Genome-wide identification and analysis of TCP family genes in Medicago sativa reveal their critical roles in Na+/K+ homeostasis
BMC Plant Biology volume 23, Article number: 301 (2023)
Medicago sativa is the most important forage world widely, and is characterized by high quality and large biomass. While abiotic factors such as salt stress can negatively impact the growth and productivity of alfalfa. Maintaining Na+/K+ homeostasis in the cytoplasm helps reduce cell damage and nutritional deprivation, which increases a salt-tolerance of plant. Teosinte Branched1/ Cycloidea/ Proliferating cell factors (TCP) family genes, a group of plant-specific transcription factors (TFs), involved in regulating plant growth and development and abiotic stresses. Recent studies have shown TCPs control the Na+/K+ concentration of plants during salt stress. In order to improve alfalfa salt tolerance, it is important to identify alfalfa TCP genes and investigate if and how they regulate alfalfa Na+/K+ homeostasis.
Seventy-one MsTCPs including 23 non-redundant TCP genes were identified in the database of alfalfa genome (C.V XinJiangDaYe), they were classified into class I PCF (37 members) and class II: CIN (28 members) and CYC/TB1 (9 members). Their distribution on chromosome were unequally. MsTCPs belonging to PCF were expressed specifically in different organs without regularity, which belonging to CIN class were mainly expressed in mature leaves. MsTCPs belongs to CYC/TB1 clade had the highest expression level at meristem. Cis-elements in the promoter of MsTCPs were also predicted, the results indicated that most of the MsTCPs will be induced by phytohormone and stress treatments, especially by ABA-related stimulus including salinity stress. We found 20 out of 23 MsTCPs were up-regulated in 200 mM NaCl treatment, and MsTCP3/14/15/18 were significantly induced by 10 μM KCl, a K+ deficiency treatment. Fourteen non-redundant MsTCPs contained miR319 target site, 11 of them were upregulated in MIM319 transgenic alfalfa, and among them four (MsTCP3/4/10A/B) genes were directly degraded by miR319. MIM319 transgene alfalfa plants showed a salt sensitive phenotype, which caused by a lower content of potassium in alfalfa at least partly. The expression of potassium transported related genes showed significantly higher expression in MIM319 plants.
We systematically analyzes the MsTCP gene family at a genome-wide level and reported that miR319-TCPs model played a function in K+ up-taking and/ or transportation especially in salt stress. The study provide valuable information for future study of TCP genes in alfalfa and supplies candidate genes for salt-tolerance alfalfa molecular-assisted breeding.
Alfalfa is the important forage world widely, and is characterized by high quality, large biomass and strong stress tolerance. While abiotic stresses such as salinity stress can severely affect alfalfa development and production. Thus, it is crucial to breed alfalfa varieties with high abiotic tolerance. In 2020, the genome information of alfalfa was published , numerous genes have been identified that may act in response to abiotic stress. Some TFs in alfalfa have been reported responding to salinity, such as Q-type C2H2 zinc-finger protein (C2H2-ZFP) , MADS-box , and SPL family , which provide important genetic resources to breed salinity-resistant alfalfa varieties.
TCP (Teosinte Branched1/ Cycloidea/ Proliferating Cell Factors) gene family was firstly documented in 1999 , they are a group of plant-specific genes encoding TFs (transcription factors) with TCP domain. The TCP proteins are characterized with a 59-amino acid basic helix-loop-helix (bHLH) motif, and are considered to be involved in DNA binding, protein–protein interaction and nuclear targeting . According to the amino acid sequences of the TCP domain, TCPs can be divided into two main classes: class I (also known as TCP-P class or PCF class) and class II (or TCP-C) . TCPs belonging to Class II can be further subdivided into two clades, the CIN (CINCINNATA) and the CYC/TB1 (CYCLOIDEA/TEOSINTE BRANCHED 1) subclades . Generally, the class I genes are mostly involved in promoting cell division and differentiation in diverse biological processes ranging from seed germination, leaf and floral organ development and senescence [9,10,11]. Class II TCP genes are mainly related to the development of lateral organs, part of them participate in plant stress resistance. TCP members belonging to CYC/TB1 clade mainly involved in regulating floral development, shoot branching and organ development [12,13,14]. It has been proved that the mRNAs of several CIN TCPs could be targeted and degraded by microRNA319 (miR319, one kind of small non-coding RNA) [15,16,17]. miR319-TCPs model is an essential genetic regulator in plants and play vital roles in plant development. Such as overexpressing miR319 or repressing its target TCPs both show abnormally wavy rosette leaves and serrated leaves , and miR319-TCP4 has been reported in regulating LOX2, which encode a key enzyme in jasmonate (JA) synthesis, and regulates plant leaf senescence [17, 19].
