Identification and characterization of MADS-box gene family in flax, Linum usitatissimum L. and its role under abiotic stress

Jianyu Lu; Hanlu Wu; David Michael Pitt; Xinyang Liu; Xixia Song; Hongmei Yuan; Yuntao Ma; Shuyao Li; Zhenyuan Zang; Jun Zhang; Michael K Deyholos; Jian Zhang

doi:10.1016/j.isci.2024.111092

. 2024 Oct 1;27(12):111092. doi: 10.1016/j.isci.2024.111092

Identification and characterization of MADS-box gene family in flax, Linum usitatissimum L. and its role under abiotic stress

Jianyu Lu ¹, Hanlu Wu ¹, David Michael Pitt ¹, Xinyang Liu ¹, Xixia Song ², Hongmei Yuan ², Yuntao Ma ⁴, Shuyao Li ¹, Zhenyuan Zang ^1,^∗, Jun Zhang ^1,^∗∗, Michael K Deyholos ^3,^∗∗∗, Jian Zhang ^1,^3,^5,^∗∗∗∗

PMCID: PMC11607607 PMID: 39618497

Summary

MADS-box transcription factors play critical roles in plant development and stress responses. In this study, we identified 114 LuMADS genes in flax (Linum usitatissimum L.) and analyzed their phylogenetic relationships, gene structure, conserved motifs, miRNA targets, and expression patterns. We classified 45 LuMADS proteins as type I and 69 as type II, further subdivided into 11 subfamilies. Notably, lus-miR396 and lus-miR156 were identified as primary miRNA targets. Cis-acting elements in the promoter regions suggested roles in hormone and stress responses. Expression analysis revealed type II LuMADS genes were associated with floral organ formation and abiotic stresses such as cold, drought, and salt. This research provides insights for improving flax stress resistance and breeding.

Subject areas: Biological sciences, Molecular biology, Physiology, Plant biology

Graphical abstract

Highlights

•
45 type I and 69 type II MADS-box genes were identified in flax
•
Lus-miR396 and lus-miR156 are the primary miRNAs targeted by the LuMADS gene family
•
Type II MADS-box proteins possess more conserved domains than type I proteins
•
11 LuMADS genes show significant response to cold stress

Biological sciences; Molecular biology; Physiology; Plant biology

Introduction

MADS-box proteins are transcription factors found broadly in eukaryotic organisms including animals, plants, and fungi, and regulate their growth, reproduction and other processes. MADS is an acronym formed from the family’s founding members: the yeast MCMI gene¹; the Arabidopsis AGAMOUS gene²; the snapdragon DEFICIENS gene; and the human SRF gene.³^,⁴ Based on sequence and domain organization, MADS-box family genes can be categorized as either type-I or type-II.⁵ Type-I MADS-box genes usually have 1–2 exons and an SRF-like MADS conserved domain, which can be further divided into four subfamilies: Mα, Mβ, Mγ, and Mδ.⁶ Type II usually contains 6–7 exons and contains 4 conserved domains: M (MADS-box), I (intervening), K (keratin-like), and C (C-terminal), so it is also called MIKC gene.⁷ Among them, the M domain is a MADS-box domain composed of about 60 amino acids. It is usually located at the N terminus of MADS-box transcription factors and specifically binds DNA CArG motifs (CC[A/T]6GG).⁸ The K domain is a semi-conserved segment of about 70 amino acids that is involved in the interaction between proteins. The I domain is a hydrophilic, non-conserved sequence located between the MADS domain and the K domain, consisting of 31–35 amino acids. The C domain is the least conserved, hydrophobic amino acid sequence, and is located downstream of the K domain. This type of gene can be further subdivided into subfamilies such as FLC, SEP, SVP, SOC1, SHP, AP3/PI, and ANR1.⁹

The MADS-box family is one of the most widely studied superfamilies in plants and plays an important role in plant growth and development. At present, type-I MADS-box genes are not as well-characterized as type-II. In Arabidopsis, research has focused on development of female gametophytes, embryos, and seeds.¹⁰ The type-II MADS-box genes are an important part of the ABCDE model of floral organogenesis.¹¹ The rice MADS-box genes SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1 (SOC1), SHORT VEGETATIVE PHASE (SVP), AGAMOUS-LIKE 24 (AGL24) and SEPALLATA4 (SEP4) genes jointly regulate the differentiation of the branch and stem meristems of rice inflorescences,¹² while another rice MADS-box gene Paniclephytomer 2 (PAP2), positively regulates the formation of spikelet meristem.¹³ Arabidopsis AGAMOUS-LIKE 67 (AGL67) is an example of a MADS-box gene involved in regulating seed germination.¹⁴

The MADS-box gene family is not only involved in regulating plant growth and development, but also plant stress responses.¹⁵ OsMADS26 has a negative regulatory effect on disease resistance and drought resistance in rice,¹⁶ indicating that it may be involved in various stress responses.¹⁷ Tomato SlMBP8 and SlMBP11 genes both have significant salt tolerance functions.¹⁸ The seed germination rate of the ZMM7-L gene in maize transgenic plants under NaCl conditions was significantly lower than that of the control group, indicating that the ZMM7-L gene may negatively regulate the salt tolerance of maize by inhibiting seed germination.¹⁹ Knocking out the SlMADS23-like gene in tomatoes reduces the plant’s resistance to cold stress, proving that the SlMADS23-like gene positively regulates tomato low-temperature stress response.²⁰ MADS-box transcription factors play important functions in plant stress resistance. However, the specific biological functions of many MADS-box transcription factors are still unclear.

Flax (Linum usitatissimum L.) is an ancient crop that is cultivated around the world, particularly in temperate regions.²¹ According their use, flax varieties can be divided into three categories: petroleum, fiber, and dual-use.²²^,²³ Flaxseed is rich in nutrients, such as lignans, dietary fiber, and α-linolenic acid, which is one of the omega-3 fatty acids necessary for the human body.²⁴ So far, there are no reports about flax MADS-box genes. This study systematically characterized the flax MADS-box family genes through bioinformatics methods, analyzing their evolution and distribution, physical and chemical properties, sequence characteristics, phylogeny, promoter cis-acting elements, protein interaction network, and gene expression patterns. Specific MADS-box gene can respond to low temperature and drought stress conditions, which provides a basis for further research on the biological functions of flax MADS-box genes. This is the first comprehensive research report on flax MADS-box genes.

Results

Identification of MADS-box gene family members in flax

Based on the SRF-TF domain (PF00319) and Kbox domain (PF01486) Hidden Markov Model (HMM) profiles, a total of 114 MADS box genes were identified in the flax genome (longya10), and were named LuMADS1-LuMADS114 according to their chromosomal positions (Table 1). By analyzing the physical and chemical properties of their predicted proteins, the longest proteins found were LuMADS31 and LuMADS102, each containing 390 amino acids. The shortest protein was LuMADS47, which contained only 47 amino acids. The molecular weight of LuMADS protein ranged from 6.96 to 44.24 kDa. Among all the proteins encoded by the LuMADS gene family, 78% of the proteins had a pI greater than 7, proving that most of the proteins encoded by the LuMADS family genes contain an abundance of basic amino acids. The Instability Index ranged from 28.87 to 81.05, of which 13.64% were classified as “Stable”. The aliphatic index of LuMADS proteins ranged from 62.59 to 107.61. Hydrophilicity analysis predicted that all LuMADS proteins are hydrophilic. Subcellular localization prediction results showed that except for LuMADS67, which is predicted to be localized in cell wall and chloroplast, the other LuMADS genes are all in predicted to be expressed in the nucleus.

Table 1.

