https://orcid.org/0000-0001-8677-4784
Figures
Abstract
F-box proteins play critical roles in plant responses to biotic/abiotic stresses. In the present study, a total of 68 wheat F-box/Kelch (TaFBK) genes, unevenly distributed across 21 chromosomes and encoding 74 proteins, were identified in EnsemblPlants. Protein sequences were compared with those of Arabidopsis and three cereal species by phylogenetic and domain analyses, where the wheat sequences were resolved into 6 clades. In silico analysis of a digital PCR dataset revealed that TaFBKs were expressed at multiple developmental stages and tissues, and in response to drought and/or heat stresses. The TaFBK19 gene, a homolog of the Attenuated Far-Red Response (AFR) genes in other plant species, and hence named TaAFR, was selected for further analysis. Reverse-transcription quantitative real-time PCR (RT-qPCR) was carried out to determine tissue-specific, hormone and stress (abiotic/biotic) responsive expression patterns. Of interest, TaAFR was expressed most abundantly in the leaves, and its expression in response to leaf rust variants suggests a potential role in compatible vs incompatible rust responses. The protein was predicted to localize in cytosol, but it was shown experimentally to localize in both the cytosol and the nucleus of tobacco. A series of protein interaction studies, starting with a yeast-2-hybrid (Y2H) library screen (wheat leaf infected with incompatible leaf rust pathogens), led to the identification of three TaAFR interacting proteins. Skp1/ASK1-like protein (Skp1) was found to interact with the F-box domain of TaAFR, while ADP-ribosylation factor 2-like isoform X1 (ARL2) and phenylalanine ammonia-lyase (PAL) were shown to interact with its Kelch domain. The data presented herein provides a solid foundation from which the function and metabolic network of TaAFR and other wheat FBKs can be further explored.
Citation: Wei C, Zhao W, Fan R, Meng Y, Yang Y, Wang X, et al. (2021) Genome-wide survey of the F-box/Kelch (FBK) members and molecular identification of a novel FBK gene TaAFR in wheat. PLoS ONE 16(7): e0250479. https://doi.org/10.1371/journal.pone.0250479
Editor: Jian Zhang, China National Rice Research Institute, CHINA
Received: March 30, 2021; Accepted: July 1, 2021; Published: July 22, 2021
Copyright: © 2021 Wei et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: All relevant data are within the manuscript and its Supporting Information files.
Funding: CW was supported by the Postgraduate Innovation Funding Project of Hebei Province (CXZZBS2021046). XY received supports from the Natural Science Foundation of Hebei Province (C2020204050) and Scientific and Technological Research Projects of Higher Education of Hebei Province (ZD2019086). XW was supported by Natural Science Foundation of Hebei Province (C2021204008) and Provincial Supporting Program of Hebei for the Returned Oversea Scholars (C20190180). All of the funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Introduction
In eukaryotes, the ubiquitin/26S proteasome system (UPS) is responsible for the selective degradation of most intracellular proteins [1]. Together with Suppressor of kinetochore protein 1 (Skp1), Cullin 1 (CUL1) and Ring-Box 1 (RBX1), F-box proteins form a ubiquitin ligase complex, where it plays the critical role of recruiting substrates to the UPS [2]. F-box proteins carry one or more 40–50 residue F-box/F-box-like domains in their N-terminus that are in charge of binding to Skp1/Skp1-like proteins [3]. Meanwhile, one or more additional conserved domains involved in substrate specificity can be found downstream of the F-box/F-box-like domain(s), such as Kelch repeats, Leucine Rich Repeat (LRR) and WD40-repeats [4]. The F-box members form a large family of proteins in plants, and within this family, the Kelch subfamily is one of the largest groups. Furthermore, F-box/Kelch (FBK) proteins are almost found in plants. The Kelch domain, originally identified in Drosophila mutants consists of 44–56 residues [5], and one or more Kelch domain(s) can be found in an FBK protein.
The size of the FBK subfamily varies depending on the plant species. In 2009, Xu et al. reported the identification of 96 FBKs in Arabidopsis, along with 27 and 35 FBKs from rice and poplar, respectively [6]. Using this information, Schumann et al. went on to identify additional FBKs in numerous species, and found: 103, 68 and 36 FBKs from the dicot species, Arabidopsis thaliana, Populus trichocarpa and Vitis vinifera, respectively; 44 and 39 FBKs in the monocot species, Sorghum bicolor and Oryza sativa, respectively; and 71 and 46 FBKs in the non-seed embryophytes Physcomitrella patens and Selaginella moellendorffii, respectively [7]. The former study reported that members of the FBK subfamily altered their protein structures by increasing or decreasing the number of exons, and the subfamily size was expanded primarily via tandem duplications [6]. The FBKs have been found to participate in biological clock regulation, photomorphogenesis, phenylpropanoid and pigmentation biosynthesis, and biotic stress responses [6,8–12]. While the FBKs subfamily exists in plants in relatively high numbers, and participates in many important biological processes, no systematic studies of the FBK subfamily have previously been reported in hexaploid wheat species.
To initiate FBK research in hexaploid wheat and to further our understanding of their role in various biological processes, a genome-wide identification study of this subfamily of F-box proteins is presented herein with a systematic analysis of protein structure, phylogenetic relationship, chromosome distribution, and expression patterns in response to different stresses. Sixty-eight genes encoding 74 wheat FBK (TaFBK) proteins were identified. In silico expression analysis for 47 of these genes revealed that they differentially regulated in response to drought and heat stresses. One gene, TaFBK19, which shows similarities to the Attenuated Far-Red Response (AFR) gene, was selected for further investigations, and is described here as TaAFR.
AFR F-box genes are involved in light signaling but have also been shown to participate in plant stress responses. Through the course of its cultivation, wheat is subjected to many kinds of environmental and biotic stresses including salt, drought, cold, heavy metals and various pathogens. These stresses can affect crop productivity and yield, which can be mitigated if a timely and appropriate stress response is mounted in the plant. To determine whether TaAFR is involved in the plant’s response to different stress stimuli, the wheat line, TcLr15, was exposed to leaf rust pathogens, salt, drought and H2O2, salicylic acid (SA), abscisic acid (ABA) and methyl-jasmonate (MeJA), and changes in gene expression were assessed by reverse-transcription quantitative real-time PCR (RT-qPCR). Subcellular localization of TaAFR was experimentally determined, and its interactions with other proteins was investigated using a combination of yeast-2-hybrid (Y2H), bimolecular fluorescence complementation (BiFC) and co-immunoprecipitation (Co-IP) assays. While providing a glimpse into the function of TaAFR and other FBKs in wheat, the results presented herein build the foundation to further dissect the function and metabolic network of this important gene family.
Materials and methods
Genome-wide survey of wheat FBKs
Database search, sequence analysis and classification of wheat FBKs.
