Genome-Wide Investigation of the PIF Gene Family in alfalfa (Medicago sativa) Expression Profiles during Development and Stress

doi:10.21203/rs.3.rs-4612280/v1

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Genome-Wide Investigation of the PIF Gene Family in alfalfa (Medicago sativa) Expression Profiles during Development and Stress

https://doi.org/10.21203/rs.3.rs-4612280/v1

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published 02 Sep, 2024

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Background Phytochrome-interacting factors (PIFs) plays an important role in plants as hubs for intracellular signaling regulation. The PIF gene family has been identified and characterized in many plants, but alfalfa (Medicago sativa L.), an important perennial high-quality legume forage, has not been reported on the PIF gene family.

Results In this study, we presented the identification and characterization of five MsPIF gene family in alfalfa (Medicago sativa). Phylogenetic analysis indicated that PIFs from alfalfa and other four plant species could be divided into three groups supported by similar motif analysis. The collinearity analysis of the MsPIF gene family showed the presence of two gene pairs, and the collinearity analysis with AtPIFs showed three gene pairs, indicating that the evolutionary process of this family is relatively conservative. Analysis of cis-acting elements in promoter regions of MsPIF genes indicated that various elements were related to light, abiotic stress, and plant hormone responsiveness. Gene expression analyses demonstrated that MsPIFs were primarily expressed in the leaves and were induced by various abiotic stresses.

Conclusion This study conducted genome-wide identification, evolution, synteny analysis, and expression analysis of the PIFs in alfalfa. Our study lays a foundation for the study of the biological functions of the PIF gene family and provides a useful reference for improving abiotic stress resistance in alfalfa.

Medicago sativa

PIF transcription factors

Genome-wide

Abiotic stress

At present, the PIFs has been identified in many plants such as Arabidopsis thaliana^[1]、tomato (Solanum lycopersicum)^[2]、apple (Malus domestica)[1]^[3]、grape (Vitis vinifera)^[4]、carrot (Daucus carota)^[5]、peanut (Arachis hypogea)^[6]、rice (Oryza sativa)^[7]、maize (Zea mays)^[8]、Brachypodium distachyon^[9]. PIFs are a sub-family of basic helix-loop-helix (bHLH) transcription factors,so all PIFs contain conserved bHLH domains.The bHLH domain contains approximately 60 amino acids, including 15 DNA binding regions of amino acids, while the rest are HLH regions. The former is responsible for binding of PIFs with their target sequences at G-box (5’-CACGTG-3’) or E-box (5’-CANNTG-3’)^[12],thus regulating the expression of target genes.The latter participates in the homodimerization or heterodimerization^[10].The N-terminal region of PIFs contains Active Phytochrome-B (APB)-binding and/or Active Phytochrome-A (APA)-binding motifs, which are interaction sites for phyB and phyA^[11]. Among the plants that have already been reported, most PIFs contain conserved APB domains (EL××GQ), and some PIFs also contain APA domains that are not conserved.

Many reports have already shown that PIFs were the integrator of light and hormones^{[13; 14; 15; 16]}. By linking the plant hormone signaling network, members of the PIF family can work together to help plants cope with various abiotic stresses. PIFs not only play an important role in light signal transduction, but also perceive environmental signals and integrate them with various hormones, circadian rhythms, and other internal signals. They have critical functions in regulating plant seed germination^[17], flowering^[18], fruit development^[19], and plant type regulation^[20], among other growth and development processes. In biological stress response, PIFs also play an important role, not only directly regulating the expression of a series of resistance genes, but also inducing hormone signaling mediated plants such as Jasmonic acid (JA), Auxin (IAA), Salicylic acid (SA), and Gibberellin (GA). In Arabidopsis, many PIFs have been identified and functionally characterized. PIF1 has been demonstrated to play a pivotal role in the inhibition of light-dependent seed germination^[47], as well as in the regulation of key genes involved in chlorophyll biosynthesis, with the objective of optimising the greening process in a direct or indirect manner^[48].PIF3 has been demonstrated to promote hypocotyl elongation in response to ethylene and the biosynthesis of chlorophyll and anthocyanin accumulation under light conditions.^[49], negatively regulates seedling de-etiolation along with other PIFs^{[50, 51]}.