Identification and characterization prediction of LuMADS gene in Linum usitatissimum L

Gene	Gene ID in genome	Group	Length (aa)	Mol.wt. (KDa)	PI	Instability Index	Aliphatic Index	Grand Average of Hydropathicity (GRAVY)	Subcellular Localization
LuMADS1	L.us.o.m.scaffold34.26	SEP	219	25.18	9.33	43.61	92.65	−0.497	Nucleus
LuMADS2	L.us.o.m.scaffold34.128	AP1	263	30.87	8.71	67.58	76.08	−0.977	Nucleus
LuMADS3	L.us.o.m.scaffold34.129	SEP	236	26.77	8.68	33.77	87.16	−0.656	Nucleus
LuMADS4	L.us.o.m.scaffold34.193	Mδ	361	41.03	6.47	50.00	71.86	−0.687	Nucleus
LuMADS5	L.us.o.m.scaffold88.47	Mγ	308	33.42	9.28	40.71	63.73	−0.563	Nucleus
LuMADS6	L.us.o.m.scaffold91.100	AP1	239	27.50	8.66	63.06	75.48	−0.960	Nucleus
LuMADS7	L.us.o.m.scaffold91.102	SEP	219	24.92	6.92	46.97	79.68	−0.605	Nucleus
LuMADS8	L.us.o.m.scaffold1.366	AP3	228	26.47	9.41	42.99	79.96	−0.669	Nucleus
LuMADS9	L.us.o.m.scaffold37.134	STK	249	28.30	9.10	54.69	78.39	−0.694	Nucleus
LuMADS10	L.us.o.m.scaffold55.76	Mα	204	22.62	9.17	32.36	70.78	−0.661	Nucleus
LuMADS11	L.us.o.m.scaffold22.214	ANR1	144	16.57	9.80	43.97	95.49	−0.421	Nucleus
LuMADS12	L.us.o.m.scaffold22.267	Mα	254	28.33	9.26	41.21	69.06	−0.684	Nucleus
LuMADS13	L.us.o.m.scaffold50.188	STK	256	28.97	9.44	62.23	68.98	−0.968	Nucleus
LuMADS14	L.us.o.m.scaffold50.34	Mα	187	21.15	9.88	47.81	66.26	−0.674	Nucleus
LuMADS15	L.us.o.m.scaffold41.292	AP1	250	28.88	8.66	68.83	81.12	−0.816	Nucleus
LuMADS16	L.us.o.m.scaffold59.135	Mδ	378	42.94	6.37	49.35	76.61	−0.692	Nucleus
LuMADS17	L.us.o.m.scaffold59.153	Mα	202	22.93	7.06	34.62	66.19	−0.724	Nucleus
LuMADS18	L.us.o.m.scaffold113.163	SEP	259	29.85	6.51	50.07	77.26	−0.854	Nucleus
LuMADS19	L.us.o.m.scaffold113.161	FLC	182	21.43	9.25	38.50	90.55	−0.591	Nucleus
LuMADS20	L.us.o.m.scaffold257.23	Mδ	357	40.04	5.88	50.62	74.03	−0.595	Nucleus
LuMADS21	L.us.o.m.scaffold347.27	AP1	261	29.72	8.65	68.72	79.96	−0.779	Nucleus
LuMADS22	L.us.o.m.scaffold321.5	STK	284	33.09	9.54	44.91	84.54	−0.696	Nucleus
LuMADS23	L.us.o.m.scaffold320.20	Mγ	173	20.08	6.93	43.32	79.48	−0.939	Nucleus
LuMADS24	L.us.o.m.scaffold20.353	Mβ	162	17.98	7.73	34.67	90.93	−0.519	Nucleus
LuMADS25	L.us.o.m.scaffold160.21	Mγ	237	27.36	7.06	55.33	81.05	−0.645	Nucleus
LuMADS26	L.us.o.m.scaffold31.244	AP1	273	31.94	6.99	71.96	72.12	−0.969	Nucleus
LuMADS27	L.us.o.m.scaffold31.242	SEP	259	29.97	8.57	40.91	79.46	−0.846	Nucleus
LuMADS28	L.us.o.m.scaffold31.175	Mδ	283	32.15	6.32	45.63	83.71	−0.669	Nucleus
LuMADS29	L.us.o.m.scaffold139.126	Mα	193	21.32	9.73	70.70	75.28	−0.565	Nucleus
LuMADS30	L.us.o.m.scaffold201.69	Mα	307	33.74	5.83	42.27	71.79	−0.526	Nucleus
LuMADS31	L.us.o.m.scaffold64.270	Mβ	390	44.24	6.27	46.05	64.31	−0.671	Nucleus
LuMADS32	L.us.o.m.scaffold4.55	Mβ	73	8.42	9.55	46.31	86.71	−0.514	Nucleus
LuMADS33	L.us.o.m.scaffold32.134	Mγ	254	28.31	6.46	35.58	65.63	−0.632	Nucleus
LuMADS34	L.us.o.m.scaffold32.124	Mγ	258	28.78	6.96	31.39	68.02	−0.620	Nucleus
LuMADS35	L.us.o.m.scaffold214.67	SOC1	236	27.20	8.90	67.67	78.09	−0.751	Nucleus
LuMADS36	L.us.o.m.scaffold214.68	SOC1	246	28.17	8.50	55.57	78.50	−0.763	Nucleus
LuMADS37	L.us.o.m.scaffold109.95	ANR1	229	26.30	8.59	46.40	83.84	−0.819	Nucleus
LuMADS38	L.us.o.m.scaffold17.323	PI	219	25.42	9.00	60.44	76.62	−0.804	Nucleus
LuMADS39	L.us.o.m.scaffold2.211	Mα	260	28.85	7.80	40.21	69.08	−0.519	Nucleus
LuMADS40	L.us.o.m.scaffold135.117	Mβ	193	21.26	5.09	45.49	72.33	−0.741	Nucleus
LuMADS41	L.us.o.m.scaffold15.144	ABS	256	30.32	8.62	67.07	75.78	−0.889	Nucleus
LuMADS42	L.us.o.m.scaffold212.41	Mδ	251	28.62	6.45	69.93	73.39	−0.669	Nucleus
LuMADS43	L.us.o.m.scaffold206.25	ANR1	240	27.66	8.85	44.71	83.25	−0.872	Nucleus
LuMADS44	L.us.o.m.scaffold60.141	PI	261	30.60	8.43	58.47	72.87	−0.852	Nucleus
LuMADS45	L.us.o.m.scaffold68.22	SOC1	226	25.08	9.10	42.83	83.27	−0.478	Nucleus
LuMADS46	L.us.o.m.scaffold70.21	SEP	253	29.30	9.27	49.56	83.68	−0.672	Nucleus
LuMADS47	L.us.o.m.scaffold70.22	FLC	62	6.96	9.85	64.19	84.84	−0.327	Nucleus
LuMADS48	L.us.o.m.scaffold70.123	AP1	267	31.39	8.72	70.46	73.48	−1.030	Nucleus
LuMADS49	L.us.o.m.scaffold70.125	SEP	230	26.61	9.34	40.83	107.61	−0.371	Nucleus
LuMADS50	L.us.o.m.scaffold70.196	Mδ	361	41.28	8.03	48.79	76.98	−0.674	Nucleus
LuMADS51	L.us.o.m.scaffold67.147	Mα	180	19.97	7.78	43.17	95.89	−0.242	Nucleus
LuMADS52	L.us.o.m.scaffold150.49	Mγ	293	31.91	9.10	40.16	62.59	−0.589	Nucleus
LuMADS53	L.us.o.m.scaffold226.16	AP3	228	26.47	9.39	36.70	82.11	−0.714	Nucleus
LuMADS54	L.us.o.m.scaffold23.189	AP1	222	25.86	9.18	69.72	83.02	−0.876	Nucleus
LuMADS55	L.us.o.m.scaffold23.190	SEP	280	31.97	8.80	52.52	83.96	−0.631	Nucleus
LuMADS56	L.us.o.m.scaffold74.36	AP3	228	26.42	9.49	38.58	82.11	−0.742	Nucleus
LuMADS57	L.us.o.m.scaffold218.15	Mα	189	21.32	9.28	57.44	69.68	−0.782	Nucleus
LuMADS58	L.us.o.m.scaffold115.39	Mα	199	22.12	9.38	53.37	66.73	−0.561	Nucleus
LuMADS59	L.us.o.m.scaffold110.83	AP1	189	21.63	9.62	51.52	77.83	−0.839	Nucleus
LuMADS60	L.us.o.m.scaffold138.26	Mδ	350	38.98	5.00	63.11	74.40	−0.643	Nucleus
LuMADS61	L.us.o.m.scaffold138.27	SVP	354	39.32	5.47	46.48	69.38	−0.703	Nucleus
LuMADS62	L.us.o.m.scaffold145.82	Mα	296	32.96	9.33	35.87	78.41	−0.342	Nucleus
LuMADS63	L.us.o.m.scaffold131.30	Mβ	162	17.92	5.25	44.53	92.65	−0.555	Nucleus
LuMADS64	L.us.o.m.scaffold262.35	Mγ	174	20.23	8.77	28.87	93.05	−0.570	Nucleus
LuMADS65	L.us.o.m.scaffold205.77	Mγ	172	19.97	8.42	44.85	83.90	−0.785	Nucleus
LuMADS66	L.us.o.m.scaffold232.35	STK	174	19.51	9.08	60.13	81.21	−0.382	Nucleus
LuMADS67	L.us.o.m.scaffold232.33	Mβ	274	29.03	4.32	48.93	71.82	−0.426	Cell wall Chloroplast
LuMADS68	L.us.o.m.scaffold232.25	STK	297	33.93	8.33	42.68	74.92	−0.699	Nucleus
LuMADS69	L.us.o.m.scaffold204.63	SOC1	248	28.48	7.63	63.49	75.93	−0.799	Nucleus
LuMADS70	L.us.o.m.scaffold204.64	SOC1	238	27.46	8.52	72.76	76.64	−0.802	Nucleus
LuMADS71	L.us.o.m.scaffold44.178	Mα	190	21.94	9.65	52.00	71.32	−0.691	Nucleus
LuMADS72	L.us.o.m.scaffold29.350	Mγ	264	29.51	5.53	38.17	67.58	−0.551	Nucleus
LuMADS73	L.us.o.m.scaffold199.50	Mβ	314	36.07	5.35	50.66	66.75	−0.657	Nucleus
LuMADS74	L.us.o.m.scaffold146.16	AP3	113	13.12	9.62	37.16	85.31	−0.626	Nucleus
LuMADS75	L.us.o.m.scaffold65.243	SVP	266	30.06	8.14	59.79	76.28	−0.738	Nucleus
LuMADS76	L.us.o.m.scaffold69.112	Mδ	221	25.63	9.38	61.68	80.32	−0.836	Nucleus
LuMADS77	L.us.o.m.scaffold202.6	Mγ	80	9.42	10.05	44.77	73.12	−0.821	Nucleus
LuMADS78	L.us.o.m.scaffold202.7	Mγ	67	7.89	10.35	44.86	71.19	−0.885	Nucleus
LuMADS79	L.us.o.m.scaffold5.97	Mγ	70	8.08	12.57	81.05	53.14	−0.989	Nucleus
LuMADS80	L.us.o.m.scaffold108.71	Mα	259	28.83	8.49	49.55	72.32	−0.505	Nucleus
LuMADS81	L.us.o.m.scaffold278.39	Mβ	193	21.20	4.79	40.04	80.41	−0.630	Nucleus
LuMADS82	L.us.o.m.scaffold76.126	ABS	246	28.95	8.31	68.79	75.69	−0.898	Nucleus
LuMADS83	L.us.o.m.scaffold43.71	AP1	195	22.33	9.66	42.57	78.46	−0.888	Nucleus
LuMADS84	L.us.o.m.scaffold96.113	Mα	165	17.44	8.86	56.20	65.70	−0.542	Nucleus
LuMADS85	L.us.o.m.scaffold166.130	SVP	349	39.47	7.10	47.37	92.15	−0.448	Nucleus
LuMADS86	L.us.o.m.scaffold263.41 + L.us.o.m.scaffold263.42	SOC1	241	27.68	6.09	69.96	69.13	−0.959	Nucleus
LuMADS87	L.us.o.m.scaffold13.337	SOC1	226	25.28	9.20	47.07	78.10	−0.586	Nucleus
LuMADS88	L.us.o.m.scaffold71.148	ANR1	241	27.67	9.80	60.16	92.28	−0.633	Nucleus
LuMADS89	L.us.o.m.scaffold79.80	SVP	266	30.06	8.64	58.96	74.10	−0.787	Nucleus
LuMADS90	L.us.o.m.scaffold24.245	Mδ	321	36.01	8.30	45.63	69.88	−0.655	Nucleus
LuMADS91	L.us.o.m.scaffold59.199	SEP	256	29.43	7.13	52.55	78.16	−0.825	Nucleus
LuMADS92	L.us.o.m.scaffold147.1	Mα	199	22.00	9.47	39.75	68.59	−0.615	Nucleus
LuMADS93	L.us.o.m.scaffold147.23	Mδ	375	42.80	5.93	41.69	76.45	−0.634	Nucleus
LuMADS94	L.us.o.m.scaffold147.131	SEP	259	29.97	8.57	40.91	79.46	−0.846	Nucleus
LuMADS95	L.us.o.m.scaffold147.132	AP1	227	26.58	9.28	65.77	77.71	−0.926	Nucleus
LuMADS96	L.us.o.m.scaffold63.144	Mδ	65	7.18	10.01	29.69	83.85	−0.198	Nucleus
LuMADS97	L.us.o.m.scaffold83.154	Mα	224	25.62	9.54	54.49	77.46	−0.567	Nucleus
LuMADS98	L.us.o.m.scaffold83.136	Mα	281	31.31	5.79	51.75	72.14	−0.427	Nucleus
LuMADS99	L.us.o.m.scaffold39.224	Mδ	219	25.46	8.84	59.12	82.37	−0.847	Nucleus
LuMADS100	L.us.o.m.scaffold7.181	Mγ	122	14.36	9.59	49.54	67.95	−0.894	Nucleus
LuMADS101	L.us.o.m.scaffold7.183	Mγ	116	13.55	9.71	35.94	73.97	−0.690	Nucleus
LuMADS102	L.us.o.m.scaffold101.7	Mβ	390	44.27	6.27	46.05	64.05	−0.677	Nucleus
LuMADS103	L.us.o.m.scaffold122.47	Mβ	190	21.56	9.69	70.91	63.68	−0.915	Nucleus
LuMADS104	L.us.o.m.scaffold225.30	STK	249	28.27	8.80	53.74	81.12	−0.638	Nucleus
LuMADS105	L.us.o.m.scaffold14.390	Mα	252	28.25	8.98	40.99	67.30	−0.675	Nucleus
LuMADS106	L.us.o.m.scaffold14.356	ANR1	214	24.49	9.54	40.86	85.75	−0.574	Nucleus
LuMADS107	L.us.o.m.scaffold28.233	Mα	239	25.57	9.34	61.29	78.70	−0.338	Nucleus
LuMADS108	L.us.o.m.scaffold28.347	Mγ	252	28.61	7.70	53.07	84.29	−0.506	Nucleus
LuMADS109	L.us.o.m.scaffold107.174	STK	253	28.84	9.69	58.43	70.95	−0.996	Nucleus
LuMADS110	L.us.o.m.scaffold0.603	SOC1	176	20.66	9.03	54.66	94.72	−0.371	Nucleus
LuMADS111	L.us.o.m.scaffold34.399	Mα	179	19.78	6.32	40.77	94.80	−0.247	Nucleus
LuMADS112	L.us.o.m.scaffold281.2	SVP	181	20.9	9.38	46.52	93.20	−0.569	Nucleus
LuMADS113	L.us.o.m.scaffold422.8	Mδ	349	38.87	4.82	62.57	75.44	−0.642	Nucleus
LuMADS114	L.us.o.m.scaffold422.9	SVP	354	39.5	5.54	58.29	68.84	−0.655	Nucleus