Recently, there has been an increasing interest in the role of the TCP family genes in plant salt stresses adaptation [20, 21]. For class I TCPs, over-expression of OsTCP19 enhanced salt tolerance through regulating ABA signal transduction . For class II TCPs, the miR319-TCPs model also plays conserved positive roles in Medicago truncatula, switchgrass and creeping bentgrass salt tolerance [23,24,25]. And, it was interested that overexpression miR319 transgenic switchgrass and creeping bentgrass showed higher K+ content under normal condition [24, 25]. It also reported that OsPCF2 potentially activate the expression of OsNHX1, a K+-Na+/H+ antiporter gene induced by salinity . Under salt stress condition, decreasing cytoplasmic Na+ concentration and increasing K+ concentration, a suitable K+/Na+ ratio in the cytoplasm can be obtained, thus preventing cell damage and nutrient deficiency . However, it is largely unknown that whether, and how, TCPs regulate plant K+ content.
In this study, we want to give an insight on TCP family genes in alfalfa, and how they response to Na+-excess and K+-deficiency condition. And, we generated the MIM319 transgenic alfalfa, verified the miR319-MsTCP pathway could affect the salt tolerance of alfalfa by influence the K+ content through physiological experiment of salt stress, and analyzed the possible molecular mechanism.
Materials and methods
Identification of the MsTCP s in alfalfa
MsTCPs protein sequences in alfalfa were obtained from protein annotation file according to Medicago sativa Genome Database via InterProScan (v. 5.17–56.0) , and were confirmed in the Plant Transcriptional Regulatory Map (PlantRegMap) online (http://plantregmap.gao-lab.org/). The obtained MsTCPs’ sequences were applied to SMART (http://smart.embl-heidelberg.de/) to conduct domain analysis to confirm whether belongs to TCP family. The molecular weight (MW) and isoelectric point (PI) of each protein were calculated using ExPASy (http://web.expasy.org).
MsTCPs chromosomal distribution analysis
Information of chromosomal location of MsTCPs and the chromosomal length were obtained from tetraploid alfalfa genome database , and figure of the distribution of TCPs on chromosome was drawn via TBtools.
TCP Phylogenetic and domain analysis of MsTCP family
TCP protein sequences of M. sativa (MsTCPs) with A. thaliana (AtTCPs)  and M. truncatula (MtTCPs)  were used to construct an unrooted phylogenetic tree using MEGA5.0 (https://megasoftware.net). DNAMAN was used to conduct sequences alignment of MsTCPs. Multiple protein sequences alignment was carried out with Jalview software11 (http://www.jalview.org).
Gene structure and cis-element analysis of MsTCPs
The CDS and corresponding genomic DNA sequences of MsTCPs were obtained in the alfalfa genome database. The diagrams of exon, intron and conserved domains of MsTCPs genes were generated using TBtools software .
The upstream sequences (2 kb) of the MsTCPs coding region were retrieved from the alfalfa genomic database and submitted to PlantCARE (http://bioinformatics.psb.ugent.be/webtools/plantcare/html) to identify regulatory elements involved in hormone and stressed responses. Including abscisic acid (ABA)-responsive elements (ABRE), involved in ABA responsiveness; MBS, MYB binding site involved in drought-inducibility; TCA-elements and salicylic acid responsive elements (SARE), involved in salicylic acid responsive; P-box, TATC-box and GARE-motif, involved in gibberellin-responsive element; TGA-element and AuxRR-core, involved in auxin responsive; TGACG-motif and CGTCA-motif, involved in MeJA-responsiveness; low temperature responsive elements (LTR), involved in low-temperature response; and TC-rich repeats, involved in defense and stress response.
Detection of MsTCPs expression patterns in different organs
To detect the expression patterns of MsTCPs in different organs, total RNA from apical meristem (MS), young leaves (YL, top leaves), mature leaves (OL, the fourth leave form the top), young stems (YS, the first internode from the top), mature stems (MS, the fourth internode from the top) and root (R, 5 mm root tips) in alfalfa (Cultivar: Zhongmu NO.1) was extracted using Trizol reagent.