As a high-quality forage, alfalfa is widely used in the animal husbandry industry due to its high protein content and good palatability.Although the PIF gene family has been identified in many plants, it has not been reported in alfalfa.In this study, we identified five MsPIFs and analyzed the basic physicochemical properties, evolutionary tree, sequence structure, structural domains, we also analyzed cis-acting elements and expression patterns to further clarify their possible functions. The results will lay the foundation for further study of these five MsPIFs and the mining of resistance gene resources.

2.1 Identification of the PIF genes in the alfalfa genome

Five genes were identified using HMM profile obtained from the Interpro database, and named MsPIF1 to MsPIF5 based on their positional arrangement on chromosomes. Its amino acid size ranges from 239-680aa, molecular weight (MW) ranges from 26.48 to 74.7kDa, among them, MsPIF1 is the smallest and MsPIF5 is the largest. Their isoelectric point (pI) ranges from 5.33 to 8.76. MsPIFs are unevenly distributed on chromosomes 1 to 8, with one gene on chromosome 1 and the remaining four genes on chromosome 7. All MsPIFs are located on the nucleus.The results obtained in this study are consistent with those in other plants.

Table 1

Characterization of *PIF* genes in *alfalfa*
Gene name	Gene ID	Chromosome location	Gene location	Protein length(aa)	MW (kDa)	PI	Predicted localization
MsPIF1	MsG0180000277.01.T01	Chr1	3708808:3710783	239	26.48	6.29	Nuclear
MsPIF2	MsG0780038030.01.T01	Chr7	38650885:38658723	492	52.98	8.41	Nuclear
MsPIF3	MsG0780038108.01.T01	Chr7	40285873:40293893	492	53.06	8.76	Nuclear
MsPIF4	MsG0780040838.01.T01	Chr7	83219453:83223659	474	52.63	5.33	Nuclear
MsPIF5	MsG0780041453.01.T01	Chr7	91073876:91083647	680	74.75	6.11	Nuclear

2.2 Multiple sequence alignment and phylogenetic analysis of the MsPIF genes

Comparison of PIF genes in alfalfa and Arabidopsis thaliana showed that each MsPIFs has a bHLH structural domain, this sequence is approximately 60aa in length, which is consistent from the length obtained in other studies. The glutamate acid (E) and arginine (R) residues responsible for binding downstream G-box and E-box in the bHLH domain are conserved in MsPIFs. All genes except MsPIF1 have an APB structural domain, But the APB domain in MsPIF5 is incomplete. Only MsPIF5 contains APA structural domain, this is consistent with AtPIF1 and AtPIF3 (Fig. 1).

To investigate the evolutionary relationships of the PIF gene family, a phylogenetic tree of the PIF gene family was constructed in this study based on PIFs identified from four other plant species (Arabidopsis, rice, maize, and peanut). Phylogenetic analysis showed that these genes could be categorized into three groups, PIF1/4/5, PIF2/3/6, and PIF7/8, and the five MsPIFs were dispersed in each of the three groups (Fig. 2). MsPIF5 belonged to PIF2/3/6; MsPIF2 and MsPIF3 were in PIF7/8; MsPIF1 and MsPIF4 were assigned into PIF1/4/5. Moreover, the homology between the PIF gene family in alfalfa and peanut is higher than that in other plants, both of which are leguminous plants.