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Phylogenetic and evolutionary analysis

MADS genes are ubiquitous in plants. To better understand the evolution of the MADS gene family in flax, we searched for the MADS gene family in 16 representative plant species and conducted a comparative genome analysis (Figure 1). The MADS gene family has been reported in all species. The results show that type I and type II contain only one MADS gene in the algae C. reinhardti. In angiosperms, the MADS gene family has rapidly expanded in number due to the ε-whole-genome duplication event. Compared with type I, type II in angiosperms is more conservative. After a γ-genome-wide tripling event, dicots have more MADS genes than monocots. In addition, flax and longan contain the same number of MADS genes (114), which is similar to S. melongena.

Evolution and distribution of the MADS gene family in 16 species

The evolutionary relationship on the left side of the figure is obtained based on NCBI (https://www.ncbi.nlm.nih.gov/Taxonomy/CommonTree/wwwcmt.cgi). The table on the right side of the figure shows the number statistics of MADS gene families in different species. Type I MADS genes include Mα, Mβ, and Mγ, and type II MADS genes include Mδ and MIKC^c. All whole genome duplication events are given by James W. Clark et al.²⁵ The blue circle represents the whole genome diploidization event, and the orange color circles represent genome-wide tripling events, and named genome-wide duplication events are represented by Greek letters.

To better understand the evolutionary relationship of the MADS-box gene family in flax, we constructed a phylogenetic tree consisting of 216 MADS-box genes from Arabidopsis (102) and flax (114) (Figure 2). Referring to the Arabidopsis thaliana MADS-box protein subfamily classification (Table S1), we divided the 114 LuMADS genes into type I and type II, containing 45 and 69 genes respectively. The 45 type I LuMADS genes can be divided into Mα (20 genes), Mβ (10 genes), and Mγ (15 genes), among which the subgroup Mα has the largest number of LuMADS genes. The remaining 69 type II LuMADS genes were divided into 13 subgroups (Mδ, FLC, ANR1, SVP, ABS, PI, AP3, SOC1, STK, AP1, SEP) based on their homologous relationship with Arabidopsis. The Mδ subfamily contains the most type II LuMADS genes (13), followed by the SEP and AP1 subfamilies, which both contain 10 LuMADS genes. SOC1, STK, SVP, ANR1, and AP3 subfamilies contain 8, 7, 6, 5, and 4 genes, respectively. The PI, ABS, and FLC subfamilies contain the fewest LuMADS genes, each containing 2 genes.

LuMADS protein phylogenetic tree

Different colors represent different subfamilies. The asterisk represents the MADS-box gene family in flax.