The Hidden Markov Model (HMM) profiles of the F-box domain (PF00646, PF15966), F-box-like domain (PF12937, PF13013) and Kelch domain (PF01344, PF07646, PF13415, PF13418, PF13854, PF13964) were obtained from Pfam (http://pfam.xfam.org/). To identify wheat FBKs, the HMMER3.1b2 software was first used to search for F-box and F-box-like domains encoded in wheat genes deposited in the IWGSC (Wheat Genome Sequencing Consortium; RefSeq v1.0 wheat database downloaded from EnsemblPlants; https://plants.ensembl.org/index.html) (E value cut-off of 1.0) [13], and TBtools (v0.6673) was used to extract the target sequences. Sequences encoding F-box and F-box-like domains were further screened for the presence of one or more Kelch domains (E value cut-off of 1.0). Finally, Pfam, SMART (http://smart.-heidelberg.de/) and HMMER (web version 2.25.0, https://www.ebi.ac.uk/Tools/hmmer/) were adopted to confirm the presence of both the F-box (or F-box-like) and Kelch domains in each FBK protein identified, E value <1.0; sequences that did not meet this criterion were removed.
The predicted isoelectric point (pI) and molecular weight (MW) of the TaFBKs were computed using the Expasy Compute pI/Mw tool (https://web.expasy.org/compute_pi/). The intron-exon organization of wheat FBKs was obtained from EnsemblPlants. Subcellular localizations were predicted using the cropPAL2020 dataset (https://crop-pal.org/).
Analysis of conserved residues within the F-box and Kelch domains of wheat FBK proteins.
The ClustalX2.0 multiple sequence alignment tool was used to align the F-box or Kelch domains extracted from the TaFBK protein sequences, and WebLogos (http://weblogo.berkeley.edu/) were generated for each of the two domains.
Phylogenetic analysis.
In order to study the phylogenetic relationship and evolution of wheat FBKs, the obtained TaFBK sequences were compared with the orthologues from the model dicot species Arabidopsis (AtFBK), and three important monocots, namely rice (OsFBK), sorghum (SbFBK) and maize (ZmFBK). The AtFBK, OsFBK, SbFBK sequences reported by Schumann et al. and ZmFBKs reported by Jia et al. were downloaded and screened for the presence of the F-box and Kelch domains [7,14]. Sequences that did not carry both F-box and Kelch domain(s) were removed, leaving a total of 94, 31, 34 and 32 FBK protein sequences from Arabidopsis, rice, sorghum and maize, respectively. The FBKs from these four species were aligned together with the wheat FBKs using the ClustalX 2.0 algorithm and a phylogenetic tree was constructed by the Maximum Likelihood (ML) algorithm in MEGA7 using default parameters, with bootstrap value set to 1000 repetitions.
Chromosomal distribution and gene duplication analysis.
The chromosomal distribution of wheat FBK genes was obtained from the EnsemblPlants (IWGSC RefSeq v1.0). MapDraw was used to visualize the detailed location of each TaFBK gene on the wheat chromosome [15]. Greater than 70% sequence similarity was set as the criterion for determining gene duplication [16]. When the maximum distance between duplicated genes on the same chromosome was smaller than 50 kb, tandem duplication and duplicated genes on different chromosomes were delimited as segmental duplications [17].
In silico expression analysis of TaFBK genes.
FBK gene sequences obtained from EnsemblPlants were input into the WheatExp wheat database (https://wheat.pw.usda.gov/WheatExp/) to identify the corresponding WheatExp gene ID. Using the zero to one normalized scale method, heat maps were constructed for the TaFBK genes collected from different digital PCR transcriptomics datasets in WheatExp. FPKM (Fragments Per Kilobase per million Mapped reads) values were obtained for the FBKs from 5 tissues (cultivar Chinese Spring) at different development stages: leaves (z10, z23, z71), roots (z10, z13, z39), stems (z30, z32, z65), spikes (z32, z39, z65) and grains (z71, z75, z85) [18]. FPKM values were also downloaded from the wheat cultivar TAM 107 where leaves were treated with drought (DS), heat (HS) and drought+heat (HD) stresses [19]. TBtools (v0.6673) was used to draw a heat map according to their corresponding FPKM values.
Molecular identification and expression patterns of TaAFR
The wheat FBK gene, TaFBK19, was selected for further analysis. This gene is similar to the Kelch containing F-box AFR genes from other species and is therefore described here as TaAFR.
Plant material, fungal strains and inoculum preparation.
A leaf rust resistant near-isogenic wheat line of Thatcher, TcLr15, and leaf rust strains 05-5-137③ and 05-19-43② were used in the present study. Unless otherwise specified, plants were grown in a greenhouse as described in Yu et al. [20]. Urediniospore and inoculum preparation of leaf rust pathogens were carried out as previously described [20].
TaAFR cloning.
Total RNA extraction and first strand cDNA synthesis were performed as previously described [20]. A pair of gene specific primers TaAFR-F and TaAFR-R (S1 Table) and Tks Gflex™ DNA Polymerase (TaKaRa, Japan) were used to amplify the full-length coding sequences (CDS) according to manufacturer’s directions, with an annealing temperature of 56.4°C. The purity of the amplicon was verified by 1.2% agarose gel electrophoresis and the product was sequenced to confirm the identity of the clone.
The TaAFR sequence was used to pull out related sequences from the NCBI transcript database using the BLASTp tool, and sequences with an expect threshold of <0.05 were aligned together with TaAFR in MEGA 7.0 and a phylogenetic tree was constructed, as described in the section on phylogenetic analysis. The TaAFR protein sequence was also analyzed using various bioinformatics tools to predict presence of signal peptides (SignalP-4.1, www.cbs.dtu.dk/services/SignalP/), transmembrane domains (TMHMM Server v. 2.0, www.cbs.dtu.dk/services/TMHMM/), and subcellular localization (cropPAL2020 dataset). The 3D structure was predicted in Phyre2 (www.sbg.bio.ic.ac.uk/phyre2/).
Wheat treatments and sampling for RT-qPCR.
Sampling of wheat for gene expression analysis was carried out in different tissues (for tissue-specific analysis) and in response to three different types of abiotic stresses and three hormone treatments, as described below. For each experiment, samples were collected from three replicates and, unless otherwise specified, 3–5 samples were harvested for each replicate. Samples were flash frozen in liquid nitrogen and stored at −80°C prior to RNA extraction.
To detect tissue-specific expression levels of the TaAFR gene, samples were collected from TcLr15 7-day old seedlings and adult plants grown in a pot with nutrient soil (Hebei Fengyuan, China) in a greenhouse (22°C, 16 h light/8 h dark). Roots, stems and leaves were collected at z11; pistils, stamens and flag leaves were collected at z51. A large number of pistils and stamens (50–100 mg) were sampled from wheat florets.
To assess the effect of leaf rust pathogens on TaAFR expression, TcLr15 plants were inoculated with rust strains 05-5-137③ or 05-19-43②, or mock-inoculated with water, as previously described [20]. The inoculated and mock-inoculated leaves were harvested at 0, 6, 12, 24, 48 and 96 hours post inoculation (hpi). Mock-inoculated samples served as a negative control for each harvest time.
