Genome-wide Identification of PP2C Genes and Their Expression Profiling in Response to Drought and Cold Stresses in Medicago truncatula.

Type 2 C protein phosphatases (PP2Cs) represent the major group of protein phosphatases in plants and play important roles in various plant processes. In this study, 94 MtPP2C genes were identified from Medicago truncatula and further phylogenetically classified into 13 subfamilies, as supported by exon-intron organization and conserved motif composition. Collinearity analysis indicated that segmental duplication events played a crucial role in the expansion of MtPP2C gene families in M. truncatula. Furthermore, the expression profiles of MtPP2Cs under different abiotic treatments were analyzed using qRT-PCR. Results showed that these MtPP2Cs genes displayed different expression patterns in response to drought, cold and ABA stress conditions and some of the key stress responsive MtPP2Cs genes have been identified. Our study presents a comprehensive overview of the PP2C gene family in M. truncatula, which will be useful for further functional characterization of MtPP2Cs in plant drought and cold stress responses.

Introduction

Reversible phosphorylation of proteins is an important protein modification process that regulates a large number of physiological and biochemical reactions in plants. Phosphorylation and dephosphorylation are catalyzed by protein kinases (PKs) and protein phosphatases (PPs), respectively. According to the specificity of substrates, PPs are divided into serine/threonine protein phosphatase (PSPs) and tyrosine protein phosphatases (PTPs). PSPs are classified into two categories: Category 1 includes PP1, PP2A, PP2B, PP4, PP5, and PP6; while category 2 is PPM (protein phosphatase M), including PP2C and other Mg2+-dependent phosphatases[1,2].

PP2C proteins belong to monomer enzymes and the activity depends on Mg2+ and Mn2+. In eukaryotes, the catalytic domain of PP2C proteins is located at either the N-terminus or the C-terminus[3]. Further research revealed that the regions of catalytic domain in eukaryotic PP2C proteins are relatively conserved, whereas the regions of non-catalytic domain have diverse amino acid sequences[2,3].

PP2Cs are evolutionarily conserved from prokaryotes to higher eukaryotes, having been found in archaea, bacteria, fungi, plants and animals[4]. In plants, PP2Cs form the largest family of phosphatase genes, accounting for 60–65% of all phosphorylases[5,6]. The high proportion of PP2C genes is indicative of their evolutionary significance, requirement and involvement in diverse plant cellular functions[2]. As a major class of protein phosphatases, PP2Cs catalyze dephosphorylation of substrate proteins to regulate signaling pathways and participate in various physiological and biochemical processes in plants. Current studies have shown that PP2Cs play crucial roles in different processes, such as ABA signaling, biotic and abiotic stress responses, plant immunity, K+ nutrient signaling and plant development[2,7].

Drought, salt, and temperature stresses are major environmental factors that affect the geographical distribution of plants in nature, limit plant productivity in agriculture, and threaten food security[8]. Plants evolve a variety of signaling mechanisms to adapt to adverse environments, such as drought, high salt, extreme temperatures and pest attacks. Many studies have shown that some PP2C genes are involved in the regulation of the ABA signaling pathway by modulating the kinase activity of SnRK or MAPK to respond to abiotic stresses[9]. For example, PpABI1A and PpABI1B, the only two subfamily A PP2Cs in moss, are directly involved in ABA responses, including induced vegetative desiccation tolerance[10]. In higher plants, the function of PP2C in abiotic stress is more diverse. For instance, in Arabidopsis, ABI1, ABI2 and HAB1 participate in plant abiotic stress/tolerance by negatively regulating ABA signaling[11-13]. Transgenic studies in maize and Arabidopsis confirmed that ZmPP2C-A10 functions as a negative regulator of drought tolerance as well[9]. Similar results have been obtained from studies in other plants, such as tomato[14], poplar[15], Artemisia annua L[16]., Populus euphratica[17], and sweet cherry[18]. These studies show that subfamily A PP2Cs in Arabidopsis and other plants negatively regulate ABA and stress signaling pathways. On the contrary, BdPP2CA6, a subfamily A PP2C from Brachypodium distachyon, was found to be a positive regulator in both ABA and stress signaling pathways[19]. Another study has identified a potential rice subfamily A PP2C, which regulates ABA signaling negatively and abiotic stress signaling positively[20]. Most subfamily A PP2C members of Arabidopsis participate in stress tolerance via ABA-dependent signaling pathways, but in other studies, some PP2Cs can also regulate plant stress tolerance by ABA-independent signaling pathways, such as OsPPOs from rice[21]. These studies indicate that PP2Cs in different plants have diverse functions in stress signal pathways.

As genome sequencing of more species is completed, the PP2C gene family has been isolated, identified, and characterized in a number of plant species including Arabidopsis[3,22,23], rice[6,22], hot pepper[24], wild soybean[25], maize[26] and Brachypodium distachyon[4]. However, as a model legume plant, the PP2C gene family in M. truncatula has not been analyzed comprehensively and their functions remain elusive at present.

In this study, we identified 94 MtPP2C genes from M. truncatula genome and grouped them into 13 subfamilies. Comprehensive analyses of gene structures, gene duplications, chromosomal distribution, and phylogeny of these MtPP2Cs were further carried out. At the same time, their expression profiles were also investigated by qRT-PCR under drought and cold treatments. The results presented here provide a solid foundation for further functional characterization of MtPP2C genes in this model species.

