duplicated gene StKFB15 was highly expressed in immature fruits and stolon. StKFB29, the segmentally duplicated gene of StKFB15 was predominately expressed in stolon. It is possible that evolution leads to structural differences in proteins, such as the generation of degenerated Kelch motifs, and results in their divergent functions.
Expression patterns and functional prediction of the StKFB genes
KFB proteins are widely involved in multitudinous biochemical and physiological processes in plants. The accelerated evolution of the KFB family may have contributed to more complex and varied protein-degradation mechanisms to improve plant adaptation to changing environments . At present, the functions of some KFB genes have been deeply studied in Arabidopsis, rice and other model plants, while only a few of the StKFBs have been functionally characterized in potato. Therefore, the existing research results of KFB homologous genes in other species can be used as an important basis for the functional prediction of potato KFB family members. The functional annotations of StKFB members and their corresponding homologous genes in Arabidopsis are shown in Additional file 1: Table S10. According to the annotated information, we found that almost all KFBs may be involved in the degradation of specific proteins by UPS (Table 4), thus playing an important role in different plant growth stages.
Primarily, the role of KFBs in different physiological processes of plant growth and development cannot be ignored. In this study, public RNA-seq data was used to investigate the expression profiles of StKFB genes in several potato tissues and in potato plants with different treatments. The results showed that StKFB10, annotated as S-haplotype-specific F-box gene (SFB) (Additional file 1: Table S10), was specifically highly expressed in flowers (Fig. 7a), indicating that this gene may play an essential role in flower development. SFB specifically degrades non-self S-RNase through the formation of SCFSFB complex with SCF, while its self S-RNase is not degraded. This inhibits the growth of self-pollen tubes by degrading ribosomal RNA (rRNA), thus presenting self-incompatibility in potato and other plants . In addition, StKFB08, StKFB13, StKFB20, StKFB22, StKFB28, StKFB33, StKFB35 and StKFB36, were also highly expressed in stamen or other flower tissues, indicating that they may also regulate potato flowering development. These studies provide evidence and direction for functional prediction of these StKFB genes, but the specific functional mechanism needs to be further studied.
StKFB01 was a LOV blue light receptor gene (StFKF1) and was highly expressed in whole flowers, leaves and petioles in potato (Fig. 7a). It has been reported that StFKF1, StGI and StCDF1 would form a complex that mediates degradation of StCDF1 through ubiquitination pathway and ultimately induces the expression of StCONSTANS (StCO) . StCO is essential for converting light and clock signals into flowering signals, thereby promoting flowering and inhibiting tuberization by regulating the expression of StFT and its homologous genes . Therefore, StKFB01 plays an important role in photoperiodic flowering and potato tuberization. Its orthologous genes AtFKF1 (At1g68050) and OsFKF1 (Os11g34460) also serve as photoreceptors that regulates flowering in Arabidopsis and rice [47, 48]. The similar function of these three KFB proteins may be attributed to the fact that they all contain a LOV domain belonging to the Per-Arnt-Sim (PAS) superfamily (Additional file 3: Figure S6), which is a blue light sensing module . Although StKFB27 belongs to the same group as these three KFBs, it is highly expressed in shoots and mature fruits (Fig. 7a), which may show different functions due to its lack of the LOV domain (Additional file 3: Figure S6).
KFBs not only participate in the growth and development of organs and tissues, but also mediate plant defense signaling . At present, the mechanism of F-box proteins response to stresses has been well investigated, while the regulation of KFBs in stress responses is rarely studied. It has been reported that multiple F-box genes, such as ATPP2-B11 and OsMSR9, positively regulate salt tolerance in plants . A nuclear KFB member in chickpea, named CarF-Box1, was also found to have a positive response to salt stress . In this study, StKFB02/03/04/17/30/34/40 had up-regulated expression levels in salt-stressed potato plants, implying that they may participate in salt stress response. For drought stress, the expression of StKFB04/11/17/23/34/35/41 were up-regulated, while StKFB06 was down-regulated in potato treated with mannitol. These genes may play positive or negative roles in potato drought tolerance. Similar results were found in other F-box proteins, such as TaFBA1 and GmFBX176, which are positive and negative regulators of drought tolerance in plants, respectively [51, 52]. Some StKFBs were also induced by heat, ABA, IAA and GA3, but the functional mechanism remains unclear. In addition, some KFB genes were identified to be involved in plant pathogen interaction as the “susceptibility” (S) genes, contributing to the successful infection of pathogens . For example, KMD3/AtKFB39 (At2g44130), a KFB from A. thaliana, could be induced in roots by Meloidogyne incognita infection . The expression of BIG24.1 was induced by botrytis infection in grapevine . However, in this study, we did not find any StKFBs that can be induced by P. infestans (Fig. 7b). Whether and in what way these StKFB are involved in potato response to P. infestans requires further investigation.
Additionally, some studies have clarified the involvement of KFBs in secondary metabolites production. OsFBK1 (Os01g47050) negatively regulated lignin synthesis by degrading Cinnamoyl-CoA Reductase (OsCCR), and thus affected the secondary cell wall thickenings of anther and root . In Arabidopsis, Zhang et al. have elucidated that protein ubiquitination and degradation mediated by AtKFB01 (At1g15670), AtKFB20 (At1g80440), AtKFB39 (At2g44130) and AtKFB50 (At3g59940) regulated the proteolysis of PALs, thereby modulating phenylpropanoid metabolism . In 2017, they also found that another KFB, named KFBCHS (At1g23390), regulate the proteolysis of CHS and control flavonoid and anthocyanin biosynthesis in Arabidopsis . However, there is limited understanding of the types of KFB interacting proteins involved in the ubiquitination pathway during secondary metabolism. Anthocyanin is one of the main secondary metabolites in the biosynthesis of plant flavonoid, which makes flowers, fruits and other organs show various colors under different pH conditions in plant vacuole . Due to its outstanding free radical scavenging capacity, anthocyanin was demonstrated to have healthcare effects such as antioxidant, anti-aging, anti-tumor and immune activity regulation [55–57]. Purple-fleshed potato, accumulating large amounts of anthocyanin content, is regarded as high-value feedstock for food and industrial processing. To investigate which StKFBs might be involved in anthocyanin biosynthesis, transcriptomic analysis and qRT-PCR validation were performed on potato tubers of different colors. The results showed that most of the StKFB genes were differentially expressed in three colored potatoes. StKFB15 and StKFB29, which were closely related with AtKFB01 and AtKFB20, were down-regulated significantly in the purple-fleshed tubers (Xisen-8), suggesting their potential negative function in anthocyanin biosynthesis regulation. StKFB07 and StKFB23, the homologous genes of OsFBK1 and AtKFBCHS, respectively, also showed a downward expression trend in Xisen-8. Furthermore, other genes that were highly expressed in yellow fleshed tubers (Jin-16) and lowly expressed in the red- (Red Rose-2) or purple-fleshed potatoes, such as StKFB11/18/30/38/42/44, may also play a negative role in phenylpropanoid biosynthesis. Notably, no expression of StKFB43 was detected either in different potato tissues or potato plants under different treatments, indicating that this gene is likely to be a pseudogene. This result is consistent with the annotation of its homologous gene in Arabidopsis. These results provide a basis for predicting the functions of StKFB members, but their specific functions need to be verified by future experiments.