Genome-wide identification and expression analysis of the plant-specific PLATZ gene family in Tartary buckwheat (Fagopyrum tataricum)

Plant AT-rich sequence and zinc-binding (PLATZ) proteins belong to a novel class of plant-specific zinc-finger-dependent DNA-binding proteins that play essential roles in plant growth and development. Although the PLATZ gene family has been identified in several species, systematic identification and characterization of this gene family has not yet been carried out for Tartary buckwheat, which is an important medicinal and edible crop with high nutritional value. The recent completion of Tartary buckwheat genome sequencing has laid the foundation for this study. A total of 14 FtPLATZ proteins were identified in Tartary buckwheat and were classified into four phylogenetic groups. The gene structure and motif composition were similar within the same group, and evident distinctions among different groups were detected. Gene duplication, particularly segmental duplication, was the main driving force in the evolution of FtPLATZs. Synteny analysis revealed that Tartary buckwheat shares more orthologous PLATZ genes with dicotyledons, particularly soybean. In addition, the expression of FtPLATZs in different tissues and developmental stages of grains showed evident specificity and preference. FtPLATZ3 may be involved in the regulation of grain size, and FtPLATZ4 and FtPLATZ11 may participate in root development. Abundant and variable hormone-responsive cis-acting elements were distributed in the promoter regions of FtPLATZs, and almost all FtPLATZs were significantly regulated after exogenous hormone treatments, particularly methyl jasmonate treatment. Moreover, FtPLATZ6 was significantly upregulated under all exogenous hormone treatments, which may indicate that this gene plays a critical role in the hormone response of Tartary buckwheat. This study lays a foundation for further exploration of the function of FtPLATZ proteins and their roles in the growth and development of Tartary buckwheat and contributes to the genetic improvement of Tartary buckwheat.