2.3 Motif composition of PIFs in alfalfa and other plant species

Through TBtools software, we predicted 10 motifs in MsPIFs, named motif 1–10. As shown in Fig. 3, MsPIFs contain 3–9 different motifs. In the PIF gene family of alfalfa, except for MsPIF1, all other genes have motif 3, which represents the APB domain. This is consistent with the results of previous research. Only three motifs, namely motif 1, motif 2, and motif 7, were detected in MsPIF1. All MsPIFs have motif 1 and motif 2, representing the bHLH domain. Moreover, in the PIF7/8 group, all genes except AtPIF7 have motif 10, which may be a characteristic of this group of members. In the PIF2/3/6 group, all genes except AtPIF2, AtPIF6, and MsPIF1 contain motif 5, which can be used to distinguish this group. The PIF1/4/5 group does not contain motif 5, but contains motif 9, which helps to distinguish them. This also demonstrates the reliability of phylogenetic trees.

2.4 Synteny analysis of the MsPIF genes

In order to determine whether replication events occurred in the PIF gene family of alfalfa during evolution, this study used TBtools for gene replication event analysis. As shown in Fig. 4A, two gene pairs were identified, there is no concatenated replication, but there are two instances of fragment replication.

In order to further elucidate the origin and evolutionary relationship of PIF genes, homologous comparisons were conducted between the PIF genes of alfalfa and Arabidopsis. The results in Fig. 4B showed that three MsPIFs and three AtPIFs together formed three gene pairs, indicating a close relationship between MsPIFs and AtPIFs. Indicating the existence of gene replication during the evolution of the gene, and the replication process is relatively conservative.They have a common ancestor.

2.5 Cis-acting elements in alfalfa PIF genes

In order to further explore the potential function of MsPIFs genes, a regulatory region of 2000bp upstream of the promoter was selected to analyze cis-acting elements. The cis-acting elements of the MsPIFs gene can be divided into three groups (Fig. 5), with stress response elements including The results indicate that the main function of the PIF family is light response, while playing an important role in hormone response and stress response.

2.6 Tissue-specific expression of MsPIFs

To investigate the possible functions of MsPIF genes, we used qRT-PCR to measure the expression levels of MsPIF genes in different tissues of alfalfa, including the roots, stems, young leaves, mature leaves and flowers (Fig. 6).The results showed that all MsPIFs expressions can be detected and had similar expression patterns. The expression levels of MsPIF1 and MsPIF5 are low in all tissues. MsPIF3 and MsPIF4 are highly expressed in leaves and stems, especially in mature leaves. Except for MsPIF1, which has a higher expression level in flowers compared to other tissues, all other genes have the lowest expression level in roots and higher expression level in leaves compared to other tissues. The results indicate that the PIF genes plays different roles in alfalfa.

2.7 Expression of MsPIFs in response to abiotic stress in leaves

To evaluate the function of MsPIFs under abiotic stress, the expression patterns of MsPIF genes after drought, cold, salt and alkaline treatments were detected by qRT-PCR (Fig. 7). All MsPIFs are expressed under all abiotic stress treatments, with most genes having the highest expression levels at 6 h and 12 h.

Under drought treatment, MsPIF2, MsPIF3, and MsPIF4 showed similar changing trends, reaching their maximum expression level at 1 h, followed by significant downregulation, and then rebounding at 24 h. Under cold stress, all five genes showed a significant upregulation followed by a significant downregulation trend. MsPIF1 and MsPIF3 reached their maximum values at 3 h, MsPIF2 reached their maximum values at 6 h, and MsPIF4 reached their maximum values at 1 h, which was 3.9 times higher than before treatment. MsPIF5 also reached its maximum value at 1 h. Under salt stress, the expression levels of MsPIF1 and MsPIF4 were low and did not show significant changes, while other genes showed significant changes. Among them, the expression levels of MsPIF2 and MsPIF3 reached their maximum levels after 1 h of treatment, about three times the level before treatment, and then significantly decreased to 0.1 times. Returned to the pre-treatment level at 24 h. Under alkaline stress, gene expression levels reached their maximum levels within 1 h, approximately three times higher than before treatment.