Result

Genome-wide Identification of PP2C Family Members in M. truncatula

To identify the PP2C genes, we searched the M. truncatula genome database (Plaza3.0 database) using the InterPro PP2C domain “IPR001932” as the key word and found 95 putative PP2C genes. After confirming the presence of PP2C domains using Pfam and Batch CD-search, we found that one putative PP2C gene lacks the PP2C catalytic domain. Therefore, 94 genes were identified as PP2C members in M. truncatula and were named as MtPP2C1 to MtPP2C94, based on their locus ID.

All of the basic information on these 94 MtPP2C genes is provided in Table 1. Sequence analysis revealed that the lengths of the deduced MtPP2C proteins vary from 118 amino acids (MtPP2C71) to 1,256 amino acids (MtPP2C23), with an average of 419 amino acids. The predicted molecular weights (MW) and isoelectric points (pI) range from 13.047 kDa (MtPP2C71) to 133.232 kDa (MtPP2C23) and from 3.80 (MtPP2C23) to 9.82 (MtPP2C84), respectively. Subcellular localization prediction showed that most of the MtPP2C proteins might be located in chloroplasts, nuclei or cytoplasm, followed by mitochondria, extracellular compartments and vacuoles (Table 1).

Table 1

List of identified PP2C genes in M. truncatula with their detailed information and localization.