The effect of abiotic stress treatments on TaAFR expression was evaluated in TcLr15 plants grown in Hoagland’s solution [21]. Once plants reached the three-leaf stage (z13), Hoagland’s solution was amended with NaCl, PEG 6000 and H2O2, to a final concentration of 300 mM, 10% and 7 mM, respectively [22–24]. The second leaves were sampled at 0, 0.5, 2, 6, 12, 24 and 48 h post-treatment. Samples were also collected at the same time points from untreated negative control plants in Hoagland’s solution.
The plant hormones, SA, ABA and MeJA, are known to be involved in both abiotic and biotic stress responses [25,26]. To investigate the effects of 3 hormones on the expression of TaAFR, exogenous treatments of SA (2 mM), ABA (100 μM) and MeJA (100 μM), each dissolved in 0.1% absolute ethanol [22,26], were applied to TcLr15 seedlings (z11) grown in a pot with nutrient soil in the greenhouse. The negative control plants were sprayed with 0.1% absolute ethanol. The primary leaf of each plant from treated and control samples was collected at 0, 0.5, 2, 6, 12, 24 and 48 h post-treatment.
Gene expression analysis by RT-qPCR.
Total RNA was extracted from the TcLr15 samples collected in the previous section for gene expression analysis, using Biozol reagent (BioFlux, Japan), according to manufacturer’s instructions. To eliminate gDNA contamination, 2 μg of each RNA sample was treated with 1 μL gDNA Remover (TransGen, China). cDNA synthesis was carried out as described by Yu et al. [20]. qPCR was performed on a Bio-Rad CFX Connect™ real-time PCR system (Bio-Rad, America). cDNA was diluted 2-fold (800 ng/μL), and 1 μL was used as the template in 20 μL qPCR reactions, with TransStart Top Green qPCR Super Mix (TransGen, China) and gene specific primers RT-qPCR-TaAFR-F and RT-qPCR-TaAFR-R (S1 Table), and the reaction carried out with an annealing temperature of 58.3°C. A similar reaction was carried out using primers for the wheat reference gene GAPDH (GenBank: AF251217) (primers RT-qPCR-GAPDH-F and RT-qPCR-GAPDH-R, annealing temperature of 58.3°C) (S1 Table). Three technical replicates were conducted for each of three biological replicates per sample. The relative expression of TaAFR was evaluated as described by Yu et al. [20]. For samples where a treatment was included, in order to take into account any potential changes in expression related to the circadian rhythm, the values of the control treatments collected at the specified harvest times were subtracted from those of the treated samples prior to comparing expression with the time zero untreated controls.
Subcellular localization.
The TaAFR CDS, minus the stop codon, was inserted upstream of a GFP tag in the pSuper1300 vector (Laboratory preservation), and the recombinant construct was transformed into Agrobacterium GV3101. The strain GV3101-pSuper1300-TaAFR was injected into N. benthamiana leaves at the five-leaf stage, and then observed over a period of 30 to 80 h by fluorescence microscope (Nikon Ti 2, Japan) with an excitation wavelength of 495 nm.
Identification of TaAFR interacting proteins
Yeast-2-hybrid (Y2H).
The TaAFR CDS was cloned into the yeast bait vector pGBKT7 which carries the GAL4 DNA-binding domain (BD), and the construct, BD-TaAFR, was subsequently transformed into yeast strain Y187. A yeast cDNA library (AD-cDNA) previously constructed was used to screen for partner proteins of TaAFR [27]. Y187-BD-TaAFR was co-cultured overnight in YPDA media with AH109-AD-cDNA at 30°C with gentle agitation (50 r/min). The mated culture was spread onto Petri plates with SD-WLHA medium and incubated at 30°C for 3–5 days. Positive clones were sequenced by Beijing Zhongke Xilin Biotechnology Co., Ltd., and the identity of the partner proteins were determined by BLAST alignments.
Once the identities of the positive interactions were determined, the CDS sequences were amplified from the cDNA of TcLr15 inoculated with leaf rust strain 05-19-43②. These coding sequences were then inserted into the pGADT7 (AD) vector to generate recombinant AD-Prey for re-testing the interactions with the bait protein in the BD-TaAFR construct. The interaction combination (TaTCTP and TaSnRK1) validated by Ma et al. was used as a positive interaction control in Y2H interaction assay [28]. The strength of the positive Y2H interactions with BD-TaAFR can be further verified according to the blueness of the yeast colony by using the β-galactosidase in the SD-WLHA medium.
To determine which of the TaAFR domain(s) interact with the partner proteins, the cDNA sequences of each of the F-box (1–71 aa) and Kelch (72–383 aa) domains of TaAFR were inserted into BD vectors. AD-Prey that showed positive interactions with BD-TaAFR were then screened for interactions with each of these two domains (AD-Prey with BD-TaAFR-F-box or with BD-TaAFR-Kelch) using the Y2H assay as described above.
Bimolecular fluorescence complementation (BiFC).
Y2H positive interactions were validated by BiFC. The CDS of TaAFR and the partner proteins were inserted into pSPY CE and pSPY NE vectors (Laboratory preservation) to construct the pSPY CE-TaAFR and pSPY NE-Prey vectors, respectively. GV3101 with pSPY CE-TaAFR and with pSPY NE-Prey were combined and co-injected into the N. benthamiana leaves. Fluorescence signal was observed as described in the subcellular localization section. The interaction combination (TaTCTP and TaSnRK1) was also used as a positive interaction control for the BiFC assay.
Co-immunoprecipitation (Co-IP).
The positive interactions tested by BiFC were further validated by Co-IP. The CDS of TaAFR and the putative partner proteins were inserted into pTF101 (Laboratory preservation) with HA or FLAG tags to construct the recombinant vectors pTF101 HA-TaAFR and pTF101 FLAG-Prey, respectively. GV3101 strains with pTF101 HA-TaAFR and pTF101 FLAG-Prey were co-injected and transiently expressed in N. benthamiana. The combination of pTF101 HA-TaAFR and pTF101 FLAG-TaGFP was used as a negative control. Proteins were extracted from N. benthamiana leaves sampled 60 h after co-injection and subjected to IP by HA-magnetic beads, as described by Zhu and Huq [29]. The eluted proteins were subjected to immunoblot analysis with anti-FLAG tag polyclonal antibody (Solarbio, China).
Results
Genome-wide identification of wheat FBKs
74 Wheat FBK proteins were identified and divided into 5 categories based on their functional domains.
The seed sequences of F-box (457), F-box-like (306) and Kelch (486) domains were obtained from the Pfam database. “F-box domain” will be used henceforth to describe both F-box and F-box-like domains. A total of 192 transcript sequences containing at least one F-box and Kelch domain were identified by searching the wheat IWGSC translated transcript database with HMMER3.1b2. Among these, 68 genes encoding 74 transcripts were found to carry both the F-box and Kelch domains, predicted by SMART and HMMER. The putative protein sequences (S1 File), pI, MW, number of introns, subcellular localization, and functional domains of the 74 putative wheat FBKs are presented in S2 Table. Wheat FBK (TaFBK) proteins range from 239 to 643 residues in length, with predicted MWs of 27.41–69.41 kDa and theoretical pIs of 4.18–9.99, of which the number of acidic/alkaline proteins account for half of the proteins, and 22 of these (29.7%) are greater than 9.0. Most of the TaFBKs were predicted to localize in the nucleus, cytoplasm or plastid, while a handful were predicted to localize in various organelles (peroxisome, golgi). The intron-exon structure has been reported to be closely related to the evolution of the F-box superfamily [30]. The number of introns identified in 74 TaFBK transcripts varies from 0 to 4, among them, 37 TaFBK transcripts are predicted to have 1 intron (50.0%) and 25 transcripts are predicted to have none (33.8%).