LuMADS gene structure and conserved domains

To visualize the protein structure of the flax MADS-box family, we predicted the amino acid sequences of 114 LuMADS proteins using the online MEME website. A phylogenetic tree was constructed from multiple sequence alignments (Figure 3A), exhibiting clustering identical to that shown in Figure 2, and thereby demonstrating strong consistency. Ten conserved motifs were identified and designated as motif 1 through motif 10. (Figure 3B), with each motif sequence logo displayed (Figure 3D). According to motif analysis, motif 1 and motif 3 are typical LuMADS domains. Most LuMADS proteins contained these two domains, although 14 genes (LuMADS67, 63, 24, 103, 32, 7, 19, 61, 114, 76, 64, 40, 81, and 34) had only motif 1, two genes (LuMADS77, LuMADS78) had only motif 3, and a total of 16 genes (14%) had incomplete MADS domains. Among the 69 type II LuMADS genes, 16 genes (23.2%) lacked the k domain, which corresponded to motif 2. We also found that motif 2, motif 5, motif 8 and motif 10 are unique structural domains in type II LuMADS genes, which may be the reason why they have more biological functions than type I MADS-box genes.

Phylogenetic tree, conserved motifs and exon-intron structure analysis of flax MADS-box genes

(A) Phylogenetic tree of flax MADS-box genes.

(B) Conserved motifs of LuMADS proteins. The 10 conserved motifs are represented by different colors.

(C) Exon-intron structure of the *LuMADS* gene. The blue box indicates the CDS region of the gene, the orange box indicates the UTR region, and the black line indicates the intron.

(D) A conserved motif of the flax MADS gene. Motif1–Motif10 represents different conserved motifs, the numbers on the x axis represent the amino acid position, and the font size represents the relative frequency at the position.

Analysis of the exon-intron structure of LuMADS genes found that the number of exons ranged from 1 to 11 (Figure 3C), of which 24 genes (LuMADS102, 31, 63, 24, 103, 58, 107, 84, 92, 111, 51, 14, 57, 71, 42, 79, 101, 100, 72, 33, 78, 77, 23, and 65) have only one exon, LuMADS4, LuMADS50, LuMADS90, and LuMADS16 contain 11 exon. Most type I LuMADS genes (45 out of 95.56%) contain 1 to 2 exons, while most type II genes (69 out of 73.91%) contain 6 to 9 exons. It is worth noting that most genes under the same branch have similar exon numbers, indicating that these genes are functionally similar.

Chromosome location and gene duplication events of the LuMADS gene family

The chromosomal location of the LuMADS gene was determined based on the flax reference genome. Here, we removed 4 genes (LuMADS111-LuMADS114) located on scaffold. It was found that 110 LuMADS genes were unevenly distributed on 15 chromosomes (Figure 4). Among them, chromosomes 2 and 13 had the most LuMADS genes, both with 12 (10.9% of the total). Followed by 11 (10%) genes distributed on chromosome 7. Chromosome 8 had the least number of genes, containing only 2 (1.8%) LuMADS genes.

Chromosomal distribution of *LuMADS* genes

Set the sliding window size to 100 kb, red to blue indicates gene density from high to low.

We performed gene duplication analysis of the LuMADS gene family by BLAST and MCScanX. Three pairs of tandemly repeated genes (LuMADS35 and LuMADS36, LuMADS60 and LuMADS61, LuMADS69 and LuMADS70) were identified, located on chromosomes 4, 9, and 10, respectively (Figure 4). Gene duplication often leads to gene mutations that form new functions and play a key role in plant environmental adaptation. To study the gene duplication events of the LuMADS gene family, a Circos map was constructed (Figure 5A). This showed 47 pairs of LuMADS gene pairs in flax, consistent with the occurrence of a flax WGD (whole genome duplication).

Collinearity analysis of *LuMADS* genes

(A) Collinearity analysis among *LuMADS* genes.

(B) Collinearity study of MADS-box genes between flax and rice, *Arabidopsis*, maize and wheat. The gray line represents all collinear gene pairs, and the red line represents collinear *LuMADS* gene pairs.

To further analyze the evolutionary relationships between flax and other species, we compared the collinearity between MADS genes in flax and four other representative species, including rice, Arabidopsis, maize, and wheat (Figure 5B). The results showed that there were 24, 58, 23, and 61 pairs of collinear genes between flax and the four species, respectively. The collinear gene pairs of rice MADS were located on chromosomes 1, 2, 7, 12, and 15 of flax; the 5 chromosomes of arabidopsis and flax are collinear except chromosome 14; the MADS-box collinear gene pairs of maize chromosomes 1, 5, 6, 7, and 9 are on flax chromosomes 1, 2, 7, and 9; Genes on chromosomes 2A, 2B, 2D, 3A, 3B, 3D, 3D, 4A, 4B, 4D, 4D, 5A, 5B, 5D, 7D, 7A, 7B, and 7D of wheat were collinearity with MADS-box genes on chromosomes 1, 2, 4, 7, 9, 10, and 15 of flax. In summary, most of the MADS-box genes of the four control species show conserved collinearity between chromosomal regions.

Analysis of cis-acting elements in promoter of flax MADS-box gene family and miRNA prediction

To further study the regulatory mechanism of the flax MADS-box gene family in abiotic stress response, we extracted the 2000 bp sequence upstream of the promoter region of each MADS-box gene in flax and analyzed it using the PlantCare service (Figure 6). We removed common element analysis such as CAAT-box and TATA-box, and the results predicted 2939 cis-acting elements, which can be divided into four categories: development-related elements; environmental stress-related elements; hormone-responsive elements and light-responsive elements (Figures 6B and 6C; Table S2).

Analysis of *cis*-acting elements of the *LuMADS* gene family

(A) Distribution of *cis*-acting elements of *LuMADS* genes.

(B and C) Number statistics of *LuMADS* gene promoters. Red represents development-related elements; light blue represents environmental-stress-related elements; dark green represents hormone-responsive elements and dark blue represents light-responsive elements.

Among them, light-responsive elements were the most abundant cis-elements in the promoter region of the LuMADS gene family, including 1285 components (43.72%), such as Box 4, G-box, and GT1-motif elements. The second largest category is hormone responsive elements, with 940 components (32%), including abscisic acid responsive element (ABRE); auxin responsive element (TGA-element); gibberellin responsive element (P-box); MeJA aresponsive element (TGACG-motif and CGTCA-motif) and salicylic acid responsive element (TCA-element). The third largest category was environmental stress-related elements, with 481 components (16.37%), including anaerobic induction element (ARE), defense and stress-responsive element (TC-rich repeats) and low-temperature responsive element (LTR). The fourth major category was development related elements, with 233 components (7.93%), including circadian responsive element (Circadian), endosperm expression element (GCN4_motif), meristem expression element (CAT-box), zein metabolism regulatory element (O₂-site), cis-acting regulatory element (A-box), wound-responsive element (WUN-motif) and seed-specific responsive element (RY-element) (Figure S1). We also found that type II LuMADS genes have more hormone response elements, these findings demonstrate that type II MADS-box genes in flax are involved in defense responses to various stresses and plant hormones.

The miRNA target prediction results showed that only 22 genes (19.3%) among all 114 LuMADS genes contained 27 miRNA targets (Table 2). Among them, LuMADS28 had the most gene targets, with 9. We found that different LuMADS genes were targeted by the same miRNA. For example, LuMADS17, LuMADS10, and LuMADS112 can be targeted by MiR396 at the same time; LuMADS35, LuMADS36, and LuMADS69 can be targeted by MiR156 at the same time. The results showed that lus-miR396 and lus-miR156 are the main miRNAs targeting of the LuMADS gene family.

Table 2.

miRNA target prediction of LuMADS gene

miRNA	Target	Expectation	miRNA Length	Target_start	Target_end	Inhibition	Multiplicity
lus-miR408a	LuMADS114	4.5	21	11	31	Cleavage	1
lus-miR408a	LuMADS61	4.5	21	11	31	Cleavage	1
lus-miR395e	LuMADS28	3.5	21	304	324	Cleavage	1
lus-miR395e	LuMADS55	5	21	396	416	Cleavage	1
lus-miR395e	LuMADS7	5	21	255	275	Cleavage	1
lus-miR399 b/d	LuMADS80	3.5	21	171	190	Cleavage	1
lus-miR394a/b	LuMADS84	5	20	335	354	Translation	1
lus-miR159b/c	LuMADS1	5	21	554	574	Translation	1
lus-miR396a/b/c/e	LuMADS17	3	21	382	402	Cleavage	1
lus-miR396a/b/c/e	LuMADS10	4	21	406	426	Cleavage	1
lus-miR396a/b/c/e	LuMADS112	4.5	21	82	102	Cleavage	1
lus-miR396a/b/c/e	LuMADS28	4.5	21	82	102	Cleavage	1
lus-miR396a/b/c/e	LuMADS85	4.5	21	82	102	Cleavage	1
lus-miR396a/b/c/e	LuMADS96	4.5	21	82	102	Cleavage	1
lus-miR396a/b/c/e	LuMADS89	5	21	82	102	Cleavage	1
lus-miR396a/b/c/e	LuMADS92	5	21	388	408	Cleavage	1
lus-miR395a/b/c/d	LuMADS28	4	21	304	324	Cleavage	1
lus-miR156b/c/e/f/h/i	LuMADS35	5	21	100	120	Cleavage	1
lus-miR156b/c/e/f/h/i	LuMADS36	5	21	100	120	Cleavage	1
lus-miR156b/c/e/f/h/i	LuMADS69	5	21	100	120	Cleavage	1
lus-miR156b/c/e/f/h/i	LuMADS70	5	21	100	120	Cleavage	1
lus-miR160a/b/d/e/f/h/i/j	LuMADS40	5	21	510	529	Cleavage	1

