Identification And Expression Analysis of FORMIN Gene Family In Rice (Oryza Sativa)

doi:10.21203/rs.3.rs-1132176/v1

Download PDF

Research Article

Identification And Expression Analysis of FORMIN Gene Family In Rice (Oryza Sativa)

https://doi.org/10.21203/rs.3.rs-1132176/v1

This work is licensed under a CC BY 4.0 License

Version 1

posted

You are reading this latest preprint version

Background: FORMIN proteins, which are composed of proteins containing FH1 and FH2 domains, play crucial roles in the growth and development of organisms. However, the functions of FORMINs in rice (Oryza sativa) remain largely unclear.

Results: In this study, a total of 17 FORMIN genes were identified in rice, OsFH17 was the first time identified in this study. In addition, the distribution on chromosomes, gene structure, as well as conserved motifs of rice FORMINs was investigated. According to their protein structural and phylogenetic features, these 17 rice FORMIN genes were classified into two distinct subfamilies. Subcellular localization prediction showed that rice FORMINs were located in cytosol, golgi, endoplasmic reticulum, extracellular, and vacuole. Protein protein interaction (PPI) prediction results shown that FORMIN protein might answer hormone signals and be involved in cytoskeleton dynamics regulation and cell wall morphology regulation. The results of silico analysis and qRT-PCR confirmation of the gene expression showed that the expression of rice FORMINs were related to their tissue distribution. Moreover, OsFH3, OsFH5 and OsFH7 were upregulated under phytohormone treatments.

Conclusions: Overall, our research may shed light on the understanding and further investigation of the biological functions of rice FORMINs.

Plant Molecular Biology and Genetics

Plant Physiology and Morphology

FORMINs

bioimformatic

rice

gene structure

expression profiles.

Actins play critical roles in cellular activities such as cytoskeleton construction, cell division, and cell polarized growth[1–7]. As actin-binding proteins (ABPs), FORMINs funtion as actin-filament nucleators that bind to the barbed ends of actin filaments to promote the polymerization and elongation of actin filaments[8]. Therefore, FORMINs are extremely paramount to regulate cell polarized growth, cytokinesis, and cell migrations [1, 5, 9, 10].

Plant FORMINs are much different from the ones in animals or fungi[11, 12]. For example, FORMINs in plants do not have GTPase binding domain (GBD), diaphanous inhibitory domain (DID), or diaphanous auto-regulatory domains (DAD)[13]. Instead, phosphatase tensin (PTEN) domain could be found in some plant FORMIN proteins but not in animal or fungal FORMIN proteins[10, 14]. Besides, comparing to the GTPase binding and activation of the animal FORMIN model, how plant FORMINs are activated remains unclear, as no GTPase binding domain has been found in plant FORMINs yet[15].

Although some studies have been done to identify the members or to explore the functions of FORMIN genes, The knowledge of plant FORMIN genes is far to be clear[16]. For example, it has been reported that there are 21 and 16 FORMIN genes that have been identified in arabidopsis and rice genomes respectively[13]. But only 3 of the 16 rice FORMIN genes have been experimentally explored for theirs functions. OsFH1 is involved in the polarized elongation of root hairs, loss-of-function of OsFH5 gene resulted in irregular cell growth in leaf sheath and shorter cells in inner husks, and OsFH15 is involved in seed size regulation by interacting with actin filaments and microtubes[17–19]. Therefore, the functions of rice FORMIN genes are far to be clear. To further elucidate the functions of rice FORMINs, both bioinformatics and molecular biological strategies were employed in this study. The cis-acting elements, temporal and spatial expression pattern and the conserved protein domains of rice FORMIN genes or proteins were analyzed along with the prediction analysis of the proteins that interact with rice FORMIN proteins. Our study may provide new insights for understanding how FORMINs function during rice growth and development.

Identification and Phylogenetic Analysis of FORMIN Genes

We used the Formin homology 2 (FH2) profile of FH2 domain (PF02181) from Pfam (https://pfam.xfam.org/) database as reference sequences to extract rice FORMIN family members by using TBtools and GenomeNet with default parameters. The FORMIN proteins of Cyanidioschyzon merolae (red alga), Saccharomyces cerevisiae (baker's yeast), Physcomitrella patens (P. patens), Selaginella moellendorffii (spike moss), Medicago truncatula (barrel medic), Lotus japonicus (L. japonicus), Zea may (maize) and Arabidopsis thaliana (arabidopsis) were downloaded from genome database and verified by TBtools software with default parameters. A total of 17 proteins were identified belonging to the FORMIN gene family in rice, OsFH17 was the first time identified in this study. The results shown that the number of rice FORMIN’s amino acids ranged from 364 to 1511 (Table 1). Furthermore, we identified 3, 2, 8, 5, 15, 14, 20 and 21 FORMIN proteins in red alga, baker's yeast, P. patens, spike moss, barrel medic, L. japonicus, maize and arabidopsis, respectively. In order to explore the evolutionary relationships of the FORMIN gene family, an unrooted phylogenetic tree was constructed using the full length FORMIN protein sequences from these nine selected species (Figure 1). Obviously, all 105 proteins were divided into two major clades, clade I including type I FORMIN subfamily and clade II including type II FORMIN subfamily (Figure 1). Regarding the 17 rice FORMIN proteins, they were divided into two types (type I, type II) as well. Type I FORMIN family consists of OsFH3, OsFH5, OsFH6, OsFH7 and OsFH12. Type II consists of OsFH1, OsFH2, OsFH4, OsFH8, OsFH9, OsFH10, OsFH11, OsFH13, OsFH14, OsFH15, OsFH16 and OsFH17 (Figure 1 ).