Background
Transcription factors (TFs) are sequence-specific binding proteins that can activate or inhibit the expression of target genes by recognizing and binding to cis-acting elements in their promoter regions of target genes to affect diverse biological processes at the transcriptional level [1]. Zinc finger proteins are an important class of TFs. Based on previous reports, more than 1500 TFs exist in Arabidopsis, accounting for approximately 5% of the Arabidopsis genome [2], of which approximately 15% are zinc finger proteins [3]. The zinc-finger protein consists of two cysteines and two histidines tetrahedrally coordinated with zinc atoms to form a compact fingerlike structure. These proteins participate extensively in plant growth and development and actively respond to various stresses [3,4]. Plant AT-rich sequence and zincbinding (PLATZ) proteins are a novel class of plant-specific zinc-dependent DNA-binding proteins that preserve the unique structure of the zinc-finger protein family and contain two distantly conserved domains: C-x 2 -H-x 11 -Cx 2 -C-x (4)(5) -C-x 2 -C-x (3)(4)(5)(6)(7) -H-x 2 -H, and C-x 2 -C-x (10)(11) -Cx 3 -C [5]. Although the first PLATZ gene, PLATZ1, was isolated from peas in 2001 [5], it has attracted increasing attention from researchers. PLATZ1 can nonspecifically bind to A/T-rich sequences and inhibit transcription, as demonstrated by a transient assay [5]. Previous studies have shown that PLATZ proteins play essential roles in several biological processes in plants. For example, Li et al. reported that Floury3 (FL3) encodes a PLATZ protein in maize, which interacts with RNA polymerase III subunit 53 (RPC53) and transcription factor class C 1 (TFC1) to affect endosperm development and filling in seeds [6]. GL6, a PLATZ protein in rice, has been demonstrated to regulate grain length and spikelet number through the same interaction mechanism [7]. SHORT GRAIN6 (SG6) regulates the division of spikelet hull cells and determines seed size in rice by interacting with DP proteins and cell division regulators [8]. Plant regulation by PLATZ is not restricted to the seeds. In Arabidopsis, the PLATZ protein ORESARA15 (ORE15) could regulate leaf growth and senescence by promoting the rate and duration of early cell proliferation [9]. ABA-INDUCED expression 1 (AIN1) represses the elongation of the primary root of Arabidopsis upon ABA induction [10]. In addition, Chao et al. illustrated via transcriptome analysis that PLATZ TFs are important for the secondary growth of Populus stems [11]. Moreover, PLATZ proteins are extensively involved in the response to numerous abiotic stresses, including heat [12], drought [13,14], salt and osmotic stresses [15,16], and in response to hormones [17,18].
Tartary buckwheat (Fagopyrum tataricum, 2n = 2x = 16) is primarily cultivated in Asia, Europe and North America [19]. As a traditional medicinal and edible crop, its grain has a balanced essential amino acid composition and is rich in phytochemicals and soluble fiber [20]. In particular, flavonoids, which have many important biomedical functions, are more abundant in Tartary buckwheat than in other main crops [21][22][23][24]. Tartary buckwheat has been recognized as a green food for humans in the twenty-first century, and has gained popularity among consumers. However, its low yield severely limits its industrial applications [25]. Therefore, identifying PLATZ proteins in Tartary buckwheat is necessary because of their functional potential, particularly their regulatory roles in the growth and development of plant seeds and their relationship to plant resistance, which could provide new insights into the yield improvement of Tartary buckwheat. The PLATZ family has been identified in several other plant species. To date, 12 members have been identified in Arabidopsis thaliana [26], 15 in Oryza sativa [26], 17 in Zea mays [26], 62 in Triticum aestivum [1] and 24 in Brassica rapa [27]. However, to the best of our knowledge, identification and functional characterization of the PLATZ gene family in Tartary buckwheat have not yet been reported. Highquality, chromosome-scale genome sequencing of Tartary buckwheat has recently been completed [28], laying the foundation for a systematic genome-wide study of the PLATZ gene family in Tartary buckwheat. In the present study, 14 PLATZ proteins were identified in Tartary buckwheat genome. We investigated the evolutionary relationships of FtPLATZs together with a comprehensive study of gene structures, conserved motif composition, and cis-acting elements in the promoter regions of FtPLATZs. Gene duplication events and their syntenic relationships with the six representative species were investigated. For functional characterization, we examined the expression profiles of FtPLATZs in different tissues of Tartary buckwheat and in grains at different developmental stages using real-time quantitative polymerase chain reaction (qRT-PCR). In addition, the responses of FtPLATZs to various exogenous hormones were investigated. This study aimed to form a foundation for further exploration of the functional mechanisms of FtPLATZs and contribute to the improvement of plant varieties and innovation of the germplasm in Tartary buckwheat.

Identification of FtPLATZ proteins in Tartary buckwheat
Combining the results of the hidden Markov model (HMM) search and BLASTP operations and further examination of the conserved PLATZ domain, 14 putative FtPLATZ proteins were identified in Tartary buckwheat ( Fig. S1 and Table S1). They were unevenly distributed on six chromosomes of Tartary buckwheat (Fig. 1). Chromosome Ft4 contained the largest number of FtPLATZ genes (four genes), followed by Ft1 and Ft8, both of which contained three genes. Ft3 contained two genes, whereas Ft2 and Ft6 contained only one. The FtPLATZ genes were not found on chromosomes Ft5 and Ft7. We designated these as FtPLATZ1 to FtPLATZ14 based on their location on the chromosomes. As shown in Table 1, the fulllength cDNAs, predicted protein products and Mw of FtPLATZ genes varied greatly, ranging from 444 to 1590 bp, 148 to 530 aa, and 16.59 to 58.82 kDa, respectively. The average coding sequence (CDS) length, predicted protein products, and molecular weight (Mw) were 805 bp, 268 aa, and 30.20 kDa, respectively. The data clearly showed that FtPLATZ3 was the smallest, and FtPLATZ10 exhibited the largest size with the maximum level of CDS length, predicted protein products, and Mw among FtPLATZs. The difference in the theoretical isoelectric point (pI) values among FtPLATZ genes was relatively small, with an average of 8.80.