Locus ID	Gene name	Size (aa)	Mass (Da)	pI	Subcellular localization	Chromosome location
Medtr1g013400	MtPP2C1	337	36552.76	5.02	vacu	chr1:3433635..3439406 reverse
Medtr1g014190	MtPP2C2	396	43773.30	5.08	extr	chr1:3077750..3082294 forward
Medtr1g014640	MtPP2C3	337	36552.76	5.02	vacu	chr1:3407349..3413810 forward
Medtr1g015110	MtPP2C4	553	59927.62	4.59	chlo	chr1:3710663..3716307 reverse
Medtr1g016620	MtPP2C5	344	37956.09	5.62	cyto	chr1:4473372..4477359 forward
Medtr1g019760	MtPP2C6	428	46615.23	5.35	vacu	chr1:6004898..6010964 reverse
Medtr1g022030	MtPP2C7	508	54464.98	6.83	cyto	chr1:6777081..6782301 reverse
Medtr1g028300	MtPP2C8	396	42990.51	6.54	mito	chr1:9502076..9505086 forward
Medtr1g041475	MtPP2C9	274	31191.74	8.83	chlo	chr1:15564924..15567717 reverse
Medtr1g050520	MtPP2C10	654	72818.36	6.28	chlo	chr1:19689741..19697531 reverse
Medtr1g067210	MtPP2C11	375	41560.88	6.90	nucl	chr1:28937740..28940160 reverse
Medtr1g071370	MtPP2C12	1071	121605.42	5.88	vacu	chr1:31666298..31681379 forward
Medtr1g075730	MtPP2C13	278	32484.43	9.48	mito	chr1:33552419..33554806 forward
Medtr1g083690	MtPP2C14	352	39057.09	5.72	nucl	chr1:37239946..37243765 forward
Medtr1g083750	MtPP2C15	423	45503.11	7.53	chlo	chr1:37276915..37280425 reverse
Medtr1g085530	MtPP2C16	892	99368.06	5.86	nucl	chr1:38194845..38201639 reverse
Medtr1g086350	MtPP2C17	390	43078.48	4.81	Nucl,cyto	chr1:38640292..38643291 reverse
Medtr1g106855	MtPP2C18	379	42381.22	6.32	chlo	chr1:48356084..48359019 forward
Medtr1g110210	MtPP2C19	327	35527.82	8.12	cyto	chr1:49705150..49706843 forward
Medtr1g112840	MtPP2C20	397	43841.86	8.18	chlo	chr1:51132582..51136407 forward
Medtr1g115570	MtPP2C21	347	37648.21	5.10	cyto	chr1:52256072..52260436 forward
Medtr1g116260	MtPP2C22	379	41987.94	8.08	mito	chr1:52552351..52555813 forward
Medtr2g008590	MtPP2C23	1256	133232.52	3.80	chlo	chr2:1541912..1549840 reverse
Medtr2g008850	MtPP2C24	281	30779.91	6.00	nucl	chr2:1663259..1666800 forward
Medtr2g020970	MtPP2C25	439	47485.12	5.13	chlo	chr2:7064747..7067687 reverse
Medtr2g033000	MtPP2C26	368	40549.97	4.86	nucl	chr2:12442268..12444743 forward
Medtr2g033910	MtPP2C27	373	41406.27	7.76	chlo, cyto	chr2:12929002..12934577 reverse
Medtr2g040500	MtPP2C28	545	60033.03	4.80	nucl	chr2:17771507..17774251 forward
Medtr2g078760	MtPP2C29	333	36857.77	7.82	chlo	chr2:32964994..32968595 forward
Medtr2g090190	MtPP2C30	470	51809.99	5.01	nucl	chr2:38268714..38271536 reverse
Medtr2g093685	MtPP2C31	219	23980.67	8.21	extr	chr2:39942441..39943100 reverse
Medtr2g435550	MtPP2C32	385	43439.06	6.72	chlo	chr2:13732574..13737504 reverse
Medtr3g031360	MtPP2C33	309	33670.96	5.12	chlo	chr3:26807600..26811666 forward
Medtr3g032590	MtPP2C34	438	49453.31	9.27	chlo	chr3:10298146..10299844 forward
Medtr3g032660	MtPP2C35	432	48461.45	6.88	nucl	chr3:10318461..10320276 forward
Medtr3g032700	MtPP2C36	432	48503.53	7.21	nucl	chr3:10335705..10337392 forward
Medtr3g068200	MtPP2C37	388	42920.03	5.32	nucl	chr3:30835692..30837501 forward
Medtr3g074610	MtPP2C38	282	31073.13	7.75	chlo	chr3:33724920..33727721 forward
Medtr3g091060	MtPP2C39	364	40395.37	6.34	chlo	chr3:41371122..41377528 reverse
Medtr3g101540	MtPP2C40	429	46753.33	5.77	vacu	chr3:46733738..46738711 forward
Medtr3g104710	MtPP2C41	549	59510.52	4.79	chlo	chr3:48269729..48274062 forward
Medtr3g105730	MtPP2C42	299	32301.73	5.11	cyto	chr3:48767036..48770816 reverse
Medtr3g105880	MtPP2C43	362	39957.92	5.12	chlo,nucl	chr3:48831424..48836500 forward
Medtr3g107880	MtPP2C44	381	41655.12	5.96	nucl	chr3:49775982..49778302 reverse
Medtr3g451410	MtPP2C45	177	19629.19	9.00	chlo	chr3:18557087..18557752 forward
Medtr3g464650	MtPP2C46	318	34776.24	6.02	chlo	chr3:26001759..26003904 forward
Medtr3g464700	MtPP2C47	334	36410.85	6.08	chlo	chr3:26016801..26018299 forward
Medtr3g491830	MtPP2C48	390	43503.68	7.06	chlo	chr3:41806436..41810585 forward
Medtr4g007440	MtPP2C49	364	40021.16	5.23	nucl	chr4:1091195..1100704 reverse
Medtr4g013295	MtPP2C50	357	39237.48	6.01	extr	chr4:3704225..3706827 forward
Medtr4g037470	MtPP2C51	479	53223.74	5.11	chlo	chr4:14958614..14963747 forward
Medtr4g063905	MtPP2C52	704	78658.40	5.44	chlo	chr4:23796219..23800131 reverse
Medtr4g076560	MtPP2C53	491	54609.98	5.99	chlo	chr4:29279043..29281997 forward
Medtr4g094208	MtPP2C54	278	30311.06	6.71	nucl	chr4:37425469..37430693 reverse
Medtr4g094542	MtPP2C55	364	40340.89	4.90	chlo	chr4:38196224..38198698 forward
Medtr4g098650	MtPP2C56	779	85337.58	5.43	nucl	chr4:40668226..40674476 forward
Medtr4g113210	MtPP2C57	257	29091.05	7.07	cysk	chr4:46533626..46535392 reverse
Medtr4g113345	MtPP2C58	341	39320.28	8.91	cyto	chr4:46588605..46592821 reverse
Medtr4g113480	MtPP2C59	554	62973.99	5.61	cyto	chr4:46648593..46652101 forward
Medtr4g116420	MtPP2C60	384	42990.49	4.89	nucl	chr4:48228380..48231422 forward
Medtr4g118340	MtPP2C61	399	44042.09	5.43	chlo	chr4:49023234..49028186 forward
Medtr4g119830	MtPP2C62	500	54688.56	5.02	nucl	chr4:49656992..49659929 reverse
Medtr4g120410	MtPP2C63	362	40623.83	8.81	cyto	chr4:49916027..49918669 forward
Medtr4g123080	MtPP2C64	381	42258.97	5.20	nucl	chr4:50809344..50811831 reverse
Medtr4g125810	MtPP2C65	513	56572.79	5.05	chlo	chr4:52217353..52220723 reverse
Medtr5g005810	MtPP2C66	583	65595.22	5.66	chlo	chr5:629189..635487 forward
Medtr5g009370	MtPP2C67	334	36584.56	5.96	cyto	chr5:2222866..2224264 forward
Medtr5g019790	MtPP2C68	450	48234.48	8.32	chlo	chr5:7501168..7504361 reverse
Medtr5g024340	MtPP2C69	379	41209.84	5.71	cyto	chr5:9794141..9796708 reverse
Medtr5g063940	MtPP2C70	282	30692.76	8.26	chlo	chr5:26529712..26533899 forward
Medtr5g065180	MtPP2C71	118	13047.33	8.86	nucl	chr5:27391180..27392171 forward
Medtr5g071550	MtPP2C72	378	41099.37	7.05	chlo	chr5:30372747..30375121 forward
Medtr5g080680	MtPP2C73	391	42953.52	4.98	nucl	chr5:34535790..34538105 forward
Medtr6g081850	MtPP2C74	321	35296.14	8.19	cyto	chr6:30528814..30534664 reverse
Medtr6g087000	MtPP2C75	1072	119581.77	4.94	nucl,cyto	chr6:33528353..33537341 reverse
Medtr7g021530	MtPP2C76	452	49512.02	5.40	nucl	chr76831281..6837332 reverse
Medtr7g025640	MtPP2C77	202	22598.21	6.64	chlo	chr7:8548988..8549923 forward
Medtr7g029240	MtPP2C78	318	46612.52	8.56	chlo	chr7:10320773..10322053 forward
Medtr7g060770	MtPP2C79	555	61760.66	5.77	chlo	chr7:21968314..21971174 reverse
Medtr7g070510	MtPP2C80	447	49886.78	5.31	nucl	chr7:26032600..26035783 reverse
Medtr7g080170	MtPP2C81	502	55957.88	5.80	chlo	chr7:30476473..30479559 forward
Medtr7g081020	MtPP2C82	387	42953.65	8.97	nucl	chr7:30892633..30894709 forward
Medtr7g090530	MtPP2C83	440	49223.49	8.85	nucl	chr7:35628987..35630695 reverse
Medtr7g090540	MtPP2C84	271	30518.97	9.82	nucl	chr7:35632158..35633285 reverse
Medtr7g090550	MtPP2C85	438	49414.62	6.69	cyto	chr7:35635422..35637149 reverse
Medtr7g093240	MtPP2C86	129	14742.86	9.40	mito	chr7:37040877..37041934 forward
Medtr7g100240	MtPP2C87	370	41248.73	8.53	nucl	chr7:40325079..40328089 reverse
Medtr7g112430	MtPP2C88	428	46356.15	7.97	chlo	chr7:46222163..46225753 forward
Medtr7g112490	MtPP2C89	395	44396.30	7.17	mito	chr7:46255834..46259283 reverse
Medtr8g017240	MtPP2C90	373	41209.76	8.51	cyto	chr8:5799043..5800524 reverse
Medtr8g074930	MtPP2C91	392	43542.87	8.16	chlo	chr8:31676759..31682907 reverse
Medtr8g102550	MtPP2C92	402	43717.00	4.90	nucl,cyto	chr8:43176042..43184129 reverse
Medtr8g463130	MtPP2C93	282	30775.81	5.90	cyto	chr8:22192735..22196250 forward
Medtr0015s0140	MtPP2C94	387	43570.61	6.95	chlo	scaffold0015:69714..74393 reverse