Interestingly, type II FORMIN proteins were not found in red alga, baker's yeast and spike moss. Besides, P. patens contained 6 type II FORMIN proteins. Moreover, rice, barrel medic, L. japonicus, maize and arabidopsis all contained over 6 type II FORMIN proteins. In addition, the survey of eumycophyta and plant genomes for FORMIN genes indicated that the numbers of type I and type II FORMIN genes in the angiosperm were more than the ones in gymnosperm and eumycophyta (Figure 2). These results imply that FORMIN genes had been diverged during evolution, probably deriving from family expansion and domain acquisition.

Gene structure or conserved domains of FORMIN Genes or FORMIN proteins

The analyses of gene structure and gene phylogenetic relation could help us to better understand the function or evolution of less explored genes. Therefore, an NJ tree was generated with MEGA7 using all the FORMIN protein sequences from rice genome, and the structure of these genes was constructed as well (Figure 3). As shown in Figure 3, the distribution of exons and introns of rice was variable, and the number of exons and the length of sequences of these FORMIN genes were widely ranged between two subclasses. Type I FORMIN subfamily includes 2 genes with 1 exon, 8 genes with 3 exons, 1 gene with 4 exons and 1 gene with 5 exons. Whereas the type II includes 1 gene with 5 exons, 1 gene with 12 exons, 1 gene with 13 exons and 2 genes with 15 exons (Table 1).

Besides the study of gene structure features, the full-length sequences of rice FORMIN proteins were submitted to MEME website for architecture analysis. The results indicated that ten conserved motifs were identified in these rice FORMIN family proteins, and the differences of motifs between type-I and type-II FORMINs were significant. Such as motif 5 and motif 9 were unique to type-I, FORMINs while motif 10 was specific to all type-II FORMINs. Meanwhile, in the same subfamily, the same type and distribution mode of motifs were shared, indicating that the protein architecture of these rice FORMIN proteins were highly conserved within a specific subfamily.

Table 1

The gene and protein features of FORMIN family members in rice
Gene name	RAP-ID	Chromosome localization	CDS length (bp)	Exon number	Protein length (aa)	Protein MW (kDa)	Isoelectric Point (pI)	Subcellular localization	GOterms
OsFH1	Os01g0897700	Chr1	2883	3	961	101642	9.1195	cytosol	C:extracellular
OsFH2	Os04g0461800	Chr4	2502	3	834	88899.1	8.5935	golgi	C:extracellular
OsFH3	Os10g0119300	Chr10	1161	13	387	43312	5.9619	cytosol	C:nucleus
OsFH4	Os10g0347800	Chr10	2550	3	850	90280.9	9.7161	cytosol	C:extracellular
OsFH5	Os07g0596300	Chr7	1914	4	638	70922.8	4.3667	cytosol	C:chloroplast thylakoid lumen
OsFH6	Os08g0280200	Chr8	2682	15	894	95782.6	9.2704	cytosol	C:nucleus
OsFH7	Os02g0794900	Chr2	1092	12	364	41156.7	7.7994	cytosol	C:nucleus
OsFH8	Os03g0204100	Chr3	1629	1	543	55704.4	10.7337	extracellular	C:extracellular
OsFH9	Os08g0431200	Chr8	2649	3	883	97860.1	8.6902	cytosol	C:extracellular
OsFH10	Os02g0161100	Chr2	2646	5	882	96463.3	5.7992	golgi	C:extracellular
OsFH11	Os07g0545500	Chr7	2790	5	930	100990	8.0425	golgi	C:extracellular
OsFH12	Os04g0245000	Chr4	4533	15	1511	165148	5.4272	cytosol	C:nucleus
OsFH13	Os07g0588200	Chr7	2325	3	775	83810.3	8.7229	vacuole	C:extracellular
OsFH14	Os05g0104000	Chr5	2493	3	831	90221.6	8.4292	endoplasmic reticulum	C:nucleus
OsFH15	Os09g0517600	Chr9	2367	3	789	84580.3	8.8049	endoplasmic reticulum	C:extracellular
OsFH16	Os02g0739100	Chr2	2721	1	907	95565.3	10.3198	endoplasmic reticulum	C:extracellular
OsFH17	Os04g0100300	Chr4	2721	3	907	95753.4	8.338	golgi	C:extracellular

In addition, the conserved FH2 domains in all rice FORMIN proteins were also analyzed by using multiple sequence alignment, and the results showed that all conserved motifs were present in FH2 domains (Figure 3c). It is noteworthy that OsFH12 (belonging to type II FORMIN subfamily) contained a PTEN structure in its N-terminus (Figure 3C). It was also found that these 17 rice FORMINs were not evenly distributed on the 12 chromosomes of rice (Figure 3D). Twelve of the rice FORMIN genes were localized on the first ten chromosomes, including Chr 1, Chr 2, Chr 3, Chr 4, Chr 5, Chr 7, Chr 8, Chr 9 and Chr 10. None of the rice FORMINs was found on the chromosome 6, 11 or 12 and no FORMIN gene cluster was found on rice chromosomes (Figure 3D).