Phylogenetic analysis and classification of FtPLATZ proteins
To clarify the evolutionary relationship between the PLATZ proteins of Tartary buckwheat and the PLATZ proteins of two model plants, Arabidopsis and rice, we constructed a maximum likelihood (ML) tree with the 14 identified FtPLATZs, 12 AtPLATZs and 15 OsPLATZs (Fig. 2). The 41 PLATZ proteins were divided into five groups (I to V), and the FtPLATZ proteins were distributed in the four main groups (II to V). Group II contained the largest number of FtPLATZ members (6 of 14, 42.86%). Half of the PLATZ proteins in Group II were FtPLATZs. Group V contained four FtPLATZs, whereas the remaining 10 proteins were from Arabidopsis and rice. Group IV contained one FtPLATZ, one AtPLATZ member, and five OsPLATZs. In particular, group III was only composed of three FtPLATZ members, indicating no homology to AtPLATZs and OsPLATZs. Group I contained two AtPLATZs and three OsPLATZs but no FtPLATZ proteins. In addition, a phylogenetic tree for FtPLATZs was constructed and labeled based on the grouping in the overall phylogenetic tree to analyze the differences in gene structure and motif components among groups (Fig. 3a).

Gene structure and conserved motifs analysis of FtPLATZ genes
The exon-intron structure of the FtPLATZ genes was investigated based on the genomic DNA sequence of Tartary buckwheat to understand the structural composition of the FtPLATZ genes (Fig. 3b). In general, the structures of FtPLATZ genes were distinguishable among the phylogenetic groups, and they showed similar characteristics within the groups. Most genes contained three introns (9 out of 14, 64.29%), and only five genes, FtPLATZ1/7/8/10/13, contained four introns. Group III was characterized by FtPLATZ genes with four introns, whereas groups IV and V contained only threeintron genes. In Group II, two genes, FtPLATZ1 and FtPLATZ13, had four introns, whereas the other genes had three introns.
Result similar to the exon-intron structure was also found in the motif composition of phylogenetically grouped FtPLATZ members (Table S2). As shown  Fig. 3c, motifs 1, 2, 3, and 6, which constituted the core domain of PLATZ, were universally present in the FtPLATZ members, except for one gene (FtPLATZ3) in group II, where motif 2 was not present, indicating a possible sequence loss during evolution. In addition, motifs 5 and 9 were uniquely present in group III, and motifs 4 and 8 were uniquely present in group V. Motif 10 appeared only in group II, and motifs 4 and 8 were present separately in the five FtPLATZs of group II. Only one member of group IV, FtPLATZ12, possessed motif 4 exclusively, in addition to the core domain of PLATZ.

Gene duplication events and synteny analysis of FtPLATZ genes
Possible gene duplication events among the FtPLATZs were investigated to explore the evolution of FtPLATZ genes. The results showed that tandem duplication and segmental duplication events were observed in FtPLATZs, where FtPLATZ7/FtPLATZ8 formed a tandem duplication event ( Fig. 1) and FtPLATZ1/FtPLATZ13, FtPLATZ5/FtPLATZ9 and FtPLATZ7/FtPLATZ10 formed three segmental duplication events (Fig. 4). These results indicate that duplication events widely participated in the evolution of FtPLATZs.
Furthermore, we investigated the syntenic relationships between FtPLATZs and PLATZ genes from four representative dicotyledons (A. thaliana, G. max, V. vinifera, and S. lycopersicum) and two representative monocotyledons (O. sativa and Z. mays; Fig. 5). The number of orthologous gene pairs between Tartary buckwheat and the other six species was quite different: five pairs with Arabidopsis, ten with soybean, six with grape, five with tomato, one with rice, and one with maize (Table S3). In particular, among the 14 FtPLATZ genes, FtPLATZ5 (FtPinG0008634800.01.T01) was the only gene that was collinear with PLATZ proteins of the six representative plants. FtPLATZ5 was collinear with at least two PLATZ genes in dicotyledons and one in monocotyledons. The results indicated that these orthologous genes may exist before the differentiation of the ancestors.