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LuMADS protein-protein interaction network and GO enrichment analysis

To further investigate potential biological functions of the LuMADS gene family, orthologous genes from Arabidopsis thaliana were used to predict the protein-protein interaction (PPI) network of the LuMADS gene family (Figure 7A). Results showed that orthologs of 35 LuMADS genes were predicted to interact with 17 other proteins. Most LuMADS proteins were predicted to interact with multiple other proteins. Several of these proteins were associated with stress or temperature responses, such as FBP7, FVE, GI, VRN1. Most other proteins were related to flower development, such as AP2, UFO, and SEU. GO enrichment analysis of the LuMADS genen flax showed that it is mainly involved in biological processes, such as metabolic process, nucleic acid binding, heterocyclic compound binding, organic cyclic compound binding, response to temperature stimulus, and photoperiodism of the plant (Figure 7B).

Protein interaction network and GO enrichment analysis of the MADS-box gene family in flax

(A) Protein interaction network of the *LuMADS* gene family through *Arabidopsis* orthologs. The green circle represents the flax MADS-box protein, and the red circle represents the gene reported in *Arabidopsis*.

(B) GO enrichment analysis of *LuMADS* genes.

LuMADS expression pattern analysis based on RNA-seq data

MADS-box genes play an important role in plant growth and development. To better explore the expression patterns of the LuMADS gene family, we analyzed all identified flax MADS-box genes using RNA-seq data (Figure 8). There were differences in the expression patterns of flax MADS-box genes in different tissues. The results showed that most LuMADS genes were highly expressed in flowers 5 days after flowering (67.54%) and in flowers 10 days after flowering (66.67%) (Figure 8A). Three genes were highly expressed in flowers 20 days after flowering (LuMADS96, LuMADS73, and LuMADS29), and only one gene (LuMADS90) was highly expressed in flowers 30 days after flowering. We also found that both TypeI and TypeII LuMADS gene families have the same expression pattern, which also proves that TypeI and TypeII MADS-box genes play a key role in flower development.

Expression pattern analysis of the *LuMADS* gene family

(A) Expression patterns of the MADS-box gene family in post-anthesis flowers.

(B) Expression patterns of MADS-box genes under salt treatment and heat treatment.

(C) Expression patterns of MADS-box genes in different tissues. FPKM values are log2(FPKM) normalized, and different colors represent relative expression levels from high (red) to low (blue).

Using previously published transcriptome data, the expression pattern of flax LuMADS genes in 14 different tissues was analyzed (Figure 8B). The results showed that most members were expressed at low levels in cotyledon-stage embryos and torpedo-stage embryos. Only LuMADS42 was highly expressed in seeds. Two members (LuMADS106 and LuMADS98) were highly expressed in roots, and two members (LuMADS26 and LuMADS44) were highly expressed in stems. Three members (LuMADS40, 81, and 111) were highly expressed in fruit, with 18 members (LuMADS10, 28, 35–37, 43, 66, 69–72, 85, 87–88, 93, 96, 112, and 114) highly expressed in leaves. Type I LuMADS genes were mainly highly expressed in embryos. One member (LuMADS12) was highly expressed in heart embryos, and seven members (LuMADS14, 25, 58, 64–65, 80, and 108) were highly expressed in globular embryos. 14 genes were highly expressed in flower-related tissues, of which six genes (LuMADS20, 24, 39, 60, 92, and 113) were highly expressed in anther; five genes (LuMADS31, 52, 62, 99, and 102) were highly expressed in stamen. Two genes (LuMADS63 and LuMADS105) were highly expressed in pistil; only LuMADS17 was highly expressed in ovary. We found that most type II LuMADS genes were expressed in clusters in flower-related tissues, and a few type I LuMADS genes were highly expressed in flower-related tissues. This also shows that type I and type II MADS-box genes in flax interact to promote flower development.

To better understand the potential function of LuMADS genes in responding to abiotic stress, RNA-seq data were used to analyze their expression levels under heat and salt treatment (Figure 8B). The LuMADS gene family did not change significantly under heat stress. The expression level of LuMADS gene was higher in tissues without salt treatment, but the expression level was suppressed in tissues after salt stress. Compared with the control group, four genes (LuMADS22, 27, 87, and 99) were significantly increased in root tissue under salt stress, among which the LuMADS22 gene had the highest differential expression. In leaf tissue, 16 genes were significantly increased under salt stress, among which LuMADS15, LuMADS24, and LuMADS29 genes had the highest differential expression.

Expression levels of flax LuMADS genes under abiotic stress

To ensure that the selected genes are representative of the diversity within the LuMADS family, we chose one gene from each of the 11 subfamilies of type II. The selected genes exhibited high levels of homology with known genes in Arabidopsis. Additionally, the relative expression levels of these selected genes were assessed using RT-qPCR to determine whether the LuMADS genes were responsive to the abiotic stress. We analyzed the functional differences between flax leaves and stems. Flax leaves primarily perform photosynthesis and gas exchange, whereas stems primarily provide support and serve as transport channels. Investigating gene expression in these two key tissues enables the elucidation of specific gene functions in different tissues. The distinct responses of flax leaves and stems to environmental stresses, including drought, salt, and cold, contribute to our understanding of flax’s stress response mechanisms. The relative expression of 11 genes (LuMADS2, 8, 9, 19, 27, 37, 41, 44, 74, 85, and 110) under abiotic stress (low temperature, drought, and salt) in leaf tissue and stem tissue was studied. The expression levels of genes at 0 h, 3 h, 6 h, 9 h, 12 h, and 24 h were compared (Figures 9, 10, and 11). As the time of low temperature stress increased, all LuMADS genes showed significant responses (Figure 9). Among them, the expression of LuMADS8 and LuMADS110 reached the extreme value in root tissue and leaf tissue at 3 h of low temperature stress, and then decreased. The stem tissue increased by 16.2 times and 18.8 times compared with the control, and the leaf tissue increased by 6.4 times and 23.6 times compared with the control. The stem tissue of four genes (LuMADS2, LuMADS9, LuMADS74, and LuMADS85) reached the extreme value at 3 h of low temperature stress, which increased by 7.8 times, 33.4 times, 16.1 times and 5.7 times, respectively, compared with the control group. The leaf tissue of one gene (LuMADS27) reached the extreme value after 3 h of low temperature stress, which increased by 1.6 times compared with the control. At 6 h of low temperature stress, the leaf tissue of LuMADS44 increased significantly, 5.1 times higher than the control. At 9 h of stress, the stem tissue of LuMADS41 was significantly increased by 11.4 times compared with the control group. The two genes (LuMADS41 and LuMADS85) had the same expression pattern in stem tissues, which increased significantly at 12 h and 24 h of stress, increasing 13.1 times compared to the control group.

Analysis of expression patterns of *LuMADS* genes under low temperature stress

The red lines represent stem tissue, and the blue lines represent leaf tissue. Using Student’s t test, asterisks indicate statistically significant differences (∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001).

Expression pattern analysis of *LuMADS* genes under salt stress

The red lines represent stem tissue, and the blue lines represent leaf tissue. Using Student’s t test, asterisks indicate statistically significant differences (∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001).

Analysis of expression patterns of *LuMADS* genes under drought stress

The red lines represent stem tissue, and the blue lines represent leaf tissue. Using Student’s t test, asterisks indicate statistically significant differences (∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001).

As the salt stress time increased, most LuMADS genes did not change significantly (Figure 10). We found that the leaf tissue of three genes (LuMADS27, LuMADS74, and LuMADS85) began to be downregulated at 6 h of salt stress until reaching the extreme value at 24 h, which may be related to the negative regulation of salt stress.

Drought treatment induced a significant upregulation of the expression of three genes (LuMADS9, LuMADS27, and LuMADS44) (Figure 11). The transcript level of LuMADS9 leaf tissue reached an extreme value at 6 h of cold stress and then gradually decreased, increasing 5.2 times compared with the control group. The transcript level of stem tissue increased significantly by 3.2 times at 9 h of cold stress; LuMADS27 leaf tissue and stem tissue had the same expression pattern reached the extreme value at 3 h of cold stress, which was 7.8 times and 5.9 times higher than the control group respectively; the transcript levels of LuMADS44 leaf tissue and stem tissue reached the extreme value at 9 h and 12 h of cold stress, respectively, and then gradually decreased. Increases of 7.4 times and 3.8 times, respectively.