Protein-protein Interaction Prediction

To determine the functions of rice FORMIN proteins, protein–protein interaction (PPI) prediction was performed. Among 17 rice FORMIN proteins, three of them interacted with cell wall morphology regulation-related proteins and proteins involved in auxin (IAA) signal pathways. Totally 11 rice FORMIN proteins were closely connected with actin polymerization-related proteins, but only OsFH5 interacted with actin-depolymerizing-related proteins. Except for OsFH2 and OsFH3, other rice FORMINs showed a close relationship with cell cytoskeleton-regulated proteins, which implies the importance of FORMINs in regulating cytoskeleton dynamics in plants (Table 2). Some protein-protein interaction prediction results were also consistent with previous studies that rice FORMINs are involved in cell elongation, cell wall loosening and assembly of microtubules and microfilaments[17, 19, 20]. Moreover, the interaction prediction between FORMIN family members also indicated that there might be interactions between OsFH10, OsFH2, OsFH14, OsFH11, OsFH8 and OsFH15 (see Supplementary Figure S3, Supplementary Table S2). Such as, OsFH11 was predicted to interact with OsFH14, OsFH2, OsFH15, or OsFH8. OsFH10, OsFH14 and OsFH2 were predicted to interact with each other.

Table 2

Analysis of protein-protein interaction of rice FORMINs
Name	Formins	Actin related	Microtubule Related	Cytoskeleton Structure Related	Cell Wall Formation Related	Exocytic Vesicles Plasma Membrane Docking Related	Kinesin-like Protein	Tanscription factor	Silicon Efflux Transporter	K+ Channel Related	Auxin Signal Pathway	Brassinosteroid Signal Pathway
OsFH1	OsFH5	-	KIN5A	profilin LP04	CESA1, EXPA17, BC1L4	-	-	-	-	-	IAA3	-
OsFH2	OsFH16	Act	-	-	-	SEC10	-	-	-	-	-	-
OsFH3	OsFH14	-	-	-	-	-	-	SH3, TFIIAy	-	-	-	-
OsFH4	OsFH16	ARP2	-	profilin LP04, profilin A	-	SEC10	-	-	-	-	-	-
OsFH5	OsFH1	ARP2, ADF9		profilin LP04, profilin A	-	-	-	-	-	-	BG1	-
OsFH6	-	ARP2	-	profilin LP04, profilin A	-	-	-	-	-	-	-	-
OsFH7	-	ARP2	-	profilin LP04, profilin A	-	-	-	-	-	-	-	-
OsFH8	OsFH16	ARP2	-	profilin LP04, profilin A	-	SEC10	-	-	-	-	-	-
OsFH9	-	ARP2	TUBG2	profilin LP04, profilin A	-	-	KIN13B	-	-	-	-	-
OsFH10	OsFH16	ARP2	-	profilin LP04, profilin A	-	-	-	-	-	-	-	-
OsFH11	OsFH12	ARP2	TUBG2	profilin LP04, profilin A	-	-	-	-	-	-	-	-
OsFH12	OsFH11	Act	-	profilin LP04, profilin A	-	-	-	-	LSI3	-	-	-
OsFH13	OsFH16	ARP2	-	profilin LP04, profilin A	-	SEC10	-	-	-	-	-	-
OsFH14	OsFH3	-	KIN5A	profilin LP04, profilin A	-	-	-	-	-	FIP2	-	-
OsFH15	-	ARP2		profilin LP04, profilin A	-	-	-	-	-	FIP2	BG1	GSK2
OsFH16	OsFH2, OsFH4, OsFH8, OsFH13	-	-	profilin LP04, profilin A	-	SEC10	-	-	-	-	-	-
OsFH17	OsFH16	ARP2	-	profilin LP04, profilin A	-	-	-	-	-	-	-	-

Expression profiles of rice FORMIN genes in different tissues or in response to phytohormone treatments

To understand the temporal and spatial expression patterns of different FORMIN genes, microarray data from the rice XPro database was used to assess the expression profile of these FORMIN genes across different tissues or organs of rice plants (anther, callus, leaf, panicle, pistil, root, shoot and seed), or under a number of phytohormone treatments (abscisic acid, gibberellin, IAA, brassinosteroid, cytokinin and jasmonic acid). Meanwhile, the tissue specificity of the expression of FORMIN genes was also validated through quantitative real time-PCR (qRT-PCR) by using the cDNA from different tissues or organs (whole seeding, shoot, shoot apices, leaf blade, leaf sheath, root, immature flower and mature flower).

The expression analysis data have shown that all these 17 genes were significantly differentially expressed in selected tissues or organs (anther, callus, leaf, panicle, pistil, root, shoot and seed). OsFH1, OsFH16, OsFH5, OsFH13, OsFH5 and OsFH3 showed relatively higher but variable expression levels in almost all selected tissues or organs. The following four genes had relatively higher expression in some specific tissues or organs: (i) OsFH17 in the anthers, (ii) OsFH14 and OsFH15 in the calli, and (iii) OsFH7 in the roots.

The qRT-PCR analysis results showed that all these 17 genes could be divided into five groups, which include cluster 1 with three genes (OsFH5, OsFH1 and OsFH2), cluster 2 with two genes (OsFH7 and OsFH10), cluster 3 with one gene (OsFH6), cluster 4 with six genes (OsFH9, OsFH12, OsFH3, OsFH17, OsFH5, OsFH16, OsFH15) and cluster 5 with five genes (OsFH4, OsFH11, OsFH14, OsFH8 and OsFH13) (Figure 4B). From cluster 1 to cluster 4, they exhibited specific expression in the tissues or organs such as immature flowers, shoot apices and leaves. And the genes of cluster 5 showed expression in leaves, immature flowers and mature flowers.