To further understand the relationship between MtPP2C genes and AtPP2C genes, we further annotated the Arabidopsis homologous genes of each MtPP2C by Blast search against TAIR (http://www.arabidopsis.org/index.jsp) (Supplementary Table S1).

Chromosomal location and duplication of MtPP2C genes

Based on physical locations on M. truncatula chromosomes, the 94 MtPP2C genes were displayed using the MapInspect software. Ninety-three MtPP2C genes are distributed across all eight chromosomes (Ch1–Ch8), ranging from two to 22 per chromosome (Fig. 1). The number of MtPP2Cs located on each chromosome varies dramatically; chromosomes 1 contains the largest number of MtPP2C family members with 22 genes, whereas the least number was detected on chromosomes 6, containing only two MtPP2C genes. Furthermore, one MtPP2C (MtPP2C94) is located on an unassembled genomic scaffold, thus cannot be mapped to any particular chromosome according to what we currently know about this genome. These results showed that the MtPP2C genes are unevenly distributed on different chromosomes, and that each subfamily gene is also unevenly distributed.

Figure 1

Chromosomal distribution and expansion analysis of MtPP2C genes in M. truncatula. Red lines show duplications between 94 MtPP2C genes.

Previous studies in rice, Arabidopsis and B. distachyon showed that PP2C gene families mainly expanded through whole-genome and chromosomal segment duplications[4,22]. Closely related genes located within a distance of less than 200 kb on the same chromosome are defined as tandem duplications, otherwise they are segmental duplications[27]. In M. truncatula, 25 pairs of paralogous MtPP2C genes were found to be involved in segmental duplication events and no tandem duplication gene pairs were found (Fig. 1). As shown in Fig. 1, these 25 pairs of duplicated MtPP2C genes are distributed on chromosome1, 2, 3, 4, 5 and 7, but not on chromosome 6 and 8. The ratio of Ka/Ks showed that 24 pairs of duplicated MtPP2C genes, except for MtPP2C17/26, have evolved mainly from purifying selection (Supplementary Table S2). Amino acid alignment and phylogenetic analysis indicated that two counterparts of each gene pair are from the same subgroup (Fig. 2 and Supplementary Table S2).

Figure 2

Phylogenetic relationships among 94 MtPP2C genes in M. truncatula. The unrooted phylogenetic tree was generated with MEGA 6.06 program using the full-length amino acid sequences of the 94 MtPP2C proteins by the neighbor-joining (NJ) method with 1,000 bootstrap replicates. Thirteen different subfamilies (A–L) are highlighted with different colored backgrounds.

Phylogenetic analysis

To evaluate the evolutionary relationships of 94 PP2C proteins in M. truncatula, we conducted a phylogenetic analysis using MEGA6.06 based on full-length protein sequences (Fig. 2). At the same time, we constructed another phylogenetic tree to compare the phylogenetic relationships of PP2Cs among Arabidopsis, rice and M. truncatula (Supplementary Fig. S1). Consistent with the previous studies in Arabidopsis and rice[22], all MtPP2C genes are grouped into 13 subfamilies and several independent single branches. As expected, most MtPP2Cs cluster together with those from Arabidopsis because both M. truncatula and Arabidopsis are dicotyledonous plants, while those PP2Cs from rice tend to form independent branches. As shown in Fig. 2 and Supplementary Fig. S1, there is only a little difference between the two phylogenetic trees and most of the MtPP2C proteins fall into the same subfamily. In Fig. 2, MtPP2C66 can be grouped into subfamily H, while MtPP2C71 and MtPP2C10 can be grouped into subfamily I because of relatively high bootstrap support (66% and 50%, respectively), but in Supplementary Figure S1 they cannot be grouped.

As shown in Fig. 2, 87 out of 94 MtPP2C genes are distributed in 13 subfamilies (A-L), and the remaining seven MtPP2C genes, MtPP2C9, MtPP2C10, MtPP2C13, MtPP2C48, MtPP2C66, MtPP2C71 and MtPP2C74, cannot be grouped into any subfamilies. The subfamilies D, E and A are the largest three subfamilies, containing 19, 12 and 9 members, respectively. Subfamily J is the smallest one, including only one gene, MtPP2C12. Moreover, subfamilies C and D as well as subfamilies L and H constitute sister clades in a monophyletic cluster with high bootstrap support (96% and 86%, respectively), suggesting close evolutionary relationships between the respective subfamilies.