Expression patterns of FtPLATZ genes in different tissues and grain developmental stages of Tartary buckwheat
The potential roles of the identified FtPLATZ genes in the growth and development of Tartary buckwheat were explored using qRT-PCR ( Fig. 6a and Table S4). In general, the expression patterns of FtPLATZ genes varied greatly in different tissues, indicating their potential multiple functions in the growth and development of Tartary buckwheat. Two genes (FtPLATZ4 and FtPLATZ11) showed similar expression patterns, specifically expressed in the roots and slightly expressed in grains. Three genes (FtPLATZ6, FtPLATZ9, and FtPLATZ12) showed the highest expression levels in the stems. FtPLATZ5 showed the highest expression levels in   the leaves and stems. Four genes (FtPLATZ1, FtPLATZ2, FtPLATZ7, and FtPLATZ13) were highly expressed in the flowers, whereas FtPLATZ2 and FtPLATZ13 were only slightly expressed in other tissues. In addition, four genes (FtPLATZ3, FtPLATZ8, FtPLATZ10, and FtPLATZ14) were highly expressed in the grains, reaching their highest expression levels successively in the S1, S2, S3, and S4 developmental stages of the grains.
The expression patterns of FtPLATZ genes in different developmental stages of Tartary buckwheat grains have drawn much attention. Six patterns are identified. The expression levels of FtPLATZ5, FtPLATZ11, and FtPLATZ14 increased with grain growth and development, whereas those of FtPLATZ1, FtPLATZ2, and FtPLATZ7 decreased with grain growth and development. In addition to the two monotonous expression , and grain (G-S1, initial formation stage; G-S2, green grain stage; G-S3, discoloration stage; G-S4, initial maturity stage) of Tartary buckwheat. Error bars are obtained from three biological replicates. Lowercase letters above the bars represent significant differences between different treatments, as determined by Duncan's multiple range test (p < 0.05). b. Pearson's correlation of expression patterns among FtPLATZ genes. Red and blue represent positive and negative correlations, respectively. * and ** indicate significance at the levels of 0.05 and 0.01, respectively patterns, some gene expression levels initially decreased and then increased (FtPLATZ6 and FtPLATZ9), and some initially increased and then decreased with grain development (FtPLATZ4, FtPLATZ8, and FtPLATZ12). In addition, the expression of FtPLATZ10 and FtPLATZ13 showed a wave-shaped trend, decreasing twice during stages S2 and S4 of the grains. In particular, FtPLATZ3 was highly expressed in the S1 stage, but not in the other stages. Collectively, FtPLATZ genes may play crucial roles during grain development in Tartary buckwheat.
Further correlation analysis indicated that the expression patterns of some FtPLATZ genes in different tissues of Tartary buckwheat and different developmental stages of the grain were significantly and positively correlated (Fig. 6b); that is, FtPLATZ1/FtPLATZ2 (p < 0.05), FtPLATZ4/FtPLATZ11 (p < 0.01), and FtPLATZ6/FtPLATZ12 (p < 0.01), which was consistent with the results shown in Fig. 6a, indicating that some FtPLATZ genes may act synergistically with one another during development.

Analysis of promoter cis-acting elements of FtPLATZ genes
The functional potential of the identified FtPLATZ genes was further explored by investigating cis-acting elements in the promoter regions of these genes. Various cis-acting elements were identified, as summarized in Table S5. Promoter-related elements (i.e., TATA-box and CAAT-box) and light-responsive elements (i.e., Box 4, G-Box, TCT-motif et al.) were most abundantly distributed in the promoter region of FtPLATZ genes. Notably, stress-related elements (i.e., ARE, LTR, and MBS) and hormone-responsive elements (i.e., ABRE, CGTCAmotif, and TCA-element) were also widely distributed in the promoter region of the FtPLATZ genes. In particular, the number of hormone-responsive elements varied considerably among the FtPLATZ genes (Fig. 7), suggesting that the 14 FtPLATZs may function specifically in response to different hormone stimulation. In addition, some development-related elements (i.e., O2-site, MSAlike and CAT-box) and site-binding-related elements (i.e., CCAAT-box, HD-Zip 3 and MBSI) were identified in the promoter region of the FtPLATZ genes, but not all FtPLATZs contained such elements.