Phytohormones play crucial roles during rice growth and development. Coincidentally, actin as a downstream element of auxin to regulate plant morphological changes has been elucidated[21]. To verify whether rice FORMINs were regulated by IAA or other phytohormones, the expression profiles of rice FORMINs were systematically established by using microarray data from the RiceXPro. As shown in Figure 4C and D, most rice FORMIN genes were ubiquitously but differentially expressed under phytohormone treatments. For instance, OsFH3 was induced by all phytohormones in the shoots. OsFH5 was significantly induced by jasmonic acid (MeJA), gibberellin (GA), IAA and abscisic acid (ABA) in the shoots. OsFH9 showed higher expression levels when treated with cytokinin (CTK) but was repressed by brassinosteroid (BR), GA, IAA and MeJA. However, it has been noticed that some rice FORMIN genes showed different expression patterns in roots and shoots. Such as, the expression of OsFH6 was induced by BR in the roots, but it was repressed by GA, IAA, ABA and MeJA in the shoots. OsFH7 showed positive expression patterns when treated with ABA, GA and MeJA, but it was repressed by IAA in the roots. In the roots, OsFH10 was upregulated under GA treatment but showed negative expression patterns when treated with IAA, MeJA or BR.

In this study, a total of 17 FORMIN family genes from rice have been identified. Syntenic analysis of the FORMIN gene family in species revealed that FORMIN genes in the plant kingdom were increased during evolution, showing that these genes played a critical role in plants during evolution. As most of the Ka/Ks ratios were less than 1.0, suggesting the purifying selection within the gene family. All these 17 FORMIN genes were divided into two subfamilies based on their protein structure. Meanwhile, structural analysis of the FORMIN gene family showed that just 1 gene (OsFH12 of type II) contained PTEN. As research has shown, PTEN is a potent tumour suppressor found in human cancers[22]. Moreover, the PTENs form dimers in order to bind to the cell membrane[23]. For the plants, the studies of PTEN in moss have shown that the PTEN domain is able to bind to PI(3,5)P2 to recruit For2 to the plasma membrane, and located in discrete cortical dots[24]. OsFH12 contain a PTEN domain, which implies that OsFH12 may possess some funciton which is mediated by the ability of lipid-binding .

The molecular weights and the isoelectric point (pI) of these 17 FORMIN proteins were different (Table 1), Which implied that the difference of rice FORMINs in the physical and chemical properties. Based on the MultiLoc analysis, we found that the FORMIN family genes were localized in various intracellular compartments, including endosomes, cytosol, golgi bodies, endoplasmic reticulum, endoplasmic reticulum, extracellular space and the vacuoles (Table 1). As can be seen, the localization of OsFH5 is in the range of previously reported experimental results, while the results of OsFH1 did not agree with the previous reports[25]. It was shown earlier that OsFH1 was located on the plasma membrane, but in this research was reported located in the cytosol[17]. We could not exclude the possibility that these PM-located FORMINs are recruited to different sites by other proteins or that FORMINs may exhibit variable subcellular localization while at different devleopment stages or under different environmental stimuli. But these results remind us that we cannot ignore the inreliability of the predicted results by bioinformatics method, which may caused by the complexity of what happen in the cells.

Gene duplication is an important mechanism for increasing genetic variability and creating novel genes. In plants, this mechanism is a significant force in increasing gene family size and the functional expansion of genomes. In our study, a total of 14, 6, 6, or 2 homolog gene pairs were identified between rice, maize, barrel medic, L. japonicus and arabidopsis, respectively (Supplementary Figure S2). These results indicated that there was a strong genetic relationship between FORMIN genes in angiosperm. However, according to colinear analysis, there was no collinearity between rice with other species.

Studies have shown that the rice morphometric variation depends on the dynamic changes of the cytoskeleton[10, 26]. Actually, genetic association studies also identified FORMIN’s crucial role in cytoskeleton regulation, such as Bni1p in baker's yeast and AtFH5 in arabidopsis[27, 28]. Moreover, gene expression patterns can provide important insights into the function of plant FORMIN genes. In our study, rice FORMIN gene family members showed differential expression patterns, no matter based on the data from the rice XPro database or qRT-PCR analysis. XPro database showed that except OsFH16, OsFH17, OsFH3 and OsFH15 which were highly expressed in differential tissues, other rice FORMIN genes showed low expression levels in all tissues. qRT-PCR results showed that most genes had higher expression in leaf (OsFH3, OsFH4, OsFH8, OsFH9, OsFH11, OsFH12, OsFH13, OsFH14, OsFH15, OsFH16 and OsFH17). The expression levels of OsFH3 and OsFH13 in the qRT-PCR analysis were consistent with in silico analysis results, with both high expressions in leaf. Recently, the AtROP6 have been demonstrated to control interdigitated growth of pavement cells to form jigsaw-puzzle shapes[29, 30]. The ROP-FORMIN regulation pathways have been clearly clarified in baker's yeast[27]. Thus those genes which were highly expressed in the leaf may be invovled in the pavement cells development. Concurrently, OsFH6 was not expressed in leaf but in leaf sheath, which also suggested the potential function of OsFH6. The function of FORMINs in the pollen tube has been elucidated in arabidopsis, such as AtFH3 and AtFH5 are involved in the regulation of apical actin polymerization and actin array construction in pollen tubes[31]. Our qRT-PCR results showed that OsFH1 and OsFH2 were highly expressed in the immature flower. However, comparing to the mature flower, OsFH1 and OsFH2 were relative less expressed in other tissues or organs, implying that OsFH1 and OsFH2 might play crucial roles in the process of rice flower development.