As shown in Supplementary Fig. 1, the number of MtPP2C genes in each subfamily is similar among M. truncatula, Arabidopsis and rice except for subfamily D. We found that the number of subfamily D genes in M. truncatula (19) is significantly higher than that of other plants, such as Arabidopsis (9), rice (11), maize (13) and B. distachyon (9)[4,6,22,26]. MtPP2C57, MtPP2C58, MtPP2C59 and MtPP2C86 are grouped into an independent branch, of which no PP2Cs from Arabidopsis and rice exist (bootstrap, 88%). Similarly, the other six genes, MtPP2C34, MtPP2C35, MtPP2C36, MtPP2C83, MtPP2C84 and MtPP2C85 also form an independent branch (bootstrap, 89%). These MtPP2C genes belonging to independent branches may have specific functions in M. truncatula. The remaining six MtPP2C genes from M. truncatula are clustered together with the PP2C genes from Arabidopsis and rice.

Gene structure and conserved motifs distribution analysis

In order to better understand the conservation and diversity of motif compositions and gene structures of MtPP2Cs, the conserved motifs and exon-intron organization of MtPP2Cs were analyzed. By comparing the CDS and the genomic DNA, the MtPP2C gene structures were obtained (Fig. 3). The number of introns is highly divergent, from zero to 19, which is consistent with PP2C genes in Arabidopsis and rice. Of the 94 MtPP2C genes, only four genes (MtPP2C6, MtPP2C31, MtPP2C43 and MtPP2C78) have no introns, whereas MtPP2C12 contains 19 introns. In the same subfamily, most members share similar exon/intron structures, such as intron phase, intron number and exon length (Fig. 3). For example, in the largest subfamily D, 16 MtPP2C genes harbor three introns, with the exception of MtPP2C57 and MtPP2C84, which have two introns, and MtPP2C59, which has five introns. In subfamily F2, all five members have seven introns. A great degree of variation in the number of introns exists in subfamilies I, H, E and K.

Figure 3

The exon-intron structure of MtPP2C genes. Exon-intron analyses of MtPP2C genes were carried out with GSDS. Lengths of exons and introns of each MtPP2C gene are exhibited proportionally. Gene families are grouped and color-coded based on the phylogenetic tree. For all genes, black lines represent introns, yellow boxes represent exons and purple boxes represent UTRs.

The conserved motifs of MtPP2C proteins were analyzed using the software MEME, and 15 distinct conserved motifs were identified (Supplementary Fig. S2). The composition patterns of motifs tend to be consistent with the results from our phylogenetic tree, that is to say, the MtPP2Cs within each subfamily share similar motif compositions, but among different subfamilies, the motif compositions vary (Fig. 4). Motif 1, 2, 3, 4, 6, 7, 8 and 13 are present in most subfamilies, among them, motif 2 is present in 91 MtPP2C proteins except for MtPP2C66, MtPP2C86 and MtPP2C84. In contrast, some other motifs exist only in specific subfamilies. For instance, motif 12 and motif 14 is present only in subfamilies E and D, respectively, while motif 9 is present in both subfamilies F1 and D. These results suggest that the specific functions of different subfamily genes may be due to specific motifs. This indicates that patterns of introns and motifs, which correlate well with the phylogenetic clades, strongly support their close evolutionary relationships among the MtPP2C genes within the same subfamilies.

Figure 4

Conserved motifs of MtPP2C proteins. The conserved motifs in MtPP2C proteins were identified by MEME software. Grey lines represent the non-conserved sequences, and each motif is indicated by a colored box numbered on the right side of the figure. The length of motifs in each protein is presented proportionally.

Cis-element analysis in the promoter regions of MtPP2Cs

Cis-elements in combination with transcription factors regulate the transcription level of a gene. To investigate the possible roles of MtPP2Cs in abiotic stresses, corresponding promoter regions (1.5 kb upstream ATG) of 94 MtPP2C genes was subjected to cis-element analysis by PlantCARE online.

Fourteen putative cis-acting elements were investigated in this study (Supplementary Table S3), including six abiotic stress-responsive (ARE, C-repeat/DRE, HSE, LTR, MBS and TC-rich repeats) and nine hormone-responsive (ABRE, CGTCA-motif, ERE, GARE-motif, P-box, TATC-motif, TCA element and TGA-element) cis-acting elements. Overall, cis-elements responsive to abiotic stresses and hormones are widely present in the promoters of the MtPP2C genes and the number of cis-elements ranges from 3 to 18 (Supplementary Table S4), suggesting that these MtPP2Cs are involved in responses to different stresses in M. truncatula.

Expression Profiles of the MtPP2C Genes in Different Tissues

Sixteen MtPP2C genes (MtPP2C9, 13, 16, 25, 31, 45, 46, 50, 55, 56, 57, 67, 77, 78, 79 and 86) do not have their corresponding probe sets in the dataset, but the expression profiles of the rest 78 MtPP2C genes were analyzed (Supplementary Fig. S3). Different MtPP2C genes show different expression patterns in each tissue. Some genes are highly expressed in all eight tissues, such as MtPP2C20, MtPP2C29, MtPP2C39, MtPP2C73, MtPP2C91 and MtPP2C93. In contrast, the expression of some genes is low in all eight tissues, such as MtPP2C34, MtPP2C35 and MtPP2C36. Some MtPP2C genes show significantly distinct tissue-specific expression patterns across the eight tissues examined. For instance, MtPP2C32 is preferentially expressed in roots but lowly expressed in other seven tissues. In another example, the expression of MtPP2C11 in roots and nodules is much lower than that in the other six tissues, but the expression of MtPP2C5 is exactly the opposite of MtPP2C11. The results revealed that different PP2C genes from M. truncatula might function in different tissues.

