MIKC-type genes play crucial roles in the growth and development stages of plants. Identifying MIKC-type genes in wheat and exploring their expression patterns under low-temperature conditions lays a foundation for subsequent research on gene function. Bioinformatics websites and software were utilized to identify MIKC-type genes in the wheat transcriptome under low-temperature conditions, and the expression changes of these genes were verified using RT-qPCR methods. A total of 90 MIKC-type genes were identified, which could be classified into two major categories and nine subfamilies, distributed across 21 chromosomes. There were numerous inter-chromosomal duplications of MIKC-type genes in wheat. Transcriptome analysis revealed that under low-temperature conditions, the expression of 14 MIKC-type genes was altered, with seven genes significantly upregulated and three genes significantly downregulated. The validation results for TaMIKC30 and TaMIKC68 were consistent with the transcriptomic data, and the results also indicated that the expression of these two genes differs slightly between different tissues. These findings suggest that MIKC-type genes may be involved in the response of wheat to low-temperature stress.
Research Article
Identification and Low-Temperature Stress Expression Analysis of Wheat MIKC-Type MADS-box Gene Family
https://doi.org/10.21203/rs.3.rs-4304376/v1
This work is licensed under a CC BY 4.0 License
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MIKC gene
Low temperature
Bioinformatics
Wheat
MADS-box gene family is an important class of transcription factors in eukaryotes, which plays a regulatory role in many aspects of plant growth. MADS-box genes contain a highly conserved MADS-box domain composed of approximately 60 amino acids, which can be divided into type I and type II(Becker et al.). Type II genes include MEF2 genes of animals and fungi, and MIKC genes characteristic of plants. The MIKC genes of plants are named for the conserved MIKC structure of their encoded proteins, which usually contains four domains, namely MADS-box (M), Intervening domain (I), Kertain-like domain (K) and C-terminal domain (C). The MIKC genes are divided into MIKCC and MIKC* types. Most MIKC genes in plants are part of MIKCC type, including 12 subfamilies (SVP, SEP, FLC, TM3, AP1/FUL, AG, AGL6, AGL12, AGL15, ANR1, AP3/PI and BS)(Schilling et al. 2020).
MADS-box genes are expressed in various parts of plants, and play fundamental roles in developmental control and signal transduction, including flower and seed development, flowering time control, fruit ripening regulation, and response to abiotic and biological stresses(Bai et al. 2019). For example, MADS-box genes in Physic nut (Jatropha curcas) exhibited significant induction or inhibition of expression in response to drought and salt stresses(Tang et al. 2020). Previous studies have found that the down-regulated expression of OsMADS26 in rice, enhanced the drought resistance of transgenic plants(Khong et al. 2015), and the down-regulated expression of SlMBP8 in tomato also enhanced the resistance of transgenic plants to salt stress(Wencheng et al. 2017). In addition, it is reported that the expression levels of MADS-box genes were significantly induced or inhibited by low temperature treatment in Arabidopsis, wheat and rice, such as FLC, SOC1, AGL19 and SVP in Arabidopsis(Hemming and Trevaskis 2011), and VRN1/TaVRT-1, VRN2 and TaVRT-2 in wheat(Tardif et al. 2007; Winfield et al. 2009). It was found that the expression of OsMADS18, OsMADS22, OsMADS26 and OsMADS27 were significantly up-regulated in seedlings of indica rice (IR64) under drought and low temperature stresses(Rita et al. 2007). And it was showed that overexpression of OsMADS26 in rice and Arabidopsis could reduce the stress response of plants to the external environment, while a series of genes related to stress resistance were significantly up-regulated in OsMADS26 overexpressing lines(Lee et al. 2008; Lee et al. 2003). The silencing of SlMADS23-like in tomato weakened the cold resistance of plants, which means SlMADS23-like plays a positive regulatory role in cold resistance(Wencheng et al. 2017). Transcriptome sequencing on wheat spike under low temperature treatment revealed that there were significant differences in the MIKC gene family(Zhang et al. 2014). The above results show that MADS-box genes play an important role in plant growth and development and in response to low temperature stress.
In recent years, the research on MADS-box genes has been increasingly deepened. Much more studies were conducted in dicotyledonous plants, especially in Arabidopsis and snapdragon, and less in monocot plants(Par̆enicová et al. 2003). In addition, the current studies are mainly limited to the reproductive development process of plants, and there are few studies on the stress resistance process. In this study, the cold-resistant winter wheat variety Dongnong Dongmai No.1 (Dn1) was selected to be exposed under cold stress at different temperatures. The differentially expressed genes (DEGs) of MADS-box family under cold stress were screened, and the expression trend of DEGs in Dn1 was detected. This study will provide a new idea for analyzing the cold stress acclimation and cold resistance molecular mechanism of winter wheat.
1.1 Experimental Materials
The experiment utilized the cold-tolerant winter wheat cultivar "Dongnongdongmai 1" . This cultivar exhibits strong stress resistance and a high regreening rate in high-altitude and cold regions such as Heilongjiang. The experimental materials were field-grown wheat plants, During the planting and growth process, the combined application of nitrogen, phosphorus, and potassium fertilizers is used.Samples were collected from the tillering nodes and leaves after ten consecutive days of average minimum temperatures reaching 5℃, -10℃, and -25℃. Triplicate samples were taken, cleaned, and then snap-frozen in liquid nitrogen before being stored in a -80℃ freezer. After the collection of all samples, some were sent to Novogene Biological Technology Co., LTD for transcriptome sequencing, while others were used for RNA extraction and subsequent analysis.