Each of the 74 TaFBK transcripts carry only one F-box domain at the N-terminus, with up to four Kelch domains at the C-terminus. A few members were also found to carry PAS and PAC domains upstream of the F-box domain. According to their different domain structures, TaFBKs can be divided into 5 categories as follows: F-box+1 Kelch, F-box+2 Kelch, F-box+3 Kelch, PAS+F-box+4 Kelch, and PAS+PAC+F-box+4 Kelch (S1 Fig). The F-box+2 Kelch is the largest category, accounting for 40.5%, followed by F-box+1 Kelch (35.1%), while PAS+PAC+F-box+4 Kelch is the least represented with only 2 proteins in this group.
Wheat FBKs show conservation of F-box and divergence of Kelch domain sequences.
MEGA7 was used to align the F-box or Kelch domains of TaFBKs. WebLogo’s were generated, where the height of each stacked letter represents the probability that a given amino acid will occur at each position (S2 Fig). In the wheat F-box domain (S2A Fig), L-16 and R-18 are relatively tall, indicating a high probability that those residues would be found at those positions. In the F-box domain alignment, 69 and 65 of the 74 proteins analyzed, respectively carry L residues at the 16th position and R residues at the 18th position, which indicates that these 2 amino acids are conserved in the F-box domain of identified wheat FBKs. In addition, L-6 (82.4%), P-7 (81.1%), V-30 (87.8%) and W-34 (81.1%) were shown to be fairly conserved, followed by P-20 (73.0%), D-8 (68.9%), R-28(63.5%), R-32 (60.8%), D-9 (56.8%), C-31 (59.5%), V-19 (54.1%), C-15 (52.7%) and A-11 (51.4%).
The Weblogo of the Kelch domain (S2B Fig) shows that G-19 (85.8%), G-20 (86.4%), W-53 (97.9%) and M-59 (54.1%) are highly conserved. Although the height of some additional residues, such as R-2 (14.11%), H-5 (11.5%), L-10 (15.5%), G-12 (25.0%) and D-45 (20.3%), are relatively tall in the Kelch domain, according to the statistical assessment these are poorly conserved. Compared to the other protein sequences, TaFBK65 has 3 additional residues (PVP) at the N-terminus of the F-box motif; these 3 residues were removed from the analysis in order to prepare the Kelch WebLogo. In general, the amino acid sequences within the Kelch motif are more divergent than that observed within the F-box domain.
Phylogenetic grouping of the wheat, Arabidopsis, rice, sorghum and maize FBK subfamilies occurs according to the number of Kelch domains.
To understand the evolutionary relationship of the 74 TaFBKs members, a phylogenetic tree was constructed using the protein sequences from 94 Arabidopsis FBKs (AtFBKs), 31 rice FBKs (OsFBKs), 34 sorghum FBKs (SbFBKs) and 32 maize FBKs (ZmFBKs) (Fig 1). The tree resolved into 7 clades, where the AtFBKs are mainly distributed in clade G, all of TaFBKs, OsFBKs, SbFBKs and ZmFBKs, with the exception of three OsFBKs (22, 14 and 9) and three SbFBKs (23, 16 and 8), distributed in clades A to F. All members in clade C belong to the F-box+1 Kelch type, and among them, only 5 members are from Arabidopsis, while the remaining 37 are from graminaceous species. In clades D and F, the FBKs of F-box+2 Kelch type account for the largest group, containing only a few members of F-box+1 Kelch and F-box+3 Kelch types. OsFBK31 and OsFBK28 (F-box+1 Kelch+RING) are also grouped into clade D. Clade E mainly consists of F-box+3 Kelch type FBKs from Gramineae and 3 members of F-box+2 Kelch proteins from Arabidopsis. FBKs with 4 Kelch domains (F-box+4 Kelch, PAS+F-box+4 Kelch, PAC+F-box+4 Kelch, and PAS+PAC+F-box+4 Kelch) are represented in 5 species and grouped in clade B. AtFBK54 (LSM14+F-box+2 Kelch) together with other members of Arabidopsis F-box+2 Kelch are in clade G. The phylogenetic analysis indicates that the number of Kelch domains is a key classification criterion within the FBK subfamily.
The full-length amino acid sequences were aligned by ClustalX 2.0 and the Maximum Likelihood (ML) tree was constructed using MEGA7. FBK proteins were grouped into 7 distinct clades named A-G.
TaFBK genes are unevenly distributed on the wheat chromosomes and mainly expanded its size by segmental duplications.
The chromosomal position of 68 TaFBK genes were retrieved from EnsemblPlants and a chromosomal distribution map was generated (S3 Fig). The TaFBK genes are unevenly distributed on the wheat 21 chromosomes. The chromosomes of 4A (5), 4B (5), 6A (6), 6B (8), and 6D (6) have relatively higher distribution densities, whereas only one TaFBK gene was found on each of chromosomes 1B, 3B and 1D, and none were detected on chromosome 2B.
In animals, the number of F-box proteins is relatively low compared with plants, with only 68 and 74 F-box genes in the human and mouse genomes, respectively [31]. Incidentally, the wheat genome encodes the same number of the Kelch subfamily proteins, which represents only a portion of the F-box proteins encoded in this species. Gene duplication is thought to be the main driving factor in the expansion of the F-box family in plants [17]. To explore the evolutionary mechanism of the wheat FBK subfamily, the present study investigated tandem duplication and segmental duplication events in the wheat FBK subfamily of the F-box family by observing similarities among 68 FBK sequences. A total of 57 TaFBKs were identified to be segmental or, to a lesser extent, tandem duplications. Most of the segmental duplications, which are distributed on 20 chromosomes, included 1 or 2 duplication events (ie. 2 or 3 genes in the group), although as many as 7 segmental duplication events were observed. Tandemly duplicated genes affected 8 TaFBK genes, and each of these occurred on chromosome 4. These results indicate that both segmental and tandem duplications played a role in the expansion of the TaFBK subfamily, and unlike the results of Xu et al. in Arabidopsis and rice species [6], segmental duplications were more prolific in wheat.
Tissue-specific and abiotic stress response in silico expression of TaFBKs.