Nowadays, studies show that actin dynamics is also regulated by phytohormones such as auxin[21]. Thus, plant FORMINs may be involved in this process as well. Our results showed that the expression patterns of OsFH3, OsFH5 and OsFH9 were altered under phyto-hormones treatments in the shoots. The expression patterns of OsFH6, OsFH7 and OsFH10 changed under phyto-hormones treatment in the root. Among the FORMIN genes altered in shoot, the function of OsFH5 has been demonstrated in early studies[25]. Studies show that OsFH5 participates in the regulation of rice morphogenesis by modulating the expansion of diffusely growing cells[20]. In contrast, reported studies have also suggested that IAA regulates rice cell expansion[32]. Moreover, the cis-element analysis showed that the upstream sequence of OsFH5 included IAA-related cis-elements (Supplementary Figure S1). Consequently, OsFH5 was likely involve in the regulation of IAA-mediated cell expansion. Further, through gene promoter element analysis, the upstream sequence of OsFH3 included ABA, MeJA and GA related cis-elements. The OsFH7 also contained MeJA and GA related cis-elements, suggesting that the expression of OsFH3 and OsFH7 might be regulated by phytohormones. Additionally, some environmental stress-related cis-acting elements, like drought-responsive elements, stress-responsive elements and low-temperature-responsive elements, were also found in the rice FORMIN promoter regions. These results indicated that rice FORMIN genes might be closely related to the responses to multiple abiotic stresses. Moreover, all rice FORMINs contained a number of light-responsive elements, suggesting that rice FORMIN genes are important players in the light response of rice.

Overall, in this study, we systematically identified rice FORMIN genes in the rice genome, established a foundation for further investigation of rice FORMIN genes, and provided novel insights into their biological functions.

Identification of FORMIN Sequences

Rice sequence-related information (gene sequences, reference genome, protein sequences, annotation and features) was retrieved from the Ensembl plants database (https://plants.ensembl.org/index.html)[33]. To identify FORMIN proteins in rice, we downloaded the FORMIN homology 2 domain proteins sequences from Pfam databases as reference sequences. Then the Formin proteins were extracted from the rice genome database by using TBtools (version 1.089) and GenomeNet with default parameters[34]. For phylogenetic analysis, the FORMIN proteins sequences of baker's yeast was downloaded from the saccharomyces genome database (SGD) (https://www.yeastgenome.org/)[35]. The red alga, P. patens, spike moss, barrel medic, L. japonicus, maize and arabidopsis were downloaded from the GreenPhyl database (https://www.greenphyl.org/)[36]. All FORMIN proteins used for phylogenetic analysis were verified through alignment against the FORMIN homology 2 domain proteins sequences.

Sequence Analysis

The length of sequences, molecular weights (MW), and isoelectric point (pI) of FORMIN genes were extracted from RAP-DB (https://rapdb.dna.affrc.go.jp/)[37]. The rice FORMIN genes IDs was used to predict the GOterms of Fomrin genes through the online bioinformatics tool GENE ONTOLOGY (http://geneontology.org/)[38]. Furthermore, the subcellular localization of rice FORMIN genes were predicted by MultiLoc (http://www-bs.informatik.uni-tuebingen.de/Services/MultiLoc/)[39]. Then the MEME website (https://meme-suite.org/) was used to identify the conserved motifs of FORMIN genes[40]. To obtain the cis-acting elements information in rice FORMINs promoters, 2-kb upstream sequences of each rice FORMIN genes were intercepted and submitted to the PlantCARE database (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/) for further analysis[41].

Multiple Alignments And Phylogenetic Analysis

For phylogenetic analysis, rice FH2 domain sequences of candidate proteins were extracted and aligned with the FH2 domain sequences of red alga, baker's yeast, P. patens, spike moss, barrel medic, L. japonicus, maize and arabidopsis using ClustaW. The evolutionary tree was constructed by MEGA7.0 with Neighbor-Joining (NJ) method (1000 bootstrap replications) and visualized by using the iTOL (http://itol.embl.de/)[42].

Chromosome Localization and Gene Duplication Analysis

The physical location information of rice FORMIN genes in the chromosomes was obtained from the genome annotation files of rice. The genome sequences information of baker's yeast, P. patens, barrel medic, L. japonicus, maize and arabidopsis FORMIN was downloaded from the Ensembl plants database. Besides, the duplication events within the subgenome was detected by the Multiple Collinearity Scan toolkit (MCScanX) with default settings[43]. Then the TBtools were used to exhibit the chromosomes distribution and the synteny relationship of orthologous FORMIN genes that identified from baker's yeast, P. patens, barrel medic, L. japonicus, maize and arabidopsis. Furthermore, KaKs Calculator 2.0 was employed to estimate the non-synonymous (Ka) and synonymous (Ks) substitution rates of the duplicated FORMIN genes.

Expression Analysis of rice FORMIN Genes in silico

The public available genes expression data of rice in different tissues and in response to different phytohormones were downloaded from the RED database (http://expression.ic4r.org/) and the rice Xpro database (https://ricexpro.dna.affrc.go.jp/)[44, 45]. Then the values were performed treatment of log2 to construct the heatmap by the TBtools.

Expression Analysis of rice FORMIN Genes by qRT-PCR

Oryza sativa spp. Japonica cultivar ‘Dongjin’ was used in this study. The full shape and disease-free seeds were selected and planted on one layer of vinyl mesh that touched the surface of the 1/2 MS solutions (pH 5.8). The growth conditions were set at 28℃ with 16 h light/8 h dark cycle.