Salt and K+ deficiency treatment
Alfalfa plants were propagated via stem-cutting. 7-week-old plants were transferred into 1/4 Hoagland’s solution (containing 1 mM KCl, 0 mM NaCl) for cultivated for 48 h as preculture. After precultured, plants were transferred into 1/4 Hoagland’s solution containing 200 mM NaCl for 12 h as salt treatment, or 1/4 modified Hoagland’s solution containing 10 μM KCl for 24 h as K+ deficiency treatment. About ten roots (3 cm length from tip upward) were collected at 0, 1, 3, 6, 12 and 24 h (only K+ deficiency treatment) after salt or K+ deficiency treatment to extract RNA, each treatment had 3 biological replications.
Prediction and identification of miR319 targeted MsTCPs
miR319 target site prediction was performed using CDS of candidate MsTCPs via psRNATarget (http://plantgrn.noble.org/psRNATarget). 5' RLM-RACE was used to validate predicted miR319 cleavage sites in MsTCPs experimentally, primers used in this experiment were listed in Table S2. In brief: total RNA was extracted and ligated with the 5’ adaptor ligation RNA (Sangon Biotech, Shanghai, China) by T4 RNA ligase. The ligated product was reverse‐transcribed into the first-strand cDNA using primer complementary to the 5’ adaptor ligation RNA. The cDNA was subsequently PCR‐amplified using GeneRacer 5’primer and MsTCP_GSP_R primer pairs. The PCR products were purified, ligated into the pMD19-T vector and sequenced. Finally, the sequencing results were analyzed to verify the miR319 cleavage site in MsTCPs .
Obtain and identification of transgenic alfalfa plants
The miR319 precursor genes of Arabidopsis, Medicago truncatula and rice were obtained from miRBase database (http://www.mirbase.org/), and were used as templates to blast in genome of alfalfa (https://www.alfalfatoolbox) to obtain MsMIR319 precursor genes. And the miRBase database was used to predict the mature miR319 sequence produced by MsMIR319s. To blocking miR319 expression, we transferred pZh01:MIM319B plasmid into alfalfa by Agrobacterium-mediated transformation as our previous report . Transgenic alfalfa plants were obtained and identified by stem-loop qRT-PCR.
Salt treatment of MIM319 alfalfa
MIM319 transgenic plants and WT plants were propagated by stem-cutting. Two-month old plants were cultured in 1/4 Hoagland’s solution containing 250 mM NaCl for 3 d. Leaves and roots of WT and transgenic plants were collected respectively to measure the concentration of K+ and Na+. Briefly, dried samples were grinded, then about 50 mg of powder was taken into a 15 ml glass test tube with cover, 10 ml of deionized water was added in a boiling water bath and extract for 2 h, fix the volume into a 50 ml volumetric flask, filter and then determine the concentration of Na+ and K+ in the filtrate with flame spectrophotometer. Each treatment had three replications.
MIM319 plants under different level of salt stress
Seedlings grown for 4 weeks via stem-cutting were cleaned and transferred to 100 mL brown bottles, containing 100 mL of the following reagents: NaCl concentrations of 0, 140, 160, 180, 200 mM solution (1/4 Hoagland nutrient solution), then were treated for 48 h (photoperiod 14 h light/8 h dark; temperature 25 °C; humidity 50%).
Prediction of TCP s binding sites
According to the annotation file of alfalfa genome, genes related to K+ up-taking and transportation were selected. The promoters of these genes were analyzed on JASPAR (https://jaspar.genereg.net/) to predict the presence of TCPs binding site. Genes containing TCP binding sites were named after the blast result on NCBI (https://blast.ncbi.nlm.nih.gov/).
RNA extraction and expression pattern detection
Total RNA was extracted using Trizol reagent. One microgram of total RNA was reverse transcription into cDNA following the protocol of a reagent kit (Takara RR047 A), the kit can remove the genomics contamination. For miR319 stem-loop qRT-PCR, One microgram of total RNA was reverse transcription using stem-loop PT primer (Table S1). Using cDNA as template, qRT-PCR reactions were performed using Starlighter SYBR Green qPCR Mix (Beijing Qihengxing Biotechnology Co., LTD, FS-Q1002 kit) with a qTOWER3G (analytik jena). The calculation of the relative expression levels following 2−ΔΔCT method . MsActin was used as an internal control for normalizing. Primers used in qRT-PCR test were listed in Table S1.
All statistical analyses were performed with the IBM SPSS Statistics program (Version 24). Values are presented as the mean ± standard deviation (SD). For multi-group comparison, P values were derived from one-way ANOVA (continuous variables). For all comparisons, P < 0.05 was considered as statistically significant.
Seventy-one MsTCP s genes were identified in alfalfa
Seventy-one MsTCPs genes which have intact TCP domains were obtained from alfalfa genome (Table 1). The validated TCP genes were named from MsTCP1 to MsTCP24 based on the phylogenetic relationship with AtTCPs and MtTCPs, and the lowercase a, b, c, or d were used to distinguish allele genes which located on homologous chromosome (Table 1).