To gleam insights into the putative functions of the identified wheat FBKs, in silico expression analysis of these genes was evaluated in different wheat tissues at different developmental stages, and in wheat leaves in response to environmental stresses. The FPKM values of TaFBKs from five different tissues and three stress combinations were downloaded from digital PCR data available in WheatExp (S3 Table) and were used to construct a heat map using the zero to one normalized scale method. Tissue-specific expression data (cultivar Chinese Spring) was available for 47 TaFBKs (Fig 2). In general, TaFBK genes exhibited differential expression in all five wheat tissues, suggesting that these genes may be involved in the developmental regulation of multiple tissues. There were two conditions where tissue-specific expression at specific developmental stages showed significantly less transcript accumulation; these are leaf (z10) and grain (z75). Meanwhile, most TaFBK genes were generally more abundantly expressed in the spikes (z32, z39, z65) and grains (z71, z85). TaFBK3 transcripts specifically accumulated in root tissues, TaFBK8 and TaFBK29 were dominantly expressed in mature leaf (z71), while TaFBK60 and TaFBK61 showed highest expression in root tissues followed by grain samples.
The color key represents FPKM values. Identity of tissue samples and developmental stages (Zadoks scale) are provided at the top of each lane.
A second data set from the WheatExp database was analyzed for the effect of DS, HS and HD stresses on the expression of the same 47 TaFBK genes in seedlings of the wheat cultivar TAM 107. A heat map was generated for this dataset showing differential expression at 1 and 6 h (Fig 3). A general overview of expression of TaFBK genes affected by DS is as follows: 34.0% of the TaFBK genes were up-regulated; 47.0% of the genes showed strongly or slightly down-regulated expression; and 19.0% (9 transcripts) maintained stable expression between treatment and control. The following changes were observed in response to HS at 40°C: transcripts TaFBK60, TaFBK61 and TaFBK46 increased sharply at 1 h, and then decreased at 6 h; TaFBK10, TaFBK23 and TaFBK50 expression gradually increased from 0 (control) to 6 h; eight TaFBKs were down-regulated at both time points assessed; the transcripts of seven genes (14.9%) decreased to roughly half of the control levels at 1 h; expression of the remaining 18 (38.3%) genes sharply declined at 1 h after the stress treatment, and transcript accumulation of 7 of these genes returned to levels similar to that of the control by 6 h, while the other 9 genes increased slightly at 6 h compared with the earlier time point. Following the combined treatment HD: 25.5% of the genes increased their expression from 1 to 6 h compared with the control; transcripts from 7 genes gradually decreased from 1 to 6 h; 72.3% transcripts sharply decreased at 1 h treatment, then slightly or sharply increased at 6 h. In brief, HS caused more obvious and intense change on expression of TaFBKs compared to the DS treatment.
FPKM values are represented by color according to the legend. The method and time of treatments are provided at the top of each lane. DS, drought stress; HS, heat stress; HD, heat+drought stresses.
Molecular identification and expression patterns of TaAFR
TaAFR gene encodes a wheat FBK protein.
The heat map presented in Fig 3 shows that the expression of TaFBK19 was strongly up-regulated under DS treatment and sharply down-regulated under HS treatment at 1 h, while the HD treatment resulted in low level expression of this gene at both 1 and 6 h. TaFBK19 was selected for further analysis. The full-length (1327 bp) cDNA sequence was cloned from TcLr15 wheat seedlings inoculated with the leaf rust strain 05-19-43②. The cDNA encodes a polypeptide with 383 amino acids. The predicted MW of the polypeptide is 40.69 kDa, and the predicted pI is 5.11. BLASTx analysis shows that the sequence shares high similarity (94%) with an F-box protein, AFR-like, from Aegilops tauschii (GenBank: XP 020194469.1). TaFBK19 protein carries a single highly conserved F-box domain (32–71 aa sites) at the N-terminus and a fairly divergent Kelch domain (136–174 aa sites) found in the internal region (Fig 4A). Phylogenetic analysis indicates that the TaFBK19 protein shares 94.10% and 87.47% similarity with AFR from A. tauschii and Hordeum vulgare, respectively, followed by AFR from Brachypodium distachyon, O. sativa, Setaria italica, Panicum hallii, S. bicolor and Z. mays. Meanwhile, AFRs from woody plants (Prunus avium, Musa acuminata, Elaeis guineensis, Phoenix dactylifera) and dicots (Nelumbo nucifera, Dendrobium catenatum and A. thaliana) were grouped in different clades (Fig 4C), which indicates that these FBKs are conserved in monocots. Based on the similarities between TaFBK19 and AFR genes from other cereals and monocots, TaFBK19 will henceforth be described as TaAFR. Sequence analysis of TaAFR did not reveal any predicted signal peptide or transmembrane domains, and the protein is predicted to localize to the cytosol. The predicted 3D structure shows three distinct α-helices at the N-terminus and β-sheets at the C-terminal end. The β-sheets are predicted to form 6 triangles, which further cluster to a regular hexagonal arrangement. These secondary and ultra-secondary structures indicate that the protein folds into chair-like configuration (Fig 4B).
(A) Functional domain of TaAFR. A schematic diagram showing the positions of the F-box and Kelch domains in TaAFR; (B) 3D structure prediction of the TaAFR protein. The ribbons represent the α-helix structures, while the arrows denote the β-sheets. The colors highlight the position within the primary structure, with blue and red at the N- and the C-terminus, respectively; (C) Phylogenetic analysis of TaAFR with F-box proteins from different plants species. The phylogenetic tree was generated using the neighbour-joining method in MEGA 7. Branches are labeled with the GenBank accession number followed by species name.
TaAFR is primarily expressed in wheat leaves.
Six tissues were sampled from wheat seedlings (root, leaf and stem) and adult plants (pistil, stamen, flag leaf) of TcLr15 to analyze the tissue-specific expression of TaAFR. The young leaf was used as a control (the expression value was set 1.0) to measure its relative expression to other tissues. TaAFR was mainly expressed in young leaf, with lower expression in the flag leaf and extremely low expression was detected in young root, pistil, and stamen (Fig 5A).
(A) Expression profile of TaAFR in different tissues of TcLr15. Different letters indicate significant differences (p < 0.05). (B) Expression patterns of the TaAFR gene in incompatible and compatible combinations of TcLr15/Puccinia triticina strains 05-19-43② and 05-5-137③, respectively. (C) Effect of NaCl, PEG and H2O2 on TaAFR expression in TcLr15 leaves. (D) Effect of SA, ABA and MeJA on TaAFR expression in TcLr15 leaves. To remove the effect of the circadian cycle on TaAFR expression in B, C and D, the relative expression values for TcLr15 leaf control samples, harvested at the same time points as for the various treatments, was subtracted from the relative expression of the treatment samples at the specified harvest times. Relative expression, with the effect of the circadian cycle removed, was then compared to the 0 h untreated control; significant differences (p < 0.05) are marked with an asterisk.
Differential expression of TaAFR in incompatible and compatible wheat/leaf rust pathogen combinations.
The temporal expression profile of TaAFR in TcLr15 leaves following inoculation with the two leaf rust strains, is shown in Fig 5B. Generally, the TaAFR transcript was higher in the compatible interaction (TcLr15 inoculated with 05-5-137③) than in the incompatible one (TcLr15 inoculated with 05-19-43②), except at 48 hpi. For the incompatible interaction, the TaAFR transcripts gradually increased from 6 to 96 hpi, but apart from the initial increase from 0 to 6 hpi, no significant difference was observed across the time course. In the compatible interaction, no significant change was observed between 0 to 48 hpi, but a rapid increase was observed at 96 hpi, where the expression of TaAFR transcripts was 3.1-fold higher than in the 0 h untreated control samples.