Differential expression of FtPLATZ genes under different exogenous hormone treatments
The expression levels of the 14 identified FtPLATZ genes after treatment with five exogenous hormones and the control, were compared using qRT-PCR to investigate the response pattern of FtPLATZ genes to hormones ( Fig. 8a and Table S6). The results showed that the expression levels of 11 of the 14 FtPLATZ genes were altered significantly after treatment with at least one type of exogenous hormone. MeJA treatment had the greatest impact on FtPLATZ genes among the five hormones, with significant upregulation of FtPLATZ2, FtPLATZ4, FtPLATZ6, and FtPLATZ9 and downregulation of FtPLATZ5, FtPLATZ7, FtPLATZ12, and FtPLATZ13. FtPLATZ genes, such as FtPLATZ3, FtPLATZ4, FtPLATZ5, FtPLATZ6, and FtPLATZ14, which responded significantly to SA treatment, were primarily upregulated. Only one gene, FtPLATZ12, was downregulated. Similar results were found for IAA and ABA treatments, in which the genes were primarily upregulated. In addition, under GA treatment, three genes were downregulated (FtPLATZ5, FtPLATZ9, and FtPLATZ14), and two genes were upregulated (FtPLATZ4 and FtPLATZ6). Notably, FtPLATZ6 was significantly upregulated by all five exogenous hormones, particularly ABA, GA, and SA. Moreover, FtPLATZ5 and FtPLATZ14 responded significantly Fig. 7 Distribution of the cis-acting elements related to hormone response in the promoter region of FtPLATZ genes to four hormones, and they showed similar response patterns to GA, IAA, and SA.
Furthermore, the results of the correlation analysis showed that the expression patterns of some genes were significantly correlated after treatment with exogenous hormones (Fig. 8b). The expression patterns of FtPLATZ2 and FtPLATZ4 were significantly and positively correlated (p < 0.01), whereas FtPLATZ3 and FtPLATZ7 showed a significant negative correlation (p < 0.05).

Subcellular localization of FtPLATZ proteins
The subcellular localization prediction results of CELLO and Plant-mPLoc consistently showed that most proteins were localized in the nucleus (11 out of 14), but the prediction results for the remaining three proteins (FtPLATZ1, FtPLATZ2, and FtPLATZ12) were inconsistent between the two prediction methods (Table 1). CELLO predicted that these proteins were localized extracellularly, whereas Plant-mPLoc predicted that they were located in the nucleus. Transient expression in Nicotiana benthamiana was examined to verify its subcellular localization (Fig. 9). These results indicated that the green fluorescent protein (GFP) fluorescent signals of the three fusion proteins were primarily localized in the nucleus. By contrast, the control 35S::GFP signal was detected in whole cells. The experimental results suggest that FtPLATZ proteins may function as conventional TFs.