Gene characteristics, including length of CDS (Coding Sequence), length of amino acids, protein molecular weight, and theoretical isoelectric point (pI) were analyzed and listed in Table 1. Based on these data, the length of MsTCP proteins ranged from 107 (MsTCP23b) to 521 (MsTCP22a), and the molecular weight ranged from 11,418.15 kDa (MsTCP23b) to 55,105.21 (MsTCP22a). Furthermore, the MsTCPs were unevenly located on the chromosomes, as shown in Fig. S1. Most TCP genes were located on Chr1 and Chr8, with 6 (MsTCP7/8/11/19/12/21) and 5 (MsTCP6/15/18/4/19) genes, respectively.
Phylogenetic analysis and classification of MsTCPs in alfalfa
In order to elucidate the evolutionary relationship of the TCPs among species, complete protein sequence of 71 MsTCPs, 24 AtTCPs, and 21 MtTCPs were used to construct an unrooted phylogenetic tree. The results showed that 71 MsTCPs can be divided into two subfamilies, they were referred to as Class I and II according to the classification of MtTCPs and AtTCPs. Class I (PCF) contained 34 members, and 37 members were classified into Class II which can be further divided into two subclasses: the CIN (28 members) and CYC/TB1 (9 members) (Fig. 1a). Alignment analysis of MsTCPs’ protein sequences revealed that all the MsTCP proteins contained a conserved basic helix-loop-helix (bHLH) domain (TCP domain) (Fig. 1b). Only the CYC/TB1 subclass members (MsTCP12/1/2/18) include the R domain (Fig. 1b). The results suggested that MsTCPs are as evolutionary conservative as other species.
Gene structure and cis-regulatory elements on MsTCPs’ promoters
To gain more insight to the evolution of MsTCPs gene family on structure, exon/ intron organization of MsTCPs genomic DNA and cis-elements on their promoters were analyzed (Fig. 2). Among non-redundant TCP genes, 17 out of 23 members had no introns. All the members among CYC/TB1 group contained one intron. Furthermore, MsTCP6 and MsTCP15 contained one intron, respectively (Fig. 2b). Exon/ intron organization within allele genes were also analyzed. Their structures were similar except for MsTCP6, MsTCP9 and MsTCP16 (Fig. S2). MsTCP6c/d had no intron, MsTCP9b and MsTCP16c had one intron, were different from the others which may due to the evolutionary changed.
Cis-elements related to phytohormone and stress responses on TCPs’ promoters were also analyzed (Fig. 2c). The varieties and locations of cis-elements on TCPs were manifold, which implied MsTCPs functions in multiple metabolic processes. In a total, 95 cis-regulatory elements related to hormones, with 39 elements involved in the abscisic acid response (ABRE), 18 involved in salicylic acid response (17 TCA-elements and 1 SARE), 13 involved in gibberellin response (6 P-box, 2 TATC-box and 5 GARE-motif), 15 involved in auxin response (10 TGA-element,4 auxRR-core and 1 TGA-box) and 15 involved in the MeJA-response (TGACG-motif/ CGTCA-motif). Besides, there were 35 cis-regulatory elements involved in stress response, with 8 involved in low-temperature response (LTR elements), 9 involved in defense and stress response (9 TC-rich repeats) and 13 involved in drought-inducibility (MBS elements). Notably, 19 TCPs except MsTCP10A/11/22/24 contained ABRE (abscisic acid response element) and the total number reached at 39, which suggested that most of them responding to ABA treatment or abiotic stresses. Among the allele genes, obvious differences were observed on their promoters. Except for MsTCP24, none of them contained identical numbers or varieties of cis-elements on their promoters (Fig. S3), which implied the evolutionary changes in their promoters are widely existed.