Expression of TaAFR is affected by salt, drought and oxidative stresses.
Three abiotic stress treatments, salt (NaCl), drought (PEG 6000) and oxidative stress (H2O2), were evaluated for their effects on TaAFR expression in TcLr15 seedlings. The expression of TaAFR was significantly affected in TcLr15 after treatment with NaCl (Fig 5C). The transcripts were strongly up-regulated from 0.5 h, and maintained a high level of expression until 12 h. Two expression peaks occurred at 0.5 h (8.2-fold) and 6 h (7.9-fold). Thereafter, the TaAFR transcripts started to down-regulate gradually, until 48 h, where transcripts dropped to half of the level detected in the 0 h untreated controls. In response to PEG 6000 treatments, the TaAFR transcript was increased in abundance at 0.5 h (3.9-fold), 2 h (2.1-fold), 6 h (4.1-fold) and 24 h (3-fold), but was down-regulated at 48 h. After treatment with H2O2, the expression of TaAFR did not differ from that of the control until 2 h after treatment when it was down-regulated, but from 6 to 24 h, TaAFR showed an upward trend, reaching a peak at 24 h where it was 7.8-fold higher than that of the 0 h untreated control. Finally, expression levels dropped below that of the 0 h control at 48 h (Fig 5C).
Exogenous SA and ABA applications significantly up-regulate the expression of TaAFR.
The expression pattern of TaAFR in TcLr15 following exogenous treatment with plant hormones is presented in Fig 5D. In response to SA treatments, TaAFR expression was down-regulated 2-fold at 2 and 6 h compared with the untreated 0 h control, and thereafter increased rapidly 4-fold at 12 h compared with the control before returning to the basal expression levels. In response to ABA, TaAFR expression increased rapidly by 3.5-fold, observed at 2 h, and continued to be up-regulated throughout the time course, but decreasing gradually until 48 h where basal level expression was observed. MeJA application resulted in significant down-regulation of TaAFR at most time points, except at 12 and 24 h, where no significant difference was observed compared with the control.
TaAFR is localized to the nucleus and cytoplasm.
N. benthamiana was injected with GV3101 containing either the empty vector 35S:GFP or the recombinant vector 35S:TaAFR-GFP, and transient expression of the recombinant proteins was observed. The fluorescence signal of 35S:GFP was visualized in both the nucleus and cytoplasm 36 h after transfection (Fig 6), whereas the fluorescence signal of 35S:TaAFR-GFP was detected after 48 h, predominantly observed in the nucleus and cytoplasm. Moreover, the nuclear dye DAPI was used to stain the tobacco leaves after transfection, and a light blue color was clearly observed in the nucleus.
The free GFP protein and TaAFR-GFP fusion protein were transiently expressed in the N. benthamiana by Agtobacterium-mediated transformation. GFP, GFP fluorescent signal channel; Bright field, ordinary light channel; DAPI, nuclei were stained by DAPI; Merge, merge of GFP, Bright field and DAPI. Bar = 20 μm.
Screening and identifying TaAFR-interacting proteins
Thirteen putative TaAFR-interactions were identified in a Y2H library screen.
To identify candidate upstream and/or downstream proteins interacting with TaAFR in wheat, we screened a yeast library carrying the cDNA of TcLr15 inoculated with the incompatible leaf rust strain against the bait construct, BD-TaAFR. Clones from positive interactions were sequenced and thirteen candidate proteins were identified from 47 clones. Candidate proteins are listed in S4 Table, and categorized into the following 5 groups: photosynthesis, stress resistance, transportation, basal metabolism, and unknown protein. Among these, 5 stress resistance related proteins were obtained: peroxidase 51-like (POD), obtusifoliol 14-alpha-demethylase (CYP51), glucan endo-1,3-beta-glucosidase 14 (GV), laccase-7 (Lac7) and leucine-rich repeat protein 1 (LRR-8 superfamily) (LRR) [32–36]. Meanwhile, transport related proteins, ADP-ribosylation factor 2-like isoform X1 (ARL2) and SEC1 family transport protein SLY1 (SLY1), and a basal metabolism related protein, Skp1/ASK1-like protein (Skp1), were also detected [3,37,38].
TaSkp1, TaARL2 and TaPAL interacted with TaAFR.
The following 11 genes identified in the Y2H library screen were selected for protein interaction validation: Rubisco, Skp1, ARL2, GV, RP, SLY1, NADH, POD, LRR, Lac7 and CYP51. The complete coding regions were obtained for each of these 11 genes from TcLr15. According to Zhang et al., Kelch repeat F-box proteins are regulated by phenylpropanoid biosynthesis by controlling the turnover of phenylalanine ammonia-lyase (PAL) [11]; therefore, in addition to the positive interactions identified in the Y2H assay, we also isolated a PAL gene from TcLr15. Basic characteristics of these 12 proteins are presented in S5 Table. The interactions were first re-verified by Y2H. Colonies with blue pigments are indicative of positive interactions, and along with the positive control, six such interactions were observed: BD-TaAFR and AD-TaSkp1, BD-TaAFR and AD-TaSLY1, BD-TaAFR and AD-TaARL2, BD-TaAFR and AD-TaCYP51, BD-TaAFR and AD-TaPAL, BD-TaAFR and AD-TaNADH. The remaining combinations, along with the negative control, did not grow on the SD-WHLA-X-α-Gal plates. These results suggest that TaAFR may physically interact with TaSkp1, TaSLY1, TaARL2, TaCYP51, TaPAL and TaNADH (Fig 7).
Yeast was cultivated on SD-WLHA+X-α-Gal plates for 3–5 days.
To further validate the above results, these six interactions were tested by BiFC. In this approach, the coding region of the TaAFR was inserted downstream of the c-Myc tag of pSPY CE vector (pSPY CE-TaAFR); meanwhile the ORFs of TaSkp1, TaSLY1, TaARL2, TaCYP51, TaPAL and TaNADH were inserted downstream of the 35S promoter in the pSPY NE vector (pSPY NE-Prey). The pSPY CE-TaAFR vector was used for co-transfection of tobacco leaves with each of the pSPY NE-Prey constructs. Among the six combinations, three were found to emit fluorescent signals (Fig 8), indicating that the gene products of those combinations were interacting. pSPY CE-TaAFR and pSPY NE-TaSkp1 emitted a fluorescent signal in the nucleus and cytoplasm 40 h after injection. The pSPY CE-TaAFR and pSPY NE-TaARL2 emitted a strong signal in the nucleus and cytoplasm 48 h after co-transfection. The pSPY CE-TaAFR and pSPY NE-PAL interaction was observed in the cytoplasm by complementary chimeric fluorescence signals 40 h after co-transfection. Thus, the BiFC assay further validated TaAFR interactions with TaSkp1, TaARL2 and TaPAL.
The fluorescence microscope (Nikon Ti 2, Japan) with an excitation wavelength of 495 nm was used to observe fluorescence signal. Three independent experiments were conducted for each combination. Bar = 20 μm.