Discussion
PLATZ TFs are a class of plant-specific zinc-dependent DNA-binding proteins that play important roles in the growth and development of plants and their response to stress [16]. In this study, we identified 14 PLATZ proteins in the Tartary buckwheat genome, all of which harbored conserved PLATZ domains. The amount of PLATZ proteins in Tartary buckwheat was similar to that identified in Arabidopsis (12) [26], rice (15) [26], and maize (17) [26]. However, the genome size of these species varied greatly (Tartary buckwheat, 489.3 Mb [28]; Arabidopsis, 125 Mb [29]; rice, 466 Mb [30], and maize, 2.3Gb [31]), implying that the amount of PLATZ proteins and the size of the genome were not closely related.
Gene duplication, including tandem duplication and segmental duplication, is regarded as a primary driving force in the evolution of genomes and genetic systems and is also a mechanism for organisms to adapt to changing environments [32,33]. Fu et al. found that 21 of the 62 TaPLATZ genes identified in the wheat genome were from tandem duplications (33.9%) and two from segmental duplications and concluded that genomic duplication was the primary cause of the expansion of the TaPLATZ family [1]. Similarly, Azim et al. found a considerable number of gene duplication events in Brassica rapa, where 20 pairs of segmental duplication genes were detected among the 24 identified BrPLATZ genes, whereas no tandem duplication events were found [27]. In our study, a pair of tandem duplicated FtPLATZ genes (Fig. 1) and three pairs of segmental duplicated FtPLATZ genes (Fig. 4) were detected in Tartary buckwheat, accounting for 50% of the FtPLATZ genes (seven out of 14 genes), implying that gene duplication was the main driving force in the evolution of FtPLATZ genes. These duplicated genes had almost the same exon-intron structure and motif composition (Fig. 3b and c), but their expression preferences seemed to differ (Fig. 6a). Subfunctionalization of duplicated FtPLATZ genes may account for their different expression patterns [34]. Furthermore, synteny analysis showed that the PLATZ genes of Tartary buckwheat shared more orthologs with dicotyledons than with monocotyledons. Tartary buckwheat and soybean had the largest number of orthologous gene pairs (Table S3), implying that they could have a closer evolutionary relationship and may have evolved from a common ancestor, which conformed to previous findings [20,35].
In the phylogenetic analysis, the 41 PLATZ proteins obtained from Tartary buckwheat, Arabidopsis and rice were classified into five groups based on their phylogenetic relationships, wherein 14 FtPLATZ proteins were distributed into four main groups (Groups II to V, Fig. 2). The exon-intron structures of FtPLATZ genes were similar, containing three or four introns (Fig. 3b), implying that FtPLATZ genes were relatively conserved during evolution [27]. FtPLATZ genes within the same group shared a similar gene structure and motif composition, whereas evident distinctions were found among different groups, particularly in motif composition, implying large functional differentiation of FtPLATZ genes. Ten motifs were detected in the FtPLATZ proteins, of which motifs 1, 2, 3, and 6 constituted the PLATZ domain (Fig. 3c). In our study, exon loss was observed in the FtPLATZ genes. In particular, FtPLATZ3 did not contain motif 2, part of the beginning of the PLATZ domain, which may be due to genetic variation that occurred during the evolution of FtPLATZ genes, thereby leading to the alteration of gene functions [36]. LOC_Os06g45540.1 (SG6; GL6) belonged to the same phylogenetic group as FtPLATZ3 (group II), which has been proven to regulate the grain size and spikelet number of rice [7,8]. Meanwhile, time-course transcriptome analysis revealed that AT3G60670.1 in group II was involved in the development and maturation of Arabidopsis grains [37]. As shown in Fig. 6a, FtPLATZ3 was significantly expressed at the S1 stage of grains, which is considered a critical developmental period for grain size [35]. Collectively, we hypothesized that FtPLATZ3 may be involved in the regulation of grain size in Tartary buckwheat, and further experimental verification is necessary. In addition, FtPLATZ4 and FtPLATZ11 may play important roles in the development of Tartary buckwheat roots. Although they have been found to be specifically expressed in the roots through tissue expression profiles, AT2G12646.1 (RITF1), located in the closest phylogenetic branch with two FtPLATZ genes, has been demonstrated to play a central role in mediating root meristem growth factor 1 (RGF1) signalling and subsequently affecting the size of root meristems [38].
Plant hormones play important roles in numerous biological processes and contribute remarkably to the adaptability of plants to changing environments [39,40]. Previous studies have shown that PLATZ genes are hormone responsive. GmPLATZ1 in soybeans [18] and PLATZ genes in Thellungiella salsuginea roots [17] could be induced by ABA. GhPLATZ1 from cotton is significantly upregulated in transgenic Arabidopsis under ABA and GA treatments [15]. Moreover, ABA can induce the expression of AIN1 in Arabidopsis, thereby affecting the elongation of the primary root [10]. PhePLATZ genes in moso bamboo were significantly regulated by GA, ABA, and MeJA treatments [41]. In the present study, hormone-responsive elements related to ABA, MeJA, SA, GA, and IAA were examined in the promoter region of FtPLATZ genes; however, their distribution across different FtPLATZ genes was diverse, which is similar to the findings of Fu et al. in the identification of TaPLATZs [1]. MeJA can activate the expression of defense genes, induce the synthesis of defensive compounds, and can also affect the antioxidant system [42]. Numerous studies have revealed that MeJA is involved in mediating defense responses against fungal pathogens [43], alleviating salt [44], drought [45], and chilling stresses [46]. The expression of nearly 80% of FtPLATZ genes (11 out of 14) changed significantly after treatment with exogenous hormones (Fig. 8a), among which MeJA treatment exhibited the widest effects in 8 of the 11 significantly disturbed genes, implying that FtPLATZ genes might be extensively involved in the stress response of Tartary buckwheat. FtPLATZ6 was the only gene that was significantly upregulated after all exogenous hormone treatments, which may indicate the critical role of FtPLATZ6 in biological processes involved in the hormone response of Tartary buckwheat. However, no abundant hormoneresponsive elements were found in the promoter region of FtPLATZ6 (Fig. 7). Previous studies have reported that the distribution pattern of cis-acting elements is not directly related to the gene expression levels [47][48][49]. Therefore, the expression of FtPLATZ genes may involve complex regulatory mechanisms that require further experimental verification.