This study identified a total of 5 PIFs and named them MsPIF1 to MsPIF5 based on their chromosomal localization order in alfalfa. Studies have found that there are 8 PIF family genes in Arabidopsis, 6 in rice, 7 in maize, and 13 in peanut, indicating that different species have different numbers of PIFs. Phylogenetic analysis shows that MsPIFs can be divided into three branches (Figure.2), which is similar to the results in maize and rice.

The sequence analysis of MsPIFs indicates that all MsPIFs have conserved bHLH domains. Research has shown that PIFs can bind to downstream gene cis elements through the basic regions of the bHLH domain ^[30]. PhyB is one of the most important photoreceptors in plants, which can interact with the APB domain of PIFs to transduce light signals. In the presentation study, all MsPIFs had a conserved APB domain except MsPIF1, suggesting that MsPIF1 may not physically interact with phytochrome B. Additionally, in the APB domain of MsPIF5, conserved E and Q residues are replaced by S and N, respectively. One MsPIF (MsPIF5) has an APA motif similar to AtPIF1 and AtPIF3, indicating that MsPIF5 may approach AtPIF1 and AtPIF3 on an evolutionary scale. Previous generations have found that all AtPIFs have an APB motif and only PIF1, PIF3 have both an APB and an APA motif. In maize, there are at least seven putative PIFs with a conserved APB motif, of which two (ZmPIF3.1 and ZmPIF3.2) additionally also contain the APA motif, but only the interaction of ZmPIF3.1 and ZmPIF3.2 with ZmphyB1 has been proved now ^[46]. In grape, the loss of the APB site in VvPIF1 may result in the inability of PHYB binding to this protein and affect the transmission of light signals. The PIF gene family has two fragment repetitions in alfalfa itself, and three gene pairs between alfalfa and Arabidopsis, indicating the conservatism of the PIF gene family during evolution. In addition, there are no gene pairs for PIFs in alfalfa and rice, which may be due to differences between dicots and monocots.

The expression of PIF genes are specific in different tissues of plants and have common and unique functions in various developmental and physiological processes. PIFs was initially considered a negative regulator of the light pathway, which promotes the morphological formation of dark growing plants ^[31]. The expression levels of six AtPIFs (PIF1, PIF3, PIF4, PIF5, PIF7, and PIF8) in seedlings and leaves are higher than those in roots, flowers, or fruits ^[32]. The four VvPIFs of grapes are highly expressed in leaves, stems, and tendrils ^[33]. In carrot, Dcpifs are also highly expressed in aboveground tissues (leaves and petioles) that directly receive light^[34]. In this study, the expression levels of MsPIFs were also higher in young and old leaves than in roots, stems, and flowers. This may be related to the response of PIFs to light.

In addition to being negative regulators of photomorphogensis, a growing body of evidence indicates that PIFs act as a signaling hub and play key roles in numerous processes, including anthocyanin synthesis ; resistance to drought ^{[35, 36]}, salt ^[37], and cold ^{[38, 45]}; signaling pathways of plant hormones(GA, BR, and auxin) [39]; and even in regulating plant immunity^[40].The AtPIFs are regulated by hormones and abiotic stress in Arabidopsis. For example, AtPIF4 is upregulated by heat and cold stress and downregulated by the ethylene precursor ACC and auxin. AtPIF3 is induced by ABA and ACC. AtPIF1 is upregulated by treatment with heat and cold and repressed by ABA. AtPIF6 is upregulated by ABA and salt stress. AtPIF5, AtPIF7, and AtPIF8 are repressed by brassinolide, cold, and methyl jasmonate (MeJA), respectively^[21]. Under long-day conditions, AtPIF3, AtPIF4, and AtPIF7 accumulate and repress the CBF genes and CBF-mediated freezing tolerance by binding directly to the G-box present in the promoters of CBFs^{[41, 42]}. Moreover, AtPIF1 and AtPIF5 also negatively regulate freezing tolerance in Arabidopsis.Our data can demonstrate that the expression of MsPIFs can be induced by several abiotic stresses. MsPIFs respond to drought, cold, salt, and alkaline stress.