Co-IP assays were performed to further validate the results tested by Y2H and BiFC in vivo. The combinations of TaAFR with three putative partner proteins TaSkp1, TaARL2, TaPAL, and negative control GFP, were successfully detected in the whole cell lysates (WCL) following transient co-expression in N. benthaminana. After IP by HA-magnetic beads, the eluted proteins were subjected to immunoblot analysis with anti-FLAG antibody. For each of the HA-purifications, an HC (IgG heavy chain) was detected at 55 kDa. An additional band was detected for each of the three candidate interacting proteins, TaSkp1, TaARL2 and TaPAL, at the expected molecular weights for interaction with TaAFR, indicating that these three proteins were co-immunoprecipitated with TaAFR. By contrast, no additional band was detected in the TaAFR-GFP interaction, as expected for the negative control (Fig 9). It should be noted that a small number of non-specific partial bands were detected in a few cases, but they did not interfere with bands of the interacting protein pairs, nor were they in the expected size range of any of the four proteins of interest. Taken together, these observations support that TaAFR interact with TaSkp1, TaARL2 and TaPAL in vivo.
Proteins were extracted from leaves of N. benthamiana 60 h after co-injection, and immunoblotting (IB) was used to detect the expression of TaAFR, TaSkp1, TaARL2, TaPAL and GFP in the WCL with HA or FLAG antibody, these proteins were immunoprecipitated by HA-magnetic beads, then the eluted proteins were subjected to IB analysis with anti-FLAG antibody. HC: IgG heavy chain. Marker: 25–90 kDa.
The F-box domain of TaAFR interacted with TaSkp1, and the Kelch domain with TaARL2 and TaPAL.
To determine which domain of TaAFR is responsible for recognizing the TaSkp1, TaARL2 and TaPAL, we further obtained the cDNA sequences of the F-box (1–71 aa) and Kelch (72–383 aa) domains of TaAFR, and then constructed recombinant BD vectors for each. Six combinations of AD-TaSkp1 and BD-TaAFR-F-box, AD-TaSkp1 and BD-TaAFR-Kelch, AD-TaARL2 and BD-TaAFR-F-box, AD-TaARL2 and BD-TaAFR-Kelch, AD-TaPAL and BD-TaAFR-F-box, AD-TaPAL and BD-TaAFR-Kelch were verified using the Y2H assay. Among them, three combinations of AD-TaSkp1 and BD-TaAFR-F-box, AD-TaARL2 and BD-TaAFR-Kelch, AD-TaPAL and BD-TaAFR-Kelch grew well on the SD-WLHA+X-α-Gal plates (Fig 10). These results indicate that TaSkp1 interacted with the F-box domain, while TaARL2 and TaPAL were recognized by the Kelch domain of TaAFR.
Yeast was cultivated on SD-WLHA+X-α-Gal plates for 3–5 days.
Discussion
The Kelch type F-box protein is one of the most common subfamilies of the F-box family proteins in plants [39]. Many wheat databases are being continuously updated, with improved annotations over recent years, making it possible for genome-wide identification and comprehensive analysis of gene families. In 2020, Hong et al. reported 41 wheat F-box/Kelch genes [40], and in our previous analysis, 59 wheat F-box/Kelch genes were identified in Phytozome 12 (v2.2) [41]. In the present study, we screened IWGSC database in EnsemblPlants with more seed sequences and identified 68 TaFBKs encoding 74 putative proteins. The TaFBK subfamily was divided into 7 categories based on differences in the number of Kelch domains present. The majority of the AtFBKs, which showed a relatively distant evolutionary relationship with the four Gramineae species evaluated, resolved to clade G. Meanwhile the TaFBKs resolved into the other six clades (A-F) together with the OsFBKs, SbFBKs and ZmFBKs, and phylogenetic grouping was found to be related to the composition of their functional domains. Compared with the Arabidopsis FBK subfamily, three types of FBKs, namely F-box+4 Kelch, PAC+F-box+4 Kelch and LSM14+F-box+2 Kelch, were not detected in wheat. Each of these types are poorly represented in Arabidopsis, with only one member for each. It may be that they are absent in wheat due to selective evolution of the species, or it may simply be that they cannot yet be detected at the current sequencing depth or annotation of the wheat protein database. F-box+1 Kelch+RING and LSM14+F-box+2 Kelch were found to be unique FBK types in rice and Arabidopsis, respectively.
Studies have shown that FBKs are localized to the nucleus, cytosol and/or organelles. For example, CarF-Box1 (chickpeas) and TML (legume) were found to localize in the nucleus, while TaKFB1 through TaKFB5 (colored wheat) were all co-localized to both the nucleus and cytoplasm [40,42,43]. The wheat FBKs identified herein, were predicted to localize in the nucleus, cytoplasm, plastid and/or other organelles. While these predictions may provide some insights into potential gene function, they are not always accurate. For example, the wheat FBK, TaAFR (TaFBK19), was predicted to localize in the cytosol, but was shown experimentally herein to localize to both the nucleus and the cytoplasm.
To gleam some insights into the potential functionality of the wheat FBKs, their expression was observed in response to different stresses. An initial in silico analysis was carried out by comparing the expression of 47 TaFBKs in their response to DS, HS, and HD. While the expression patterns varied amongst these genes in response to the different treatments, many of them were strongly down-regulation in response to HS. TaAFR was further selected for expression analysis in response to abiotic/biotic stresses and hormone treatments. NaCl and H2O2 treatments resulted in the strongest up-regulation of TaAFR. Interestingly, TaAFR expression patterns showed different trends in response to a virulent vs an avirulent strain of the leaf rust pathogen. Many previous studies have reported similar observations for the expression of different plant FBKs in response to various abiotic and biotic stresses. For example, the nuclear localized FBK gene CarF-box1 from chickpea was shown to play an important role in abiotic stress, where expression of this gene was significantly up-regulated after drought and salt treatments, but down-regulated under heat and cold stresses [42]. The grape FBK gene BIG24.1 was up-regulated by Botrytis infection, and up-regulation of this gene affected the plants response to other biotic and abiotic stresses [44]. In another example, the F-box protein containing two Kelch repeats in sugar beet homologous to Arabidopsis FBK AT1G74510, was found to interact with the beet necrotic yellow vein virus pathogenicity factor P25, and it was speculated that P25 could affect formation of the SCF complex [45].
Biotic and abiotic stress responses are often regulated by plant signaling hormones and exposure to such stresses can activate these pathways [46]. It is therefore interesting that the expression of TaAFR was also affected by three different plant hormones. SA, ABA and MeJA treatments had a medium effect on the expression of TaAFR, suggesting that this gene may regulate and be regulated by different plant hormones. A regulatory behavior in plant hormone responses would be consistent with the role of FBKs in hormone signaling pathways [22,24].