MsTCPs of the same subfamily had similar expression patterns in different organs
Expression pattern of MsTCPs were detected by qRT-PCR at different organs in alfalfa, including meristem (MS), young leaf (YL), mature leaf (OL), young stem (YS), old stem (OS) and root tip (R) (Table S3). It should be noticed that due to the highly similarity in sequences between MsTCP1 and MsTCP2, MsTCP16 and MsTCP23, MsTCP10A and MsTCP10B, their expression level cannot be divided through qRT-PCR. As is shown in Fig. 3, each subclasses had their own characteristics in addition to individual genes. MsTCPs of CIN clade were predominantly expressed in mature leaves except for MsTCP23 which mainly expressed in young leaves and young stems, implying these genes may participate in leaf development. MsTCP4, MsTCP16 and MsTCP9 mainly expressed in meristem. MsTCP9, MsTCP16, MsTCP11 and MsTCP8 presented a relatively low expression level in roots. For CYC/TB1 class TCPs predominately expressed at meristems, suggesting they play similar roles in plant developmental processes. Meanwhile, MsTCP18 also had a relatively higher expression level at mature leaves. The TCPs belonging to PCF clade were found expressed specifically in different organs. Such as, MsTCP19, MsTCP22 and MsTCP14 mainly expressed at mature leaves, while MsTCP7, MsTCP21 and MsTCP15 showed relatively high expression level at young stems. Apart from those, MsTCP24 predominantly expressed at young leaves. These results implied that TCPs function in multiple plant development processes. However, divergent functions of MsTCPs in alfalfa are remaining uncovered, and further studies still to be needed to elucidate specific function on each MsTCP gene.
MsTCP s showed different expression pattern after high Na+ treatments
Recent study has reported that TCPs response to salinity stress . And root is the first organ to feel and response to salinity stress . Thus, to decipher how MsTCPs respond to salinity stress, root expression profiles of 23 non-redundant MsTCPs under 200 mM NaCl for 0, 1, 3, 6 and 12 h were analyzed. As is shown in Fig. 4, 16 out of 23 MsTCPs were up-regulated under 200 mM NaCl treatment at first several hours then down-regulated, and reached their peaks within 3 h, except for MsTCP9/11/16/23 belonging to PCF family (Fig. 4a), MsTCP6/24 belonging to CIN family (Fig. 4b), and MsTCP1/2 of CYC/TB1 subclade (Fig. 4c). MsTCP1/2 had the same expression pattern as mentioned above, reached the peak after salt treatment for 12 h. MsTCP9 and MsTCP6 showed a consistent up-regulated pattern. Besides, the expression level of MsTCP11 and MsTCP21 did not change significantly. MsTCP3, MsTCP10A/B, and MsTCP24 showed significant decreased after salt treatment for 24 h. Their different expression patterns suggested that they may work at different stages in response to salinity stress.
Most of MsTCP genes response to K+ deficiency treatment
The wild type alfalfa were treated with 10 μM KCl, then the expression pattern of MsTCPs in roots was tested by qRT-PCR. As the result shown in Fig. 5, most MsTCPs were responded to 10 μM KCl treatment, except for MsTCP7 and MsTCP11 remained stable expression and showed no significant change, while expression pattern of the other MsTCPs were different. Most of them (MsTCP3/8/9/15/16/23/19/21/22) showed an increasing at first several hours then decreasing, and the time they reached their peaks were different (Fig. 5a-c). MsTCP8, MsTCP15 and MsTCP4 reached their peaks at 3 h post of treatment, however, MsTCP9, MsTCP16/23 and MsTCP19 reached the peak in 6 h. MsTCP10A/B had the highest expression level at both 6 and 24 h after treated with low K+ treatment. MsTCP14 and MsTCP6 showed a consistently increasing tendency. Besides, MsTCP5, MsTCP12 and MsTCP13 decreased firstly then increased after treatment in 3 h. MsTCP4 reached two peaks at 1 h and 6 h respectively. Besides, only MsTCP24 remained decreasing under 10 μM KCl treatment. Expression level of MsTCP3/14/15/18 were increased about 10 times after treatment with 10 μM KCl compared to their expression level before treatment. It should be noticed that MsTCP15/19/22 had similar expression pattern under salt treatment and K+ deficiency situation, while MsTCP16/23, MsTCP13 and MsTCP5 showed an opposite expression pattern under NaCl stress and low-concentration of K+ treatment, indicating these genes play dominant roles under stresses of high concentration of NaCl and low concentration of K+.