After 14 days of cultivation, the plant's whole seeding, shoot, root, shoot apices were selected as samples. After 42 days of cultivation, leaf blade and leaf sheath were selected as samples. Moreover, 80 days cultivated immature flower and 100 days cultivated mature flower also selected as samples.

Total RNA of the samples was extracted using the Plant RNA miniprep kit. Then the RevertAid First Strand cDNA Synthesis Kit (Thermo) was used to synthesize the first strand of cDNA from the high-quality DNAfree RNA. qRT-PCR was performed by using qTOWER3G instrument with SYBR Green. The qRT-PCR reaction conditions were as follows: 95◦C for 2 min; 40 cycles of 95◦C/15 s, 55◦C/15 s, 72◦C/20 s. Every treatment contained three biological replications, and each analysis was repeated by three times. The data from qRT-PCR was analyzed using the 2 − ΔΔCt method, and the expression levels were represented by the mean values of the three replicates. The student’s t test was carried out to determine whether the changes in gene expression were significant. The heatmap of correlations was generated by TBtools. And sequences of the primers used in this study were shown in detail in Supplementary Table S1.

Availability of data and materials

The FORMIN homology 2 domain (PF02181) sequences were downloaded from the Pfam (https://pfam.xfam.org/). The FORMIN amino amcid sequences of alga, P. patens, spike moss, barrel medic, L. japonicus, maize and arabidopsis were downloaded from GreenPhyl (https://www.greenphyl.org/). The FORMIN amino acid sequence of baker's yeast was downloaded from the saccharomyces genome database (SGD) (https://www.yeastgenome.org/). Rice sequence-related information (gene sequences, reference genome sequences, protein sequences, annotation and features) was retrieved from Ensembl plants database (https://plants.ensembl.org/index.html). The information of sequences length, molecular weight (MW), and isoelectric point (pI) of FORMIN genes and proteins were extracted from RAP-DB (https://rapdb.dna.affrc.go.jp/). The evolutionary tree was visualized by using iTOL (http://itol.embl.de/). The GO terms of FORMIN genes were analyzed by the online bioinformatics tool GENE ONTOLOGY (http://geneontology.org/). The subcellular localization of rice FORMIN genes were predicted by MultiLoc (http://www-bs.informatik.uni-tuebingen.de/Services/MultiLoc/). The conserved motifs of FORMIN genes were predicted by MEME website (https://meme-suite.org/). To obtain the cis-acting elements information of rice FORMINs promoters, 2-kb upstream sequences of each rice FORMIN genes were intercepted and submitted to PlantCARE database (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/). The publicly available expression data of rice genes were downloaded from RED database (http://expression.ic4r.org/) and the rice Xpro database (https://ricexpro.dna.affrc.go.jp/). Public access to all databases is open. All data generated or analyzed during this study are included in this published article (and its Additional files).

ABPs: actin-binding proteins; GBD: GTPase Binding Domain; DID: Diaphanous Inhibitory Domain; DAD: Diaphanous Auto-regulatory Domain; PTEN: Phosphatase Tensin; Red alga (Cm): Cyanidioschyzon merolae; P. patens (Pp): Physcomitrella patens; Spike moss (Sm): Selaginella moellendorffii; Barrel medic (Mt): Medicago truncatula; L. japonicus (Lj): Lotus japonicus; Maize (Zm): Zea may; Arabidopsis (At): Arabidopsis thaliana; Rice (Os): Oryza sativa; IAA: auxin; MeJA: jasmonic acid; GA: gibberellin; ABA: abscisic acid; CTK: cytokinin; BR: brassinosteroid;

Funding

This work was supported by the National Natural Science Foundation of China (Grant Nos. 31870383).

Author Contributions

HJ, and CJ conceived and designed the experiments and provided funding for research work. LB, DZY and HS performed most of the experiments. XK, WX and WKS performed some of the experiments. BL and SH wrote the manuscript. All authors contributed to the article and approved the submitted version.

Acknowledgements

At the point of finishing this paper, we sincerely thank all those have lent me hands while my writing this paper.

Ethics approval and consent to participate

Not applicable.