Expression Profiles of MtPP2C Genes Under Cold, Drought and ABA Stress

In plants, many PP2Cs play important roles in response to drought and cold stresses. To investigate the expression profiles of MtPP2C genes under different abiotic stress, quantitative real time-PCR (qRT-PCR) analysis was used to examine their transcription levels.

In our study, transcripts of 80 MtPP2C genes could be detected by qRT-PCR (CT vaule ≤ 35), but transcripts of 14 MtPP2C genes was barely detectable (MtPP2C45, MtPP2C50, MtPP2C53, MtPP2C55, MtPP2C57, MtPP2C59, MtPP2C77, MtPP2C78, MtPP2C79, MtPP2C83, MtPP2C84, MtPP2C85, MtPP2C90 and MtPP2C94). As shown in Fig. 5, we found that many MtPP2C genes tested in this study show similar trends under three different treatments, especially under drought and ABA treatments. On the contrary, some genes have different expression patterns under different treatments. Furthermore, the MtPP2C genes with significantly altered expression after treatments (fold change ≥ 2 than controls in all three independent treatments) were selected and listed in Supplementary Table 5.

Figure 5

Relative transcriptional expression levels of MtPP2Cs under different abiotic treatments. Each column indicates a sampling time point, and each row indicates an MtPP2C member. The expression level of the control (at 0 h; marked in gray) in every treatment for each gene is used as the rescaled value when calculating the relative expression levels. The relative expressions are log2 transformed and visualized for heatmap using Graphpad prism 7. The colors vary from blue to red representing the scale of the relative expression levels.

All together, we obtained 24 MtPP2C genes showing significant differences in expression levels under cold stress, including 14 up-regulated and 10 down-regulated genes. Three genes belonging to subfamily B, MtPP2C72, MtPP2C46 and MtPP2C47, were most significantly up-regulated under cold treatment, implying their important roles in the response to cold stress. The expression levels of five genes belonging to subfamily D changed significantly under cold treatment, four (MtPP2C34, MtPP2C35, MtPP2C36 and MtPP2C87) of which were down-regulated and one (MtPP2C18) was up-regulated. Similarly, the expression levels of the four genes belonging to subfamily A changed remarkably, three (MtPP2C4, MtPP2C41 and MtPP2C92) of which were down-regulated and one (MtPP2C8) was up-regulated. In addition, some MtPP2C genes from other subfamilies were also induced or inhibited by cold treatment.

Under drought treatment, 11 MtPP2C genes showed obviously different expression levels, including six up-regulated and five down-regulated genes. The expression levels of five genes belonging to subfamily A, MtPP2C8, MtPP2C37, MtPP2C65, MtPP2C67 and MtPP2C92, were all up-regulated obviously, and another subfamily A MtPP2C genes, MtPP2C73, was also up-regulated but at a lower degree (fold change ≥ 1.5). The expression level of MtPP2C69, which belongs to subfamily G, was the most obviously down-regulated under drought treatment.

Under ABA treatment, 14 MtPP2C genes exhibited different expression levels, including nine up-regulated and five down-regulated genes. The MtPP2C genes with increased expression levels after ABA treatment are highly correlated with those responsive to drought treatment, such as MtPP2C8, MtPP2C37, MtPP2C65, MtPP2C67, MtPP2C67, MtPP2C92 and MtPP2C30.

Among the MtPP2C genes with significantly altered expression levels after different treatments, MtPP2C8 is the only gene that was up-regulated by all three treatments. Unlike MtPP2C8, the expression level of MtPPC92 was increased significantly by drought and ABA treatments, while decreased significantly by cold treatment. The expression levels of some MtPP2C genes changed significantly by two treatments, such as MtPP2C67, MtPP2C73, MtPP2C 37, MtPP2C23, MtPP2C69 and MtPP2C80 under drought and ABA treatment, and MtPP2C34, MtPP2C35 and MtPP2C36 under drought and cold treatment. In addition, the expression level of some genes changed only by one treatment, such as MtPP2C40 by cold treatment. Different expression patterns of MtPP2C genes may indicate different roles in response to different treatments.

Discussion

Based on the completion of M. truncatula genome sequencing[28], many gene families were identified and characterized at the whole-genome level, including CCCH[29], LBD[30], WRKY[31], AP2/ERF[32], Dof [33], GH3[34], CAMTA[35], LEA[36], MAPKKK[37], U-box[38], MYB[39,40] and GRAS[41,42]. In this study, PP2C genes in M. truncatula were comprehensively studied, from genome-wide identification, chromosomal locations, evolutionary relationships, gene structure and conserved motifs analysis to expression patterns under cold and drought stresses.

Compared to other gene families, the PP2C gene family is one of the largest families in the plant kingdom. Genome-wide analyses have identified 80, 90, 91, 88, 104 and 86 PP2C gene family members in Arabidopsis[22], rice[6], tomato, hot pepper[24], maize[26], and B. distachyon[4] genomes, respectively. Evolutionary analysis showed that PP2C genes are divided into 11,12 or 13 groups in different plants. In lower plants, such as Chlamydomonas reinhardtii, Physcomitrella patens and Selaginella tamariscina, the PP2C gene family members are much less than those in higher plants. The increase and expansion of PP2C genes from lower plants to higher plants may correlate with adaptations to complex environmental conditions[43]. Here, we identified 94 MtPP2C genes from M. truncatula and divided them into 13 subfamilies (Table 1 and Fig. 1), consistent with other higher plants, such as tomato and hot pepper[24].