miR319 post-transcriptional cleaveaged MsTCP3/4/10A/B and repressed MsTCP1/2/5/13
Suppression of some TCPs by miR319 could be a conserved molecular connection among species . To elucidate this relationship within alfalfa, the supposed MsMIR319 sequences in alfalfa genome database were selected that were highly homology with MtMIR319s, AtMIR319s and OsMIR319s, and predicted the mature miR319 sequences in miRBase software. We obtained eleven MsMIR319s and produced three kind of miR319 sequences (Fig. S4a). Non-redundant MsTCPs were searched for the miR319 target sites using psRNATarget, 14 TCPs were found containing a miR319 cleavage site. Ten of them belong to PCF class, three belong to CYC/TBI family, and 1 belongs to CIN family (Fig. S4c). 5′ RLM-RACE was then conducted to detect the miR319 cleavage site in vitro. The result showed the mRNAs of MsTCP10A/B, MsTCP3 and MsTCP4 were directly cleavaged by miR319 between the 10th and 11th bases of miR319 target site with the probabilities of 16/20, 18/20, 13/20 and 18/20, respectively (Fig. 6a). These results suggested that expression of MsTCP10A/B, MsTCP3, and MsTCP4 were post-transcriptionally regulated by miR319. To further illuminate the relationship of miR319 and MsTCPs, we overexpressed a MIM319 gene in alfalfa to blocking in vivo miR319. The stem-loop qRT-PCR results showed that the expression level of miR319 significant decreased in MIM319 transgenic plants (M4 and M6) compared that in WT (Fig. 6b, c). And the expression level of miR319 cleavaged MsTCPs (MsTCP3, 4, 10A/B) showed a significant increase in Ms than that in WT (Fig. 6d). We also found that the expression level of the other TCPs containing miR319 target site but don’t cleavage were changed in MIM319 transgenic plants (Fig. 6d). MsTCP1/2, MsTCP5 and MsTCP13 were up-regulated, while the expression level of MsTCP9 and MsTCP18 were decreased, which uncovered that their transcriptional level were regulated by miR319. The expression level of MsTCPs without miR319 complementary region showed no significant difference between WT and Ms plants (Table S4).
Blocking of miR319 decreased alfalfa resistance ability of salt shock due to lower K+ content in alfalfa
To test the effects of miR319-MsTCPs model on Na+/ K+ content regulation in alfalfa, four-week old seedlings were used to test the salt tolerance under different level of salt stress. As is shown in Fig. S5, MIM319 plants showed significant salt sensitivity compared to WT plants. Then, we analyzed the salt shock resistance of WT and Ms by soaking with 250 mM NaCl for 3 d. As is shown in Fig. 7, both WT and Ms alfalfa began to wilt, while the top leaves of WT plants were less damaged compared to those of MIM319 plants, after treated with 250 mM NaCl for 3 d (Fig. 7a-c). DAB staining assay revealed that more H2O2 was accumulated in MIM319 plants than WT plants. The concentration of K+ in roots of MIM319 plants was significantly lower than that in WT plants (P < 0.05), and gradually decreased with the prolong of salt treatment hours. Within this process, the concentration of K+ in MIM319 plants remained lower compared to WT plants (Fig. 7e). However, the concentration of K+ in WT leaves was stable during salt treatment (P < 0.05). Concentration of Na+ was gradually increased in both WT and MIM319 alfalfa, but no significant difference between WT and MIM319 plants (Fig. 7f). These results indicated that MIM319 plants reduced salt tolerance in alfalfa by the reduction of K+ concentration, which resulted a lower ratio of K+/ Na+ compared to WT plants.
K+ transport genes were up-regulated in MIM319 transgenic plants
We detected the expression level of potassium-related iron transport genes which promoters contain TCP binding sites (Table S5). It can be observed that CNGCs (Cyclic Nucleotide-gated Channels), HAKs (High-affinity K+), and KEA (K+ efflux anti-porter) were up-regulated, which have been reported to be induced in K+ deficiency [32, 33]. CIPK23 (CBL-Interacting Protein Kinase) was also observed up-regulated in MIM319 plants, which can directly binds to the promoter of AKT1 (Arabidopsis K+ channel 1), and improve the influx of K+ . These results elucidated that MIM319 showed a salt sensitivity characteristic due to the K+ deficiency. However, AKT2/3 (K+ channel 2/3) was induced at in MIM319 plants, which has been reported to be inhibited in K+ free solution , which may partly explain the K+ deficiency in MIM319 plants (Fig. 7g).
The TCP transcription factors are widely exist in many monocotyledons and dicotyledons. While the number of them varies among species , for example, 23 and 22 TCP genes were identified in A. thaliana and O. sativa, respectively . 21 MtTCPs were identified in M. truncatula , 42 PvTCPs were identified in switchgrass  and 19 FvTCPs were found in strawberry . The genome of ‘Zhongmu No.1’ alfalfa assembled one set of the chromosomes, while the genome of ‘XinJiangDaYe’ assembled the whole four set of chromosomes. Considering that alfalfa is a tetraploid plant with self-incompatibility, there may be differences among genes located at homologous chromosomes, thus the genome sequence of ‘XinJiangDaYe’ was selected to perform the analysis. In alfalfa, 71 MsTCPs were identified from the genome of tetraploid (Cultivar: XinJiangDaYe), and there were 23 non-redundant MsTCPs. These TCPs anchor on chromosomes unevenly, which was also reported in MtTCPs . The MsTCP gene family were phylogenetically divided into three clades, named as clade PCF, CYC/TB1, and CIN, as that in A. thaliana and M. truncatula [7, 29], which revealed that TCPs in alfalfa was evolutionary conserved. Exon/ intron arrangement of MsTCPs also revealed that the genes in the same class/ clade have similar extron/ intron structure.