Campellone KG, Welch MD: A nucleator arms race: cellular control of actin assembly. Nat Rev Mol Cell Biol 2010, 11(4):237–251.
Pollard TD, Cooper JA: Actin, a central player in cell shape and movement. Science 2009, 326(5957):1208–1212.
Mathur J, Hülskamp M: Microtubules and microfilaments in cell morphogenesis in higher plants. Curr Biol 2002, 12(19):R669-676.
Chen J, Wang F, Zheng S, Xu T, Yang Z: Pavement cells: a model system for non-transcriptional auxin signalling and crosstalks. J Exp Bot 2015, 66(16):4957–4970.
Zhang Y, Dong J: Cell polarity: compassing cell division and differentiation in plants. Curr Opin Plant Biol 2018, 45(Pt A):127–135.
Wu J, Akhmanova A: Microtubule-Organizing Centers. Annu Rev Cell Dev Biol 2017, 33:51–75.
Lian G, Kanaujia S, Wong T, Sheen V: FilaminA and Formin2 regulate skeletal, muscular, and intestinal formation through mesenchymal progenitor proliferation. PLoS One 2017, 12(12):e0189285.
Martin SG, Chang F: Dynamics of the Formin For3p in Actin Cable Assembly. Current Biology 2006, 16(12):1161–1170.
Young KG, Copeland JW: Formins in cell signaling. Biochimica et Biophysica Acta (BBA) - Molecular Cell Research 2010, 1803(2):183–190.
Vidali L, Gisbergen P, Guérin C, Franco P, Li M, Burkart G, Augustine R, Blanchoin L, Bezanilla M: Rapid formin-mediated actin-filament elongation is essential for polarized plant cell growth. Proceedings of the National Academy of Sciences of the United States of America 2009, 106:13341–13346.
Deeks MJ, Hussey PJ, Davies B: Formins: intermediates in signal-transduction cascades that affect cytoskeletal reorganization. Trends Plant Sci 2002, 7(11):492–498.
Liu R, Linardopoulou EV, Osborn GE, Parkhurst SM: Formins in development: orchestrating body plan origami. Biochim Biophys Acta 2010, 1803(2):207–225.
Cvrcková F, Novotný M, Pícková D, Zárský V: Formin homology 2 domains occur in multiple contexts in angiosperms. BMC Genomics 2004, 5(1):44–44.
Kollárová E, Baquero Forero A, Stillerová L, Přerostová S, Cvrčková F: Arabidopsis Class II Formins AtFH13 and AtFH14 Can Form Heterodimers but Exhibit Distinct Patterns of Cellular Localization. International Journal of Molecular Sciences 2020, 21(1).
Liu W, Sato A, Khadka D, Bharti R, Diaz H, Runnels LW, Habas R: Mechanism of activation of the Formin protein Daam1. Proceedings of the National Academy of Sciences 2007.
Higgs HN, Peterson KJ: Phylogenetic analysis of the formin homology 2 domain. Mol Biol Cell 2005, 16(1):1–13.
Huang J, Kim CM, Xuan Y, Liu J, Kim T, Kim B-K, Han C-D: Formin homology 1 (OsFH1) regulates root-hair elongation in rice (Oryza sativa). Planta 2013, 237.
Yi-Ran TAO Y-ZX, Jia XIE,Wei-Jiang TIAN,Xiao-Qiong ZHANG,Xiao-Bo ZHANG,Qian ZHOU,Xian-Chun SANG,Xiao-Wen WANG: Identification and Gene Mapping of sdb1 Mutant with a Semi-dwarfism and Bigger Seed in Rice. Acta Agronomica Sinica 2018, 44(11):1621–1630.
Sun T, Li S, Ren H: OsFH15, a class I formin, interacts with microfilaments and microtubules to regulate grain size via affecting cell expansion in rice. Scientific Reports 2017, 7(1):6538.
Wang J, Xue X, Ren H: New insights into the role of plant formins: regulating the organization of the actin and microtubule cytoskeleton. Protoplasma 2012, 249(2):101–107.
Scheuring D, Löfke C, Krüger F, Kittelmann M, Eisa A, Hughes L, Smith RS, Hawes C, Schumacher K, Kleine-Vehn J: Actin-dependent vacuolar occupancy of the cell determines auxin-induced growth repression. Proceedings of the National Academy of Sciences 2016, 113(2):452-457.
Lee Y-R, Chen M, Pandolfi PP: The functions and regulation of the PTEN tumour suppressor: new modes and prospects. Nature Reviews Molecular Cell Biology 2018, 19(9):547–562.
Das S, Dixon JE, Cho W: Membrane-binding and activation mechanism of PTEN. Proceedings of the National Academy of Sciences of the United States of America 2003, 100(13):7491–7496.
Gisbergen P, Li M, Wu S-Z, Bezanilla M: Class II formin targeting to the cell cortex by binding PI(3,5)P2 is essential for polarized growth. J Cell Biol 2012, 198:235–250.
Chang S, Yang L, Liang W, Xie Q, Huang G, Wang D, Zhu W, Zhang D: RIPs Interact with OsFH5 and Control the Rice Morphology by Affecting Stability of OsFH5; 2021.
Frank MJ, Smith LG: A small, novel protein highly conserved in plants and animals promotes the polarized growth and division of maize leaf epidermal cells. Curr Biol 2002, 12(10):849–853.
Liu W, Santiago-Tirado FH, Bretscher A: Yeast formin Bni1p has multiple localization regions that function in polarized growth and spindle orientation. Molecular biology of the cell 2012, 23(3):412–422.
Ingouff M, Fitz Gerald J, Guérin C, Robert H, Sørensen M, Van Damme D, Geelen D, Blanchoin L, Berger F: Plant formin AtFH5 is an evolutionarily conserved actin nucleator involved in cytokinesis. Nature cell biology 2005, 7:374–380.
Fu Y, Xu T, Zhu L, Wen M, Yang Z: A ROP GTPase Signaling Pathway Controls Cortical Microtubule Ordering and Cell Expansion in Arabidopsis. Current Biology 2009, 19(21):1827–1832.
Lin D, Ren H, Fu Y: ROP GTPase-mediated auxin signaling regulates pavement cell interdigitation in Arabidopsis thaliana: ROP Signaling Regulates Pavement Cell Interdigitation. Journal of Integrative Plant Biology 2014, 57.
Lan Y, Liu X, Fu Y, Huang S, Yang Z: Arabidopsis class I formins control membrane-originated actin polymerization at pollen tube tips. Plos Genetics 2018, 14(11).
Li G, Liang W, Zhang X, Ren H, Hu J, Bennett MJ, Zhang D: Rice actin-binding protein RMD is a key link in the auxin–actin regulatory loop that controls cell growth. Proceedings of the National Academy of Sciences 2014, 111(28):10377.
Howe KL, Contreras-Moreira B, De Silva N, Maslen G, Akanni W, Allen J, Alvarez-Jarreta J, Barba M, Bolser DM, Cambell L et al: Ensembl Genomes 2020—enabling non-vertebrate genomic research. Nucleic Acids Research 2019, 48(D1):D689-D695.
Chen C, Chen H, Zhang Y, Thomas HR, Frank MH, He Y, Xia R: TBtools: An Integrative Toolkit Developed for Interactive Analyses of Big Biological Data. Mol Plant 2020, 13(8):1194–1202.
Skrzypek MS, Nash RS: Biocuration at the Saccharomyces genome database. Genesis 2015, 53(8):450–457.
Valentin G, Abdel T, Gaëtan D, Jean-François D, Matthieu C, Mathieu R: GreenPhylDB v5: a comparative pangenomic database for plant genomes. Nucleic Acids Res 2021, 49(D1):D1464-d1471.
Sakai H, Lee SS, Tanaka T, Numa H, Kim J, Kawahara Y, Wakimoto H, Yang CC, Iwamoto M, Abe T et al: Rice Annotation Project Database (RAP-DB): an integrative and interactive database for rice genomics. Plant Cell Physiol 2013, 54(2):e6.
Ashburner M, Ball CA, Blake JA, Botstein D, Butler H, Cherry JM, Davis AP, Dolinski K, Dwight SS, Eppig JT et al: Gene Ontology: tool for the unification of biology. Nature Genetics 2000, 25(1):25–29.
Höglund A, Dönnes P, Blum T, Adolph H-W, Kohlbacher O: MultiLoc: prediction of protein subcellular localization using N-terminal targeting sequences, sequence motifs and amino acid composition. Bioinformatics 2006, 22(10):1158–1165.
Bailey TL, Johnson J, Grant CE, Noble WS: The MEME Suite. Nucleic Acids Research 2015, 43(W1):W39-W49.
Lescot M, Déhais P, Thijs G, Marchal K, Moreau Y, Van de Peer Y, Rouzé P, Rombauts S: PlantCARE, a database of plant cis-acting regulatory elements and a portal to tools for in silico analysis of promoter sequences. Nucleic Acids Res 2002, 30(1):325–327.
Letunic I, Bork P: Interactive Tree Of Life (iTOL) v5: an online tool for phylogenetic tree display and annotation. Nucleic Acids Research 2021, 49(W1):W293-W296.
Wang Y, Tang H, Debarry JD, Tan X, Li J, Wang X, Lee TH, Jin H, Marler B, Guo H et al: MCScanX: a toolkit for detection and evolutionary analysis of gene synteny and collinearity. Nucleic Acids Res 2012, 40(7):e49.
Xia L, Zou D, Sang J, Xu X, Yin H, Li M, Wu S, Hu S, Hao L, Zhang Z: Rice Expression Database (RED): An integrated RNA-Seq-derived gene expression database for rice. J Genet Genomics 2017, 44(5):235–241.
Sato Y, Takehisa H, Kamatsuki K, Minami H, Namiki N, Ikawa H, Ohyanagi H, Sugimoto K, Antonio BA, Nagamura Y: RiceXPro version 3.0: expanding the informatics resource for rice transcriptome. Nucleic Acids Res 2013, 41(Database issue):D1206-1213.