Discussion

MADS-box proteins are plant transcription factors that are not only involved in the regulation of plant growth and development, but also in the regulation of plant stress responses. So far, there are no reports about the MADS-box gene family in flax. A total of 114 flax MADS-box genes were identified in this study, including 45 type I genes and 69 type II genes. The number of MADS-box genes in flax (114) is greater than in the monocotyledonous species maize (75) and rice (75), but similar to the number found in the dicots soybean (106) and Arabidopsis (107), and longan (114).²⁶^,²⁷^,²⁸^,²⁹^,³⁰ MADS-box gene families was compared in 16 representative species (Figure 1). Chlamydomonas reinhardtii is a primitive lower plant that contains only two type I and type II genes, which is consistent with the view that these arose through duplication events before the divergence of animals and plants.⁵ Overall, the number of MADS-box genes in angiosperms is greater than that in ferns and mosses. We found that MIKC^C genes were greatly amplified in angiosperms, and we predicted that MIKC^C genes may be endowed with new functions as plants evolve, especially in flowering, growth and development, and stress response. In monocotyledons and dicotyledons, the number of type I genes differs greatly, while type II genes are relatively conserved. Due to the recent whole-genome duplication event in flax, flax has a large number of MADS-box genes co-regulating gene networks.³¹

Previous studies have shown that Arabidopsis thaliana MADS-box proteins can be divided into five subfamilies, including Mα, Mβ, Mγ, Mδ, and MIKC.²⁹ There are currently two main types known: type I, including Mα, Mβ, Mγ and Mδ, and type II (MIKC type). The phylogenetic tree of LuMADS gene family was divided according to the classification of homologous genes in Arabidopsis. The LuMADS gene family was divided into Mα (20), Mβ (10), Mγ (15), Mδ (13), and MIKC (56) (Figure 2), which is very similar to the number of divisions in Populus trichocarpa.³² Motif analysis showed that LuMADS proteins all contain highly conserved MADS domains, while type II genes contain special I, K, and C regions (Figure 3B). They can form dimers or advanced complexes to exert transcriptional activity.³³^,³⁴ Gene structure analysis showed that most members of the flax type I MADS-box gene family contain 1-2 exons, and the gene structure is relatively simple, while most members of the type II MADS-box gene family contain 6-9 exons (Figure 3C), which is similar to the gene structure and conserved motifs of peanut and Juglans mandshurica.³⁵^,³⁶

Gene duplication events play a key role in the proliferation and evolution of MADS-box genes,³⁷ and duplicated gene pairs may produce new functions, thereby enhancing plant responses to environmental stress.¹⁷^,³⁸ In this study, 3 pairs of tandem duplication genes (Figure 4) and 47 pairs of segmental duplication genes (Figure 5A) were found in the LuMADS gene. This suggests that segmental duplication is the main cause of LuMADS gene expansion, which is consistent with findings in other species, such as Salvia miltiorrhiza and Sechium edule.³⁹^,⁴⁰ Collinearity analysis is a powerful method to analyze gene evolutionary trajectories.⁴¹ In this study, 24, 23, and 61 pairs of collinear genes were identified in the monocotyledonous plant species rice, maize, and wheat, respectively, and a total of 58 pairs of collinear genes were identified in the dicotyledonous plant Arabidopsis (Figure 5B), indicating that these homologous gene pairs may have a common ancestor that existed before their divergence. In summary, it is proved that the LuMADS gene is highly conserved in flax.

Promoter cis-elements are involved in the regulation of gene expression. At the transcription level, the interaction between transcription factors and promoter binding sites plays a key role.⁴² In the promoter region of the LuMADS gene, we found a large number of cis-acting elements that respond to plant hormones (such as abscisic acid; auxin; gibberellin; MeJA and salicylic acid) and abiotic stress responses (such as anaerobic; defense and stress and low-temperature) (Figure 6), among stress response elements, LTR is related to plant low temperature response, and ARE response element is related to anaerobic induction.⁴³ In this study, most LuMADS genes contain ARE response elements and LTR response elements, proving that MADS-box genes play an important role in plant stress adaptability and signaling pathways.⁴⁴ MicroRNAs (miRNAs) are key regulators that coordinate plant development and plant-environment interactions.⁴⁵ Our analysis showed that lus-miR396 and lus-miR156 are the main miRNAs targeting of the LuMADS gene family (Table 2). In Cymbidium ensifolium, Solexa technology was used to identify miR156, miR172, and miR5179 targeting MADS-box transcription factors,⁴⁶ while miR396 regulates rice leaf length in the form of a miR396-GRF module.⁴⁷ The regulatory mechanisms of MADS and miRNA156-MADS modules need to be further studied. The interaction between proteins is the key to maintaining the normal function of proteins and is also the key to predicting the functional diversity of proteins.⁴⁸ In this study, most LuMADS proteins were predicted to interact with FBP7, FVE, GI, and VRN1 proteins (Figure 7A). AtFBP7 is a protein widely expressed that undergoes induction in response to cold and heat temperature. Furthermore, it has been reported that FBP7 mutants exhibit defects in protein biosynthesis following exposure to cold and heat stress.⁴⁹ FVE may exist as a multiprotein complex, regulating plant flowering time and cold response by interacting with FLC and COR chromatin.⁵⁰ The AtGI gene is involved in mediating cold stress response, and overexpression of GI in Arabidopsis thaliana shows increased sensitivity to freezing stress.⁵¹ The expression of VRN 1 is induced by vernalization, and VRN 1 targets genes that are central to both low-temperature-induced frost resistance and hormone metabolism.⁵² LuMADS genes may be involved in the temperature response mechanism of plants, which is also consistent with the study in Rhododendron hainanense Merr.⁵³ MADS-box genes play an important role in plant growth and development network and stress response network.⁵⁴ Therefore, mining MADS-box genes and analyzing their interactions with stress responses plays a very important role in optimizing crop production performance.

Because the expression patterns of MADS-box genes can predict their functions, and we analyzed RNA-seq data from 24 flax tissues. The results showed that the expression pattern of LuMADS gene was tissue-specific and closely related to its gene function (Figure 8). For example, FLC plays a repressive role in the flowering time of Arabidopsis. The flax FLC homologous genes LuMADS19 and LuMADS47 are not expressed in tissues from 5 days to 30 days after flowering, and may be repressive genes for flax flowering. STK is a member of the AG subfamily of Arabidopsis thaliana and is involved in the growth and development of ovules and seeds. Its homologous gene SHELL is involved in regulating lipid synthesis.⁵⁵

The homologous genes of flax AG subfamily members, LuMADS9, LuMADS13, LuMADS22, LuMADS66, LuMADS68, LuMADS104, and LuMADS109, are actively expressed in seeds, indicating that they may play an important role in seed development and lipid synthesis. SEP1/2/3 is important regulators of the development of important floral organs (petals, stamens, carpels) in Arabidopsis.⁵⁶ The SEP homologous genes OsMADS5 and OsMADS3 in rice are involved in the formation of spikelets and flowers, respectively.⁵⁷ The SEP homologous genes in flax are all involved in the formation of floral organs. Overall, the tissue-specific analysis of flax provides a deeper insight into the functionality of LuMADS genes.

The MADS-box is a multifunctional gene extensively found in diverse plants. We further confirmed this finding by utilization of RT-qPCR. The findings reveal that, besides influencing plant growth and development, LuMADS genes also play a significant role in stress response (Figures 9, 10, and 11). Eleven flax genes (LuMADS2, 8, 9, 19, 27, 37, 41, 44, 74, 85, and 110) were found to respond positively to cold stress at different times. Among them, six genes (LuMADS2, 8, 9, 74, 85, and 110) showed the same expression trend in stem tissue after 3 h of cold stress, indicating that the stem tissue of the six genes was more sensitive at 3 h of cold stress, which is a critical time point. Under salt stress, three genes (LuMADS27, 74, and 85) were found to be significantly downregulated, indicating a negative regulatory effect of LuMADS27, 74, and 85 on salt. Under drought treatment, the expression of three genes (LuMADS9, 27, and 44) showed a trend of first increasing and then decreasing, indicating that LuMADS9, LuMADS27, and LuMADS44 play important roles in drought stress. We also found that LuMADS27 not only plays a positive regulatory role in cold stress, but also in drought stress. However, in saline alkali stress, this gene plays a negative regulatory role, demonstrating the important role of LuMADS27 gene in flax abiotic stress. In previous studies, it was found that the expression of BdMADS30, BdMADS23, BdMADS33, and BdMADS55 genes in Brachypodium patens was significantly regulated by PEG 6000, 200 mM NaCl and low temperature treatment.⁵⁸ The CaMADS gene is involved in salt, low temperature and osmotic stress, and overexpression of CaMADS can improve the stress resistance of Arabidopsis.⁵⁹ In rice, the overexpression lines of OsMADS25 exhibit higher levels of free proline and reduced MDA accumulation. The significant upregulation of genes related to salt stress suggests that OsMADS25 enhances salt tolerance by mitigating oxidative damage.⁶⁰ OsMADS23 confers drought and salt tolerance in rice by regulating ABA biosynthesis.⁶¹ A novel MADS-box transcription factor, SlMBP22, has been identified within tomatoes. Transgenic plants expressing elevated levels of SlMBP22 demonstrate enhanced drought stress tolerance compared to wild-type plants.⁶² In the common bean, transgenic plants overexpressing PvMADS31 show enhanced lateral root development, root elongation, and seed germination under stress conditions. Additionally, overexpressing PvMADS31 in Arabidopsis enhances drought resistance, possibly due to improved ROS scavenging and increased proline accumulation.⁶³ In addition, OsMADS26 has a negative regulatory effect on drought in rice.¹⁶ The AGL22 gene in Arabidopsis is a bridge between primary metabolism and the initiation of drought stress response.⁶⁴ These results indicate that MADS-box plays an important role in plant stress response. Research on the LuMADS gene family will lay the foundation for an in-depth understanding of the function and molecular mechanism of MADS-box genes in flax stress resistance.