TCP gene family can influence multiple pathways related to plant growth (such as leaf development, flower morphogenesis phytohormone biosynthesis, and lateral branching) and also evolved in abiotic stress [19, 26, 38, 39]. To predicted MsTCPs participate in which phytohormone metabolic pathways, cis-elements in MsTCPs’ promoters were analyzed, and hormone response elements and stress response were focused. Intriguingly, most of the MsTCPs’ (19 of 23 non-redundant TCPs) promoter had at least one abscisic acid responsive element (ABRE), which is responsible for ABA-mediated osmotic stresses signaling . Suggested that abiotic stress such as salinity stress would change the expression level of MsTCPs. We also noticed that the cis-elements of allele genes’ promoter changed a lot, which implied the evolutionary changes in the promoters are widely, and resulted in their functional difference.
The expression pattern of MsTCPs at organs were analyzed, and different subclasses of TCPs have their unique expression pattern. CIN-like clade TCPs are involved in regulation of leaf mororphosis, and silencing these genes will lead to an increase of leaf area [18, 41]. Such as BpTCP7-overexpressing in Betula platyphylla resulted promoted ability of reactive oxygen species scavenging under salinity and drought conditions by integrating multiple hormone metabolic pathways . TCPs of CIN clade in alfalfa were also predominantly expressed in mature leaves implying these genes may participate in leaf development. For CYC/TB1 clade, all of them showed high expression level in meristem, which implied their vital functions in floral development and branching process. In chrysanthemums, CYC/TB1 clade TCPs were associated with regulation of floral asymmetry . In Arabidopsis, this clade genes are destabilized by phytoplasma SAP11 effector, resulting in the proliferation of axillary meristems . Specifically, AtTCP1 plays an important role in the longitudinal elongation of petioles, rosettes and inflorescence stems . In M. truncatula, MtTCP1A/1B/12 were specifically expressed in flowers, suggesting that they may have similar function. However, the molecular mechanism of these transcription factors on flower development are needed to be further investigated . In cotton and Arabidopsis, both of TCP12 and TCP18 (also known as BRANCHED1 (BRC1)) are related to branching and axillary bud growth [46, 47], and is also a response factor for spring bud recovery in perennial plants , and can directly bind to a HD-ZIP gene then improve its transcription level, resulting in enhancing the expression of NCED3, and inhibiting bud development . By directly inhibiting the expression of CsPIN3, CsBRC1 inhibit auxin accumulation in axillary buds and inhibit lateral buds growing in cucumber . For CYC/TB1 class TCPs in alfalfa predominately expressed at meristem, suggesting they play similar roles in plant developmental processes, as their functions in other species. Compared with other two types of TCP transcription factors, PCF class showed less tissue-/organ-specific expression patterns, and widely expressed in various tissues, suggesting that PCF class members play various regulatory roles at multiple developmental stages in both Medicago truncula and Medicago sativa.
The expression level of MsTCPs in roots after treated with 200 mM NaCl and 10 μM KCl were tested respectively, to elucidate whether MsTCPs response to salt stress. We noticed that MsTCP9, MsTCP15 and MsTCP22 were significantly induced by 200 mM NaCl treatment, besides, MsTCP3, MsTCP14, MsTCP15 and MsTCP18 were significantly induced by K+ deficiency. The results implied that these MsTCPs may participate in salt stress through K+ up-taking or transportation. It was well known that TCP genes can be post-transcriptionally regulated by miR319 . Recent research has reported that this miR319-TCP model affect multiple development and metabolic pathways. In A. thaliana, miR319 affects leaf development and photosynthesis through TCPs . Besides it also regulates leaf growth and leaf aging through JA synthesis pathway [17, 51]. MIR319 was also found influence the elongation of internodes, which leads to the decreasing of plant height. Besides, miR319-TCPs significantly induced ethylene synthesis and downstream signaling in switchgrass . And under K+ deficiency condition, ethylene stimulates the up-regulation of the low potassium ion marker gene AtHAK5 and improves plant perception of low K+ concentration .