FBKs interact both with other members of the UPS and with downstream targets for proteasome degradation; identification of some of these interacting proteins can further provide insight into the function of this protein. A multifaceted approach was employed to identify and validate candidate interactions. First, using a leaf rust pathogen treated TcLr15 wheat leaf cDNA library, a Y2H library screen was utilized as a broad scale approach to fish for candidate interacting proteins. A total of 13 candidates were identified, and 11 of these were cloned and re-screened by Y2H for interactions with TaAFR. Additionally, a PAL gene, which was not identified in the pool, but has been shown to be involved in regulation process of FBKs, was added to the list. Among these, a total of 6 interactions, including the TaAFR-TaPAL interaction, were confirmed positives. However, since Y2H assay can pick up false positives, these 6 genes were then validated using the BiFC and Co-IP methods, and finally three partner proteins interacting with TaAFR were confirmed: TaSkp1, TaARL2, and TaPAL.
To further characterize their interactions, another Y2H assay was carried out between the F-box and Kelch domains of TaAFR with each of these proteins. TaSkp1 was shown to interact with the F-box domain but not with the Kelch domain. This was not unexpected since Skp1 is a known component of the SCF complex, and since F-box proteins interact with Skp1 via the F-box domain. This result provides preliminary evidence that TaAFR forms part of the SCF complex. Meanwhile, the other two proteins, TaARL2 and TaPAL, were shown to interact with the Kelch domain, and not the F-box domain, suggesting that these two proteins are targeted by TaAFR for ubiquitination and thus designated for proteolytic degradation.
ARL2 is an ADP ribosylation factor (ARF)-like GTPase and members of the ARF family are known to regulate a wide range of cellular processes in eukaryotes, including mitochondrial fusion and microtubule dynamics [47–49]. Most of what is known about ARL2 proteins comes from research in humans and yeast, with little information in plants. Some plant ARF genes have been linked with biotic or abiotic stress responses, either by gene expression analysis [50] or through experimental validation [51]. Guan et al. characterized three ARF genes from Switchgrass (Panicum virgatum L.), namely PvArf1, PvArf-B1C and PvArf-related [51]. Through transgenic overexpression of these genes, the authors determined that they contribute to salt-tolerance in Switchgrass and that then phenotype was linked to an increase in proline accumulation. They also found that the encoded PvARF proteins interacted with a key enzyme in the proline biosynthesis pathway, PvP5CS1. While the role of TaARL2 in wheat biological processes is yet to be determined, the identification of an interaction between TaARF and TaARL2 through the Kelch domain of TaARF, suggests that this protein might be regulated through the ubiquitination pathway.
PAL activity is modulated by abiotic/biotic stresses in plants, including infections with fungal pathogens, UV/blue light irradiation, and wounding [52]. Zhang et al. found that differential expression of an Arabidopsis FBK genes affected the stability of PAL, and PAL isozymes were shown to physically interact with FBKs both in vitro and in vivo [11]. The interaction of PAL with FBKs thereby controls phenylpropanoid biosynthesis by mediating the ubiquitination and subsequent degradation of PAL. In another study, the authors showed that the Arabidopsis FBK protein, KFB39, a homolog of AtKFB50, also interacted with PAL isozymes and regulated PAL stability and activity, thereby participating in the plant’s tolerance to UV irradiation [12]. In the work presented herein, TaAFR interacted with TaPAL, and was shown specifically to interact with the Kelch domain. Based on these results, together with what is known for homologous proteins in Arabidopsis, it is speculated that TaAFR binds Skp1 (through the F-box domain) and the downstream target, TaPAL (through the Kelch domain), forming SCFPAL, thereby regulating PAL stability and activity in the wheat response to abiotic/biotic stresses [11,12].
The detailed protein interacting assays presented indicate that TaAFR is likely to bind TaSkp1, and suggest that TaARL2 and TaPAL are likely downstream targets. Additional studies are needed to determine whether TaAFR is actively involved in the regulation of these proteins by forming an SCF complex and targeting these proteins for ubiquitination and proteosome degradation. Furthermore, how these putative activities in the regulation of TaARL2 and TaPAL participate in biotic or abiotic stress responses have yet to be investigated. The work presented in this manuscript provides a glimpse into the potential function of TaAFR and their partner proteins, and opens the door for future studies to further characterize these genes.
Conclusion
A total of 68 TaFBK genes encoding for 74 proteins were identified in wheat. The FBK proteins from wheat, Arabidopsis and three important monocots were grouped into 7 clades according to the number of Kelch domain. Sixty-eight TaFBK genes were unevenly distributed on 21 wheat chromosomes, TaFBKs differentially expressed at multiple developmental stages and tissues, and in response to drought and/or heat stresses by in silico analysis. A Kelch type F-box gene TaAFR was isolated and found to be primarily expressed in wheat leaves, and it’s expression was perturbed by various treatments, including exposure to leaf rust pathogens, exogenous plant hormone treatments, and abiotic stresses. The protein was shown to localize in the nucleus and cytoplasm. The wheat Skp1 protein was found to interact with the F-box domain of TaAFR, while ARL2 and PAL were recognized by Kelch domain suggesting that TaAFR targets these two proteins proteasomal degradation. This work provides a foundation from which to build more detailed research inquiries into the function of the numerous wheat FBKs and also to further characterize the TaAFR gene.
Supporting information
S1 Fig. Classification of FBK proteins in wheat based on different functional domains.
F-box, the protein with F-box domain; Kelch, F-box protein having Kelch domain; PAS, FBK protein with PAS domain that was named after three proteins that it occurs in: Per-period circadian protein, Arnt-Ah receptor nuclear translocator protein and Sim-single-minded protein; PAC, FBK protein with PAC domain that usually appears at the C-terminus of the PAS motif.
https://doi.org/10.1371/journal.pone.0250479.s001
(TIF)
S2 Fig.
WebLogo generated by alignments of the F-box (A) or Kelch (B) domains of wheat FBKs. The F-box or Kelch motifs were retrieved from 74 wheat F-box proteins. The overall height of every stack is indicative of sequence conservation at the given position within the motif, whereas the height of the letters within each stack is indicative of the relative frequency of the corresponding amino acid. The bit score represents the information content for each position. Asterisks mark the conserved residues.
https://doi.org/10.1371/journal.pone.0250479.s002
(TIF)
S3 Fig. Chromosomal distribution of wheat FBK genes.
The chromosomes were drafted to proportion and the chromosome numbers were indicated at the top of each stave. Chromosomal distances were given in megabases (10 Mb). The gene names were listed at the right side of each chromosome corresponding to the position of each gene. Tandemly duplicated genes were shown in pink boxes. Segmental duplications were shown in colored blocks.
https://doi.org/10.1371/journal.pone.0250479.s003
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S2 Table. Characteristics of wheat, Arabidopsis, rice, sorghum and maize FBK proteins.
https://doi.org/10.1371/journal.pone.0250479.s005
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S4 Table. Screening of the candidate proteins interacting with TaAFR.
https://doi.org/10.1371/journal.pone.0250479.s007
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S5 Table. Bioinformatics analysis of the candidate proteins.
https://doi.org/10.1371/journal.pone.0250479.s008
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S1 File. Sequences of FBK proteins in wheat, Arabidopsis, rice, sorghum and maize.
https://doi.org/10.1371/journal.pone.0250479.s009
(FASTA)
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