No competing interests reported.

AdditionalFigures.pptx
Additional File 1: Figure S1. Cis-acting element analysis in OsFHs promoter regions. Positional distribution of predicted Cis-regulatory element on OsFHs promoters were shown as vertical bars. Promoter sequences (− 2000 bp) of seventeen OsFHs genes were analyzed by using PlantCARE. Legend depicts the colour of individual cis-acting elements. Figure S2. The collinearity analysis of Formin genes between rice and six Species, including Cyanidioschyzon merolae (red alga), Physcomitrella patens (ssp. patens), Selaginella moellendorffii (spikemoss), Medicago truncatula (barrel medic), Lotus japonicus (L. japonicus), Zea may (maize), and Arabidopsis thaliana (Arabidopsis). Gray lines indicated the all collinear blocks and red the syntenic Formin gene pairs. (A) Homology and collinearity analysis between rice and Saccharomyces cerevisiae. (B) Homology and collinearity analysis between rice and red alga. (C) Homology and collinearity analysis between rice and spikemoss. (D) Homology and collinearity analysis between rice and ssp. patens. (E) Homology and collinearity analysis between rice and barrel medic. (F) Homology and collinearity analysis between rice and Arabidopsis. (G) Homology and collinearity analysis between rice and L. japonicus. (H) Homology and collinearity analysis between rice and maize. Figure S3. The protein-protein interaction network as analyzed by String software. An edge was drawn with up to two differently colored lines that represent the existence of the two types of evidence used in predicting the associations. A green line indicates neighborhood evidence, and a blue line indicated co-occurrence evidence.
AdditionalTable1.xlsx
Additional File 2: Table S1 Primers used in qRT-PCR analysis.
AdditionalTable2.xlsx
Additional File 3: Table S2 The protein-protein interaction network as analyzed by String software.

Download PDF

Version 1

posted

You are reading this latest preprint version

Identification And Expression Analysis of FORMIN Gene Family In Rice (Oryza Sativa)

Status:

Version 1

Abstract

Figures

Background

Results

Protein-protein Interaction Prediction

Discussion

Methods

Identification of FORMIN Sequences

Sequence Analysis

Multiple Alignments And Phylogenetic Analysis

Chromosome Localization and Gene Duplication Analysis

Availability of data and materials

Abbreviations

Declarations

Acknowledgements

References

Additional Declarations

Supplementary Files

Status:

Version 1