Conclusions
In this study, we systematically identified and characterized the PLATZ gene family in Tartary buckwheat. Fourteen FtPLATZ proteins were identified, which were unevenly distributed on six of the eight chromosomes in Tartary buckwheat. Based on phylogenetic analysis, the FtPLATZ proteins were classified into four groups, and each group shared a similar gene structure and motif composition. In addition, gene duplication, particularly segmental duplication, was the main driving force in the evolution of the FtPLATZ genes. We analyzed the expression levels of 14 FtPLATZ genes in different tissues and different grain developmental stages of Tartary buckwheat and their responses to five exogenous hormones. The results revealed, to a great extent, the important roles of FtPLATZ genes in the growth and development of Tartary buckwheat, such as FtPLATZ3, which might be involved in the regulation of grain size; FtPLATZ4 and FtPLATZ11, which played a role in root development; and FtPLATZ6, which was significantly upregulated after all the exogenous hormone treatments and may be critical for the hormone response of Tartary buckwheat. This study provides a foundation for further exploration of the functional characteristics of FtPLATZ genes and promotes targeted genetic breeding research for crop improvement in Tartary buckwheat.

Identification of FtPLATZ genes in Tartary buckwheat genome
The Tartary buckwheat genome was obtained from the Tartary Buckwheat Genome Project (TBGP; http:// www. mbkbase.org/Pinku1/), and the gene anotation V2 version was used for subsequent analysis [28]. To identify PLATZ genes in the Tartary buckwheat genome, the HMM profile of PLATZ (PF04640) downloaded from the Pfam database (http:// pfam. xfam. org/) was used to search against the Tartary buckwheat genome database via HMMER3.3 with default parameter settings [50]. The PLATZ genes of Arabidopsis and rice obtained from the TAIR database (https:// www. arabi dopsis. org/) and iTAK database (http:// itak. feilab. net/ cgi-bin/ itak/ index. cgi) [51], respectively, were used to perform a BLASTP operation to further retrieve possible FtPLATZ genes from the Tartary buckwheat genome with a score ≥ 100 and e-value ≤1 × 10 − 10 [52]. All putative FtPLATZ genes integrating the results of the HMM retrieval and BLASTP operations were submitted to the NCBI Conserved Domain Database (CDD, https:// www. ncbi. nlm. nih. gov/ cdd), SMART (http:// smart. embl-heide lberg. de/), and Pfam to examine the existence of the conserved PLATZ domain.