Limitations of the study

Studying the subcellular localization and function of the LuMADS gene helps to comprehensively analyze the MADS-box gene and reveal its role in plant stress resistance MADS-box is an important class of transcription factors involved in regulating plant growth and development. It plays a crucial role in the development of plant floral organs, the length of flowering period, and the sex determination of male and female flowers. Through functional verification of the 114 LuMADS genes identified in this article, the mechanism of flax flowering can be further elucidated. There are research reports that the MADS-box gene may play a crucial role in flax under salt stress and waterlogging tolerance. Therefore, studying its stress responses provides valuable insights into the LuMADS gene family.

Resource availability

Lead contact

Further information and requirements for resources and reagents should be directly contacted by the main contact person, Professor Jian Zhang, (zhangjian@jlau.edu.cn).

Material availability

This study did not generate new unique reagents.

Data and code availability

•
Data: This paper analyzes existing, publicly available data. These accession numbers for the datasets are listed in the key resources table.
•
This paper does not report original code.
•
Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request.

Acknowledgments

This work was funded by Jilin Agricultural University high-level researcher grant (JLAUHLRG20102006 and 10102028602), and Jilin Provincial Department of Human Resources and Social Security grant: No. 201020012.

Author contributions

Conceptualization, J.L., H.W., and D.M.P.; methodology, J.L., X.L., and X.S.; validation, H.W., S.L., and H.Y.; writing—original draft preparation, J.L.; writing—review and editing, H.W. and Y.M.; visualization, Z.Z.; supervision, M.K.D. and Jian Zhang.; project administration, Jun Zang. and Z.Z.; funding acquisition, Jian Zhang. All authors have read and agreed to the published version of the manuscript.

Declaration of interests

The authors declare no competing interests.

STAR★Methods

Key resources table

REAGENT or RESOURCE	SOURCE	IDENTIFIER
Biological samples

Healthy Flax Leaf and Stem Tissue	Laboratory of Bast Fiber Crops, College of Agriculture, Jilin Agricultural University	N/A
Organizations subjected to abiotic stress treatment	Incubator in the Bast Fiber Crops Laboratory	N/A

Critical commercial assays

RNAprep Pure Plant Plus Kit (Polysaccharides &Polyphenolics-rich)	TIANGEN	DP441
PrimeScript™ RT reagent Kit	TAKARA	RR047A
TB GreenTM Premix ExTaqTM II	TAKARA	RR820B
PrimeSTAR	TAKARA	R045A

Deposited data

Genome data	National Center for Biotechnology Information (NCBI) database (https://www.ncbi.nlm.nih.gov/)	QMEI02000000
Transcriptome data in pistil, stamen, fruit and shoot tip tissue	National Center for Biotechnology Information (NCBI) database (https://www.ncbi.nlm.nih.gov/)	PRJNA1002756
Transcriptome data in flower tissue 30days, 20days, 10days and 5days after flowering	National Center for Biotechnology Information (NCBI) database (https://www.ncbi.nlm.nih.gov/)	PRJNA833557
Transcriptome data in torpedo embryo, root, ovary, mature embryo, heart embryo, globular embryo, cotylden embryo, anther and seeds tissue	National Center for Biotechnology Information (NCBI) database (https://www.ncbi.nlm.nih.gov/)	PRJNA663265
Transcriptome data on root and leaf tissues after salt stress	National Center for Biotechnology Information (NCBI) database (https://www.ncbi.nlm.nih.gov/)	PRJNA977728
Transcriptome data in stem tissue after heat stress	National Center for Biotechnology Information (NCBI) database (https://www.ncbi.nlm.nih.gov/)	PRJNA874329

Oligonucleotides

qPCR primers sequences	This paper	Table S3

Software and algorithms

ClustalW	Yuan et al.⁶⁵	http://www.clustal.org
MEGA 11	Tamura et al.⁶⁶	https://www.megasoftware.net/
iTOL	Letunic et al.⁶⁷	https://itol.embl.de/
MEME v5.5.7	Bailey et al.⁶⁸	http://meme-suite.org/
TBtools	Chen et al.⁶⁹	https://github.com/CJ-Chen/TBtools/releases
MCScanX	Wang et al.⁷⁰	https://github.com/wyp1125/MCScanX
psRNATarget	Griffiths-Jones et al.⁷¹	http://plantgrn.noble.org/psRNATarget/
PlantCARE	Lescot et al.⁷²	http://bioinformatics.psb.ugent.be/webtools/plantcare/html/
SWISS-MODEL	Franceschini et al.⁷³	https://string-db.org/
Cytoscape	Shannon et al.⁷⁴	https://cytoscape.org/
Fastp	Chen et al.⁷⁵	https://github.com/OpenGene/fastp
Tidyverse	Carpenter et al.⁷⁶	https://github.com/tidyverse
Rsubrad	Liao et al.⁷⁷	https://anaconda.org/bioconda/bioconductor-rsubread
Limma	Ritchie et al.⁷⁸	https://anaconda.org/bioconda/bioconductor-limma
R 4.3.1	The R Foundation	https://www.r-project.org/
EdgeR	Robinson et al.⁷⁹	https://anaconda.org/bioconda/bioconductor-edger

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Experimental model and study participant details

Plant materials and abiotic stress treatment

Trials were conducted on the flax variety "longya10". Seeds were sterilized with 75% ethanol for 10 min, then washed with sterile water, and transplanted into soil. The incubator was set at 26/18°C temperature and 16/8 h of light. Flax seedlings were grown to 6–7 cm. The drought treatment group and salt treatment group followed previous research methods.⁸⁰^,⁸¹ Clean the soil thoroughly with clean water, and soak plants with similar growth in conical flasks containing 10% PEG solution and 100mM NaCl solution; The low-temperature treatment group placed the plants in a 4°C incubator. At 0, 3, 6, 9, 12, and 24 h, each treatment group ensured that leaf and stem tissues were collected at the same time and repeated 3 times to avoid rhythm effects caused by different sampling times. All linen samples were frozen in liquid nitrogen and stored in an ultra-low temperature refrigerator at −80°C.

Method details

Identification of LuMADS Gene family in flax

The whole genome sequence of flax was downloaded from NCBI (entry number QMEI02000000), and genome annotation file download on (https://figshare.com/articles/dataset/Annotation_files_for_Longya-10_genome/13614311). The contour hidden Markov model (http://pfam.xfam.org/) of the MADS gene PF00319) domain was obtained using the Pfam database, and the LuMADS gene was predicted. Use SMART (http://smart.embl.de/smart/batch.pl), and CDD (https://www.ncbi.nlm.nih.gov/cdd/) to filter and correct the predicted LuMADS genes. Finally, the final 114 LuMADS genes were obtained. The MADS gene of flax is prefixed with "Lu", and subsequent Arabic numerals are numbered based on the gene’s position on the chromosome. Analysis of the physical and chemical properties of LuMADS, including coding length, number of amino acids, molecular weight (MW), theoretical isoelectric point (pI) and grand average of hydropathicity index (GRAVY),⁸² was calculated using the ExPASy ProtParam tool (https://web.expasy.org/protparam/). Subcellular localization prediction was performed using the BUSCA website (http://www.busca.cn).