Most proteins in the same MtPP2C subfamily share similar parameters and the number of introns except for subfamily K. Different subfamilies of MtPP2Cs are distinguished from each other in the values of MW and pI (Table 1 and Supplementary Fig. S4). Overall, members of most subfamilies have a more concentrated MW distribution (30–60) and a wider pI distribution (4.5–10). In contrast, members in subfamilies C and L have a wider MW distribution (22.6–119.58) and a concentrated pI distribution (4.94–6.64). Neither MW nor PI distribution is concentrated in the members of subfamilies K and J (Table 1 and Supplementary Fig. S4). Similar to MW, pI, and the number of introns, MtPP2C proteins grouped into the same subfamily exhibit similar motif distributions, suggesting functional similarities for members in the same subfamily.

Subfamilies A and B PP2C genes only exist in plants. Members of subfamily A play a role in ABA-dependent stress responses, while members of subfamily B have been characterized as regulators of MAPK activities[43]. In this study, expression pattern analysis showed that subfamilies A and B exhibit the most prominent responses to abiotic stresses among all 13 MtPP2C subfamilies (Fig. 5 and Supplementary Table S5).

Studies on model organisms Arabidopsis and rice demonstrated that family A PP2C plays an important role in plant response to abiotic stress, especially in the ABA signaling pathway[2,44]. After evolutionary analysis and sequence alignment, nine PP2C genes belonging to family A in M. truncatula were identified. Consistent with reports in other plants, most members in subfamily A in M. truncatula are significantly up- or down-regulated under cold and drought stress. Furthermore, those subfamily A genes significantly up-regulated by drought are induced by ABA as well, indicating that they are regulated by ABA-dependent pathways.

MtPP2C8, MtPP2C37, MtPP2C67 and MtPP2C73, which are homologs of HAI PP2Cs (Highly ABA-Induced1,2,3) in Arabidopsis, are significantly induced by drought and ABA treatment, while MtPP2C8 is also significantly induced by cold treatment (Fig. 5 and Supplementary Table 5). Studies in Arabidopsis have shown that HAI PP2Cs have unique drought resistance functions. HAI PP2Cs have the greatest effect on ABA-independent low water potential phenotypes but have lesser effect on classical ABA sensitivity phenotypes[44].

The expression of MtPP2C92 and MtPP2C65 is increased significantly under drought and ABA treatment, but the expression of MtPP2C92 is decreased under cold treatment (Fig. 5 and Supplementary Table S5). In Arabidopsis, ABI1 (homolog of MtPP2C92) and ABI2 (homolog of MtPP2C65) are two most extensively studied PP2Cs and have been characterized as the main components of the ABA signaling pathway under abiotic stresses and during development[2,43,45]. The function of MtPP2C92 and MtPP2C65 in M. truncatula may be similar to that of ABIs in Arabidopsis, but the different expression patterns after cold treatment may indicate their differences in cold responses.

There are six members of subfamily B PP2Cs in Arabidopsis[22], four of them (AP2C1–4) maintain a kinase interaction motif at the N-terminal region of the proteins and are characterized as MAPK phosphatases[3]. Only three members of subfamily B PP2C (PP2C46, PP2C47 and PP2C72) in M. truncatula were identified. Phylogenetic analysis indicates that they are closely related to AP2C1-4 (Supplementary Fig. S1 and Supplementary Table S1). AP2C1, a homolog in Arabidopsis with MtPP2C46 and MtPP2C47, was reported as a negative regulator of stress-induced MAP kinase cascade by interacting with and inactivating Arabidopsis MPK4 and MPK6. AP2C1 modulates innate immunity and stress hormones such as jasmonic acid and ethylene in Arabidopsis[46]. In alfalfa, MP2C (homolog with AP2C1) functions as a negative regulator of the stress-activated MAPK pathway that is activated by cold, drought, touch, and wounding[47]. AP2C2, a homolog in Arabidopsis with MtPP2C72, is a regulator of stress response signaling, in particular ROS signaling activated by both biotic and abiotic stresses[48]. Expression analysis showed that the expression of MtPP2C46, MtPP2C47 and MtPP2C72 is induced by cold, drought and ABA, especially by cold treatment (Fig. 5 and Supplementary Table S5). In Arabidopsis, AP2C1 expression is strongly induced by cold, drought and wounding, but AP2C2 is slightly induced by these treatments[48]. The above studies indicate that subfamily B PP2C genes in M. truncatula may be regulators of the stress-induced MAP kinase cascade, similar to those in Arabidopsis, but the specific function may be different. In M. truncatula, MtPP2C46, MtPP2C47 and MtPP2C72 may play a vital role in cold responses.

In addition to the PP2C genes from subfamilies A and B, many PP2C genes from other subfamilies have also been reported to respond to abiotic stress in plants. Similar to reports in other plants, our study in M. truncatula also revealed that some MtPP2Cs from other subfamilies are induced by cold and drought. The expression of several genes in subfamily E is significantly altered after treatments, such as MtPP2C89 under cold and ABA treatments (Fig. 5 and Supplementary Table S5). A recent study showed three EGRs (Clade E Growth-Regulating) (homolog of MtPP2C89), which belong to subfamily E PP2C in Arabidopsis, act as negative growth regulators to restrain growth during drought[49]. However, the function of other subfamily PP2C in plant resistance to abiotic stress is poorly understood and needs to be further investigated.

The results of our study establish a foundation for future studies on the functions of MtPP2C genes in plant abiotic response, and provide a basic understanding that may allow us to elucidate the potential functions of MtPP2C genes under drought and cold stresses in M. truncatula.