In the promoter regions of MsPIF genes,we identified cis-elements related to three main categories: light responses, hormone responses, and stress responses(Fig. 5).These findings underscore the pivotal roles of MsPIF genes in both plant development and stress responses.The number of cis-elements related to photoreaction in the promoter of PIFs is the most abundant, which corresponds to the role of PIFs in the light response process. It has been found in Apple that the MdPIF gene is regulated by light mediated circadian rhythms. We also found ABA, GA, IAA, and MeJA response motifs as well as various stress response elements in the promoters of most MsPIF genes, Previous studies have shown that some hormones, such as ABA, GA, and JA, play a key role in regulating plant responses to stress conditions^{[43, 44]}. ZmPIF1 promoter region was rich in drought response (MBS) and ABRE-related elements, and that the expression level of ZmPIF1 was significantly induced by drought and abscisic acid treatments^[38]. In grapes, there is less cis-elements related to ABA response and no cis-elements related to drought response. In alfalfa, there are 8 ABRE elements related to abscisic acid in MsPIF5, and in MsPIF4, there are two drought stress responsive elements, suggesting that these two genes are related to drought stress.

In this study, we identified five PIF genes and classified into three main groups in alfalfa. MsPIFs are randomly distributed on two chromosomes, and two genes come from fragment duplication. The cis-acting elements of MsPIFs genes are involved in hormone regulation, stress regulation, and light response. The results of the regression profile analyses indicated that the MsPIF genes play a pivotal role in alfalfa development and participate in the response to abiotic stress and phytohormone-related signal transduction processes. The comprehensive analysis of PIF family members their involvement in alfalfa growth and development as well as stress response to guide further research into the functions of MsPIFs.

5.1 Identification and characterization of PIF genes in alfalfa

Using the amino acid sequences of all AtPIFs in Arabidopsis as query sequences, a blast search was performed on the genome sequence of alfalfa, where candidate genes were obtained with an E value greater than 1e-5^[22]. Submit the candidate MsPIFs protein to Interpro for domain validation to check for the presence of complete bHLH and APB domains. After removing duplicate sequences and redundant transcripts, five protein sequences encoded by PIF family genes were ultimately obtained. Using the online website ExPASyProtParam (www.expasy. org) to predict the isoelectric point and molecular weight of MsPIFs, WoLFPSORT (http://www.csbio.sjtu.edu.cn/bioinf/Cell-PLoc-2/) .Predict subcellular localization of MsPIFs.

5.2 Multiple sequence alignment and phylogenetic analysis

The identified PIFs protein sequence of alfalfa was compared with the PIF protein sequences of Arabidopsis, maize, rice, and peanut using DNAMAN tool. The genomes of Arabidopsis and other plants are sourced from the Tair and NCBI websites. Then, MEGA7.0 was used to import the alignment file and Neighbor Joining (NJ) was used to construct a phylogenetic tree of PIFs. Among them, the Bootstrap value is set to 1000, and the remaining parameters are set to default values^[23].

5.3 Motif composition of alfalfa PIF gene family members

Using MEME online software (https://meme-suite.org/meme/tools/meme) to predict 5 conserved motif positions for MsPIFs, with a motif count of 10^[26].Conserved domains were analyzed and visualized using CD-Search (https://www.ncbi.nlm.nih.gov/Structure/cdd) ^{[24, 25]}.

5.4 Synteny analysis of MsPIF genes

Using MCScanX software tool to align the entire genome sequence of alfalfa, perform replication analysis on the MsPIFs. Compare alfalfa and Arabidopsis using MCScanX software tool to obtain the collinearity relationship between alfalfa and Arabidopsis.