Phylogenetic Analysis, Chromosomal location, conserved domains and conserved motifs of LuMADS

The TAIR website was used to download (https://www.arabidopsis.org/) the Arabidopsis thaliana MADS protein sequence, and use ClustalW (version 11) in MEGA to compare the MADS proteins of plants such as Arabidopsis thaliana and flax using default parameters.⁶⁵ Based on the principle of maximum likelihood (ML), a phylogenetic tree was constructed with the help of MEGA version 11 and the default configuration (neighborhood Joining; Parameter: Bootstrap 1000).⁶⁶ Then, we visualized the constructed tree using iTOL version 6 (https://itol.embl.de/).⁶⁷ The position information of LuMADS on the chromosome was obtained from the flax genome FASTA file and gff3 file, and the NCBI CD-search website (https://www.ncbi.nlm.nih.gov/Structure/BWRPSB/BWRPSB.Cgi) predicted the conserved structure area. LuMADS protein motifs were analyzed using MEME (http://alternate.meme-suite.org/tools/meme),⁶⁸ and TBtools (version 2.069) was used for visualization.⁶⁹

Whole-genome duplication, and synteny analysis

Download genomic and annotated files including Arabidopsis, corn, rice and wheat from the public database phytozome (https://phytozome-next.jgi.doe.gov/). Use the Multiple Collinearity Scan (MCScanX) toolkit to predict collinearity relationships.⁷⁰ Duplicated LuMADS genes were identified as whole genome duplications (WGDs). Tandem repeat genes are two or more homologous genes on a chromosome that are no more than 100kb apart and have no other genes in between.⁸³ The nucleotide BLAST (BLASTN) was used to detect fragment duplicate genes (score <1e-5) and contained a 100 kb range around the coding sequence (CDS)(50 kb each upstream and downstream). Criteria for identifying duplicate genes include sequence alignment lengths greater than 200 bp and sequence similarity greater than 85%.⁸⁴^,⁸⁵

miRNA and cis-acting element prediction

The miRNA sequence of flax comes from previous research results.⁸⁶ By comparing miRNA sequences with the 5′ and 3' untranslated regions (UTRs) and coding sequences (CDS) of all LuMADS genes, the Plant MicroRNA Target Analysis Server (psRNATarget) is used to predict potential target sites for miRNA candidate molecules under default parameters (https://www.zhaolab.org/psRNATarget/analysis?function3).⁷¹ TBtools was used to extract the 2000 bp genomic sequence upstream of all LuMADS genes. To demonstrate the potential regulatory mechanisms of LuMADS genes in response to environmental stress, the PlantCARE database (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/) was used to identify cis-acting regulatory elements in the 2.0 kb nucleotide sequences upstream of the translation start site of LuMADS genes,⁷² and TBtools was used for visualization.

Protein interaction network and GO enrichment

Arabidopsis homologous proteins were used to predict PPI networks and study relationships between LuMADS proteins. The functional protein association network was analyzed through the STRING database (https://string-db.org/) with a confidence value of 0.15. The PPI network is predicted based on default parameters.⁷³ Cytoscape software was used to visualize the protein interaction network.⁷⁴ For GO enrichment analysis of LuMADS genes, we first downloaded the go-base.ob file from TBtools. Then, we used the online website eggnog (http://eggnog-mapper.embl.de/) to obtain the annotation file by inputting the protein information of the whole flax genome. Finally, we used the chiPlot website (https://www.chiplot.online/) for online visualization.

Expression pattern analysis of LuMADS

Five flax RNA-seq datasets were used in this study: (i) pistil, stamen, fruit and shoot tip tissue (NCBI SRA PRJNA1002756) (https://www.ncbi.nlm.nih.gov/sra/?term=); (ii) Flower tissue 30days, 20days, 10days and 5days after flowering (PRJNA833557); (iii) torpedo_embryo, root, ovary, mature_embryo, heart_embryo, globular_embryo, cotylden_embryo, anther and seeds tissue (PRJNA663265); (iv) salt Root and leaf tissue after stress (PRJNA977728)⁸⁰; (v) Stem tissue after heat stress (PRJNA874329). Data were filtered using fastp to map to the Longya10 reference genome and quantified using the tidyverse,⁷⁵^,⁷⁶ Rsubrad,⁷⁷ limma and edgeR R packages.⁷⁸^,⁷⁹ Finally, use TBtools to draw a heatmap of the log2 values of FPKM.

RNA extraction, reverse transcription and qRT-PCR analysis

Total RNA of flax leaves and stem was extracted using the Trizol reagent. The first-strand cDNA was generated from total RNA using PrimeScript RT reagent Kit (Takara). The upstream and downstream primers were designed by primer 5 (Table S3). Perform qRT-PCR experiment using TB Green Pemix Ex TaqTM II (TaKaRa Bio, Kyoto, Japan) fluorescence quantification kit. Real-time PCR was performed using a CFX96 Real-Time PCR System (Applied Biosystems). The PCR reaction system was 25 μL consisting of 2 μL cDNA, 1 μL forward primer, 1 μL reverse primer, 12.5 μL qRT-PCR Master Mix, and 8.5 μL sterilised ddH₂O. The PCR program was: 95°C for 30s, followed by 40 cycles of 95°C for 5s and 60°C for 30s, followed by melting curve analysis. The data were analyzed by the 2^−ΔΔCT method.⁸⁷ Each sample is repeated 3 times, with GAPDH as the internal reference gene to calculate Ct.⁸⁸

Quantification and statistical analysis

Data processing was performed using GraphPad version 9.0 software for statistical analysis. The criteria and sample sizes for each experiment are noted in the legend. Data are presented as the standard error of the mean (SEM). Using Student’s t-test, asterisks denote statistically significant differences (∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001).

Published: October 1, 2024

Footnotes

Supplemental information can be found online at https://doi.org/10.1016/j.isci.2024.111092.

Contributor Information

Zhenyuan Zang, Email: zhenyuanzang1989@163.com.

Jun Zhang, Email: zhangjun@jlau.edu.cn.

Michael K. Deyholos, Email: michael.deyholos@ubc.ca.

Jian Zhang, Email: jian.zhang@ubc.ca.

Supplemental information

Document S1. Figure S1

mmc1.pdf^{(203KB, pdf)}

Table S1. The protein sequences of MADS family genes in Arabidopsis thaliana and Linum usitatissimum L., related to Figure 2

mmc2.xlsx^{(36.7KB, xlsx)}

Table S2. The number and corresponding relationship of cis-acting elements in the LuMADS gene family, related to Figure 6

mmc3.xlsx^{(129.8KB, xlsx)}

Table S3. Primers for LuMADS gene in qRT PCR experiment, related to Figures 9, 10, and 11

mmc4.xlsx^{(11.8KB, xlsx)}

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Document S1. Figure S1

mmc1.pdf^{(203KB, pdf)}

Table S1. The protein sequences of MADS family genes in Arabidopsis thaliana and Linum usitatissimum L., related to Figure 2

mmc2.xlsx^{(36.7KB, xlsx)}

Table S2. The number and corresponding relationship of cis-acting elements in the LuMADS gene family, related to Figure 6

mmc3.xlsx^{(129.8KB, xlsx)}

Table S3. Primers for LuMADS gene in qRT PCR experiment, related to Figures 9, 10, and 11

mmc4.xlsx^{(11.8KB, xlsx)}

Data Availability Statement

•
Data: This paper analyzes existing, publicly available data. These accession numbers for the datasets are listed in the key resources table.
•
This paper does not report original code.
•
Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request.

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PERMALINK

Identification and characterization of MADS-box gene family in flax, Linum usitatissimum L. and its role under abiotic stress

Jianyu Lu

Hanlu Wu

David Michael Pitt

Xinyang Liu

Xixia Song

Hongmei Yuan

Yuntao Ma

Shuyao Li

Zhenyuan Zang

Jun Zhang

Michael K Deyholos

Jian Zhang

Summary

Graphical abstract

Highlights

Introduction

Results

Identification of MADS-box gene family members in flax

Table 1.

Phylogenetic and evolutionary analysis

Figure 1.

Figure 2.

LuMADS gene structure and conserved domains

Figure 3.

Chromosome location and gene duplication events of the LuMADS gene family

Figure 4.

Figure 5.

Analysis of cis-acting elements in promoter of flax MADS-box gene family and miRNA prediction

Figure 6.

Table 2.

LuMADS protein-protein interaction network and GO enrichment analysis

Figure 7.

LuMADS expression pattern analysis based on RNA-seq data

Figure 8.

Expression levels of flax LuMADS genes under abiotic stress

Figure 9.

Figure 10.

Figure 11.

Discussion

Limitations of the study

Resource availability

Lead contact

Material availability

Data and code availability

Acknowledgments

Author contributions

Declaration of interests

STAR★Methods

Key resources table

Experimental model and study participant details

Plant materials and abiotic stress treatment

Method details

Identification of LuMADS Gene family in flax

Phylogenetic Analysis, Chromosomal location, conserved domains and conserved motifs of LuMADS

Whole-genome duplication, and synteny analysis

miRNA and cis-acting element prediction

Protein interaction network and GO enrichment

Expression pattern analysis of LuMADS

RNA extraction, reverse transcription and qRT-PCR analysis

Quantification and statistical analysis

Footnotes

Contributor Information

Supplemental information

References

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

PERMALINK

RESOURCES

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