Methods

Database Searches and Identification of PP2C Genes in M. truncatula

The InterPro PP2C domain “IPR001932” was used to search the Plaza3.0 database (http://bioinformatics.psb.ugent.be/plaza/) in order to identify PP2C candidate genes in M. truncatula[50]. Amino acid sequences (Supplementary Data 1), CDS sequences (Supplementary Data 2) and Genomic sequences (Supplementary Data 3) of PP2C genes in M. truncatula were downloaded from the Phytozome12.1 database (https://phytozome.jgi.doe.gov/pz/portal.html)[51]. All protein sequences were manually checked individually using Pfam (http://pfam.xfam.org/) and the online Batch CD-search (https://www.ncbi.nlm.nih.gov/Structure/bwrpsb/bwrpsb.cgi) to confirm the presence of the PP2C domains[52,53]. All candidate PP2C genes with no PP2C domains were removed.

Proteins of PP2Cs in Arabidopsis and rice were downloaded from the TAIR database (https://www.arabidopsis.org) and the Rice Genome Annotation Project Database (https://rice.plantbiology.msu.edu/), which was described in previous reports[4,22].

Analysis of protein features and chromosomal locations

The Compute pI/MW tool of the ExPASy server (http://web.expasy.org/compute) was used to calculate the molecular weight (MW) and the theoretical isoelectric point (pI) of MtPP2C proteins. The WoLF PSORT program (https://wolfpsort.hgc.jp/) was used to predict protein subcellular localization[54].

According to the starting positions on chromosomes, the MapInspect software was used to draw the chromosomal distribution images of of MtPP2C genes.

Duplications between the PP2C genes were identified and complemented using the PGDD database (http://chibba.agtec.uga.edu/duplication/)[55,56]. The number of nonsynonymous substitutions per nonsynonymous site (Ka) and the number of synonymous substitution per synonymous site (Ks) of duplicated genes were obtained from PGDD database. Ka/Ks < 1 means purifying selection; Ka/Ks = 1 means neutral selection; while Ka/Ks > 1 means positive selection[57].

Phylogenetic tree, gene structure and conserved motifs

The protein sequences of MtPP2C genes were aligned by ClustalW[58] and used for phylogenetic analysis using MEGA6.06[59], and an unrooted phylogenetic tree was constructed using the neighbor-joining (NJ) method with the following parameters: Poisson correction, pair-wise deletion, and 1,000 bootstrap replicates.

The exon-intron structures of MtPP2C genes were determined by comparing the coding sequences and the corresponding genomic sequences on the GSDS website (http://gsds2.cbi.pku.edu.cn)[60].

The MEME software (Version 4.11.4) was used to identify conserved motifs in MtPP2C protein sequences according to the following parameters: -protein, -oc, -nostatus, -mod zoops, -nmotifs 15, -minw 6, -maxw 50[61].

Cis-elements analysis

The 1,500 bp sequences upstream from the initiation codon (ATG) of all MtPP2C genes (Supplementary Data 4) were obtained from Phytozome v12.1[51]. The putative stress and hormone responsive cis-elements in the promoter regions were identified using the PlantCARE (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/) program. The details of six abiotic stress-responsive and nine hormone-responsive cis-elements investigated in this study were list in Supplementary Table 6.

Expression profiling of the MtPP2C genes in different tissues

The expression profile of MtPP2C genes in eight tissues (root, stem, leaf, vegetative bud, petiole, flower, pod and nodule) were analyzed using M. truncatula microarray data[62].

The genome-wide microarray data were obtained from the M. truncatula Gene Expression Atlas (MtGEA) Project website (http://mtgea.noble.org/v2/). The relative expressions were log2 transformed and visualized for heat map using Graphpad prism 7.

Plant materials, growth conditions and abiotic stress treatments

M. truncatula ecotype Jemalong A17 was used in this study. The seeds were first treated with sulfuric acid and washed with sterilized water, then sown in a mixture of peat soil and vermiculite (1:1, V/V). Seedlings were grown at 22–24 °C in a growth chamber with a 16/8 h (day/night) photoperiod until they were used for treatment at eight weeks old. The method of stress treatment is in accordance with Shu’s report[29]. For cold stress treatment, the seedlings were transferred to the 4 °C incubator. For drought stress treatment, the seedlings growing under normal conditions were watered with 300 mM mannitol solution. For ABA treatment, the seedling leaves were sprayed with 100 μM ABA solution. The seedlings were harvested at 0, 1, 3 and 12 hours after treatment. For each treatment, five randomly chosen whole seedlings were pooled to form a biological replicate. All samples were frozen immediately in liquid nitrogen after harvest and stored at −80 °C until used for RNA extraction.

Expression analysis of MtPP2C genes response to abiotic stress

Total RNA was isolated from all of the samples using the total RNA extraction kit (Tiangen, China). The quality and quantity of RNA was evaluated by agarose gel electrophoresis and Quawell micro volume spectrophotometer (Q5000, USA), respectively. Then, 1 µg of total RNA after DNase I digestion was reverse transcribed into cDNA using the PrimeScript™ II 1st Strand cDNA Synthesis Kit (TaKaRa, Japan).

The cDNA was amplified using LightCycler 480 SYBR Green Master, with a Roche LightCycler 480 Real Time PCR system (Roche, Switzerland). The thermal cycling program was 95 °C for 30 s, followed by 40 cycles of 95 °C for 5 s, 60 °C for 30 s and 72 °C for 15 s. The melting curves were analyzed at 60–95 °C after 40 cycles. All qRT-PCRs were carried out for three technical replicates. The relative expression levels of MtPP2C genes were calculated according to the method of Livak and Schmittgen[63]. MtActin (Medtr2g008050) and MtGapdh (Medtr3g085850) were used as reference genes. The primers used in this study were listed in Supplementary Table S6. The relative expressions were log2 transformed and visualized for heat map using Graphpad prism 7.

Supplementary Figures

Supplementary Tables

Dataset