5.5 Analysis of cis-element within the MsPIF gene promoter

Extract the upstream 2000bp sequence of the starting codon of five members of the PIF family in alfalfa using TBtools software, and then upload it to the PlantCARE online website (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/). Predict and analyze all promoters to obtain cis acting elements^[27]. The cis-acting elements of each gene are arranged by type in Excel software and visualized through TBtools.

5.6 Plant materials and treatments

This study used Medicago sativa L.‘Zhonglu No.1’ as the material.Select alfalfa seeds with uniform morphology and full seeds, cultivate them in vermiculite, and irrigate them with 1/10 Hoagland nutrient solution. At 4 weeks of seed age, plants with uniform growth were treated with 4℃, 15% PEG-6000, 150 mmol/L NaCl, 150 mmol/L NaHCO₃. Samples were collected at 0 h (CK), 1 h, 3 h, 6 h, 12 h, and 24 h after treatment^[28].

5.7 RNA extraction and gene expression analysis

RNA was extracted using CoWin Biotech, Beijing, China,and first stranded cDNA was synthesized using the reverse primer oligo (dT) 10 using the instructions of the HiScript II Q Select reverse transcriptase kit (Vazyme Biotech, Nanjing, China). Through ChamQ ™ Universal SYBR qPCR Master Mix (Vazyme, China) was used for quantitative qRT-PCR. Using the GAPDH gene of alfalfa as an internal reference. The reaction process is as follows: react at 95℃ for 30 seconds; 40 cycles, 95℃ for 5 seconds, 60℃for 34 seconds; And 95℃ for 15 seconds ^{[29, 30]}.

5.8 Statistical analyses

Design qPCR primers based on the cDNA sequences of the existing PIF gene family in alfalfa, using MsActin as an internal reference gene and qRT PCR technology. After 2^−ΔΔCT method was used to analyze the relative expression levels of PIF gene family in various tissues of alfalfa. Each treatment has 3 biological replicates. Draw the final data graph using GraphPad Prism 5.0.

Acknowledgements

This study was financially supported by Breeding Technology Innovation and New Provenance Creation of Cold-resistant and High-yield Alfalfa (2022ZD040120401).

Authors’ contributions

Qianning Liu wrote the manuscript and Jinqiu Yu revised it. Qianning Liu, Jinqiu Yu and Guowen Cui designed the experiments. Qianning Liu and Baiji Wang conducted experiments. Wen Xu and Yuanyu Ying prepared figures. All authors read and approved the final manuscript.

Funding

This research was supported by Breeding Technology Innovation and New Provenance Creation of Cold-resistant and High-yield Alfalfa (2022ZD040120401). The funding body had no influence on the experimental design, data analysis and interpretation, or writing of the manuscript.

Data Availability

The data does not involve sequencing and are listed in the article.

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.

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Genome-Wide Investigation of the PIF Gene Family in alfalfa (Medicago sativa) Expression Profiles during Development and Stress

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Journal Publication

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Abstract

Figures

1 Introduction

2 Results

2.1 Identification of the PIF genes in the alfalfa genome

2.2 Multiple sequence alignment and phylogenetic analysis of the MsPIF genes

2.3 Motif composition of PIFs in alfalfa and other plant species

2.4 Synteny analysis of the MsPIF genes

2.5 Cis-acting elements in alfalfa PIF genes

2.6 Tissue-specific expression of MsPIFs

2.7 Expression of MsPIFs in response to abiotic stress in leaves

3 Discussion

4 Conclusion

5 Materials and methods

5.1 Identification and characterization of PIF genes in alfalfa

5.2 Multiple sequence alignment and phylogenetic analysis

5.3 Motif composition of alfalfa PIF gene family members

5.4 Synteny analysis of MsPIF genes

5.5 Analysis of cis-element within the MsPIF gene promoter

5.6 Plant materials and treatments

5.7 RNA extraction and gene expression analysis

5.8 Statistical analyses

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References

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Status:

Journal Publication

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