It has been reported miR319-TCPs model functions in salt stress in many species such as Medicago truncula, Panicum virgatum and Solanum lycopersicum [23, 37, 52]. In this study, we identified four MsTCP genes (MsTCP3/4/10A/10B) can be degraded by miR319, and MsTCP3 significantly induced by K+ deficiency. Which we considered as a candidate gene that will regulate the tolerance of alfalfa via influencing the iron balance. Furthermore, MIM319 plants were conducted, and it turned out that salt tolerance was reduced in MIM319 plants compared to wild type alfalfa, which could be caused by the lower content of K+ in root and shoot. We also noticed that K+-deficiency induced genes were upregulated in MIM319 compared to wild-type (WT) plants, such as CNGCs, KEA5, HAKs and CIPK23. Interesting, a K+-efflux channel MsAKT2/3 was up-regulated in MIM319 plants, which has been reported as a down-regulated gene under K+ deficiency. Which may be part of explanation that salt sensitivity and K+ deficiency in MIM319 plants. Therefore, it is possible that miR319-MsTCPs module play a significant role in salt-tolerance by regulating the K+ up-taking and transportation pathway.
In conclusion, we identified 71 (23 non-redundant) MsTCPs in tetraploid alfalfa genome, which located on different chromosome and belong to PCF (37 members), CIN (28 members) and CYC/TB1 (9 members) subfamily. And, MsTCPs of the same subfamily had similar expression patterns in different organs, but with different expression pattern under Na+-excess and K+-deficiency situation, suggesting that MsTCP genes involved in growth and development regulation and keeping the homeostasis of iron under salt tolerance with function redundancy and specificity. Four MsTCPs (MsTCP3/4/10A/10B) were targeted and degraded by miR319 at the post-transcriptional level, and the expression levels of MsTCP1/2, MsTCP5 and MsTCP13 (containing miR319 target site but do not degraded by miR319) were also up-regulated in MIM319 plants. MIM319 plants showed a sensitive to salt stress, and low concentration of K+ in roots and leaves, demonstrating that miR319-TCPs module involved in the regulation of salt stress via K+ up-taking and/ or transportation, at least partly. And, the expression of potassium transported related genes showed higher expression level in MIM319 transgenic plants than that in WT. The study provide valuable information for future study of TCP genes in alfalfa and supplies candidate genes for salt-tolerance alfalfa molecular-assisted breeding.
Availability of data and materials
All data generated or analyzed during this study are included in this published article and its supplementary information files. The datasets analysed during the current study are available in Medicago Analysis Portal (https://v1.legumefederation.org/data/v2/Medicago/sativa/genomes/).
Teosinte Branched1/ Cycloidea/ Proliferating cell factors
CYCLOIDEA/TEOSINTE BRANCHED 1
MYB binding site involved in drought-inducibility
Abscisic acid (ABA)-responsive elements
Salicylic acid responsive elements
Low temperature responsive elements
Polymerase Chain Reaction
Quantitative Real-time Polymerase Chain Reaction
Cyclic Nucleotide-gated Channels
High-affinity K +
K+ efflux anti-porter
CBL-Interacting Protein Kinase 23
Arabidopsis K+ channel
Protein isoelectric point
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We are grateful to Prof. Dayong Li of Beijing Vegetable Research Center (BVRC), Beijing Academy of Agricultural and Forestry Sciences for providing the MIM319 expression vector.
This work was supported by the National Natural Science Foundation of China (32201454) to Yanrong Liu.
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Chromosomal distribution of MsTCP genes. Fig. S2. Structure analysis of allele TCP genes. Fig. S3. Prediction of cis-elements of promoters among allele TCP genes. Fig. S4. The sequences of miR319 in alfalfa and prediction of miR319-targeted MsTCPs. a. The phylogenetic analysis of MsMIR319, MtMIR319, AtMIR319 and OsMIR319 and their mature miR319 sequences. b. Comparison of MIM319 sequence with miR319 in alfalfa. c. Prediction of target regions for miR319 in MsTCPs. Fig. S5. Comparison of MIM319 and WT plants under different level of salt stress. Table S1. Primers used for qRT-PCR. Table S2. Primers used in 5'RLM-RACE. Table S3. The expression profiling of MsTCP genes in different organs. Table S4. The expression level of MsTCPs in MIM319 plants. Table S5. Prediction of the binding region of TCP3 and TCP4 on the promoter of potassium-related iron-transport genes.
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Zhang, M., Qin, S., Yan, J. et al. Genome-wide identification and analysis of TCP family genes in Medicago sativa reveal their critical roles in Na+/K+ homeostasis. BMC Plant Biol 23, 301 (2023). https://doi.org/10.1186/s12870-023-04318-4