Sequence characterization
We collected chromosomal location information for the identified FtPLATZ genes from the Tartary buckwheat genome database and visualized them using TBtools [53]. The properties of the identified FtPLATZ genes, including CDS length, protein length, Mw, and pI, were investigated using Expasy (https:// web. expasy. org/ compu te_ pi/). The exon-intron structure of the FtPLATZ members was investigated using TBtools based on Tartary buckwheat genome annotation information. Conserved motifs of FtPLATZ proteins were identified using the MEME Suite (https:// meme-suite. org/ meme/ tools/ meme) with default parameters, except that the maximum number of motifs was set to 10. Moreover, the cis-acting elements within the 2000 bp sequence upstream of FtPLATZ genes, usually regarded as the promoter region of a gene [1], were analyzed using PlantCARE (http:// bioin forma tics. psb. ugent. be/ webto ols/ plant care/ html/) [54].

Phylogenetic analysis
The identified genes, together with AtPLATZs and OsPLATZs, were used to construct a phylogenetic tree using the ML method with the MEGA X software [55]. The Jones-Taylor-Thornton (JTT) model combined with a discrete gamma distribution (+ G) was selected as the optimal model for constructing the phylogenetic tree. Sequences with more than 20% alignment gaps were removed, and a bootstrap test was conducted with 1000 replicates. A phylogenetic tree containing only FtPLATZs was constructed using these parameters. The classification of FtPLATZs based on the phylogenetic tree was referred to the method described by Wang et al. [26].

Gene duplication and synteny analysis
Possible gene duplication events among FtPLATZ genes were probed using multiple collinear scanning toolkits (MCScanX) [56]. Syntenic analyses were conducted using TBtools between the identified FtPLATZ proteins and PLATZ protein sequences of Glycine max, Vitis vinifera, Solanum lycopersicum, Oryza sativa, and Zea mays obtained from the iTAK database and the AtPLATZs obtained from the TAIR database. To investigate the response of FtPLATZ genes to exogenous hormones, 21-day-old seedlings (Fig. S2) were treated with different exogenous hormones, including 100 μM methyl jasmonate (MeJA), abscisic acid (ABA), salicylic acid (SA), 10 μM indole-3-acetic acid (IAA), and gibberellin (GA) by foliar spraying. The control group was sprayed with equal amounts of water. After 6 h of treatment, the second leaves of the seedlings were collected separately [57]. All the samples were collected from at least three healthy plants, immediately frozen with liquid nitrogen, and then stored at − 80 °C for RNA extraction and subsequent qRT-PCR analysis.

Expression analyses of FtPLATZ genes by qRT-PCR
Total RNA was extracted from all samples using a Min-iBEST Plant RNA Extraction Kit (TaKaRa). First-strand cDNA was synthesized using the PrimeScript ™ II 1st Strand cDNA Synthesis Kit (TaKaRa). qRT-PCR was performed using TB Green ™ Premix Ex Taq ™ II (TaKaRa) on a Q7 Real-Time PCR System (Applied Biosystems ™ , Foster City, CA, USA) following the manufacturer's instructions. The primers for qRT-PCR analysis were designed using Primer3 software (version 4.1.0, https:// prime r3. ut. ee/) based on the CDSs of the identified FtPLATZ genes obtained from TBGP, and the information of all primer sequences are listed in Table S7. FtH3 was selected as the internal reference gene, which has been proven to be stably expressed in Tartary buckwheat under any condition [58]. Expression data were analyzed using the 2 −ΔΔCT method [59].

Statistical analysis
Comparisons of the expression levels of the FtPLATZ genes in different tissues were statistically evaluated by one-way analysis of variance (ANOVA) using IBM SPSS Statistics 25 (IBM Corporation, Armonk, NY) [62]. Duncan's multiple range test was used to determine significant differences between groups. Student's t-test was carried out using R software (version 4.0.2) to examine whether the expression of FtPLATZ genes changed significantly after stimulation with exogenous hormones.