1.2 Identification and Physicochemical Analysis of MIKC Family Members
The protein sequences of Arabidopsis thaliana MIKC genes were downloaded from the TAIR website based on the transcriptome database. BLAST functionality within TBtools was used to align these sequences against the wheat transcriptome data, identifying potential MIKC gene sequences in wheat. The conserved domain model PF00319 for MADS transcription factors was downloaded from the InterPro website, and HMMER software was employed to identify MADS genes within the transcriptome(Marchler-Bauer et al. 2014). The intersection of the two alignment results was taken, and the NCBI's CD-Search and SMART tools were utilized to further verify the presence of the MADS domain in the obtained sequences. Finally, the Plant Transcription Factor Database (PFDB) was used to predict and classify the transcription factors, obtaining the complete set of MIKC transcription factor sequences in the transcriptome. The sequences were renamed to facilitate subsequent analysis. The physicochemical properties of all sequences were analyzed using the Protein Paramter Calc tool in TBtools.
1.3 Classification, Conserved Motifs, Domains, Gene Structure, and Evolutionary Analysis of MIKC Proteins
Protein sequences of Arabidopsis thaliana MIKC-type transcription factors were downloaded from the PFDB and combined with the identified protein sequences from wheat. MEGA11 software and the Evolgenius website were used to construct phylogenetic trees and classify the sequences. The protein sequences were uploaded to the MeMe website for conserved motif analysis using default parameters with the number of conserved motifs set to 13, and the results were downloaded(Nystrom and McKay 2021; Tamura et al. 2021). Structural domain information and promoter information for the sequences were obtained using the CD-Search website and PlantPAN 3.0 website, respectively. The transcriptome files were organized to obtain gene structure information, and the results were visualized using plugins in TBtools(Gao et al. 2015).
1.4 Chromosome Localization and Collinearity Analysis
Gene density information and the distribution of MIKC sequences on chromosomes were extracted from the transcriptome data, and the results were visualized using plugins TBtools(Chen et al. 2020). The collinearity of MIKC family members in wheat was analyzed using MCScanX.
1.5 Expression Pattern Analysis
FPKM (fragments per kilobase per million) values corresponding to MIKC genes under different temperature conditions were obtained from the sequencing transcriptome data. After processing the results, they were visualized using the Weishengxin platform.
1.6 Gene Expression Analysis
To ensure the reliability of the transcriptome results, primers were designed for two genes TaMIKC68 and TaMIKC30 with significant expression changes based on the expression pattern analysis (Table 1). qPCR was used to verify the results. Additionally, the expression changes of these genes in wheat leaves under different temperature conditions were also experimentally analyzed to compare their expression patterns in different tissues.
2.1 Identification and Physicochemical Properties Analysis of MIKC Family Members
A total of 178 MADS transcription factors were identified from wheat, which was consistent with previous identification results. Of these, 90 are MIKC-type transcription factors, named TaMIKC1-TaMIKC90. Analysis of their physicochemical properties revealed that the wheat MIKC-type protein sequence length ranged from 164 to 274 amino acids, with average molecular weights ranging from 18,145.07 to 31,307.45 kDa, and theoretical isoelectric points (pI) spanning a broad range from 5.12 to 9.77. Of these, 23 proteins are acidic (pI < 7) and 67 are basic (pI > 7). The instability index of the three protein sequences is less than 40, indicating they are stable proteins (TaMIKC65, TaMIKC80, TaMIKC89), and the remaining 87 sequences are unstable proteins. The lipophilicity coefficient is (64.97-96.29), and the average hydrophilicity index for all proteins is less than zero, suggesting that the MIKC proteins are hydrophilic.
Table 2 Physicochemical properties of the MIKC gene family
|
Gene ID |
Gene Name |
Number |
kD |
pI |
InsTability Index |
Aliphatic Index |
Grand Average of Hydropathicity |
|
|
TraesCS1A01G045000 |
TaMIKC1 |
198 |
22832 |
8.77 |
49.09 |
88.13 |
-0.687 |
|
|
TraesCS1A01G199900 |
TaMIKC2 |
228 |
25671.47 |
8.97 |
44.84 |
92.72 |
-0.578 |
|
|
TraesCS1A01G044900 |
TaMIKC3 |
243 |
27695.23 |
7.87 |
51.67 |
87.9 |
-0.705 |
|
|
TraesCS1B01G058300 |
TaMIKC4 |
236 |
26516.87 |
7.11 |
47.97 |
87.2 |
-0.692 |
|
|
TraesCS1B01G058100 |
TaMIKC5 |
243 |
27658.14 |
7.86 |
48.89 |
85.06 |
-0.76 |
|
|
TraesCS1B01G275000 |
TaMIKC6 |
208 |
24361.9 |
9.08 |
65.88 |
73.61 |
-0.876 |
|
|
TraesCS1B01G273300 |
TaMIKC7 |
255 |
28281.09 |
9.41 |
51.37 |
85.49 |
-0.469 |
|
|
TraesCS1B01G214500 |
TaMIKC8 |
218 |
24588.38 |
9.51 |
44.98 |
87.25 |
-0.645 |
|
|
TraesCS1B01G058200 |
TaMIKC9 |
223 |
25613.14 |
8.31 |
57.5 |
91.79 |
-0.625 |
|
|
TraesCS1D01G441200 |
TaMIKC10 |
190 |
21255.96 |
5.61 |
44.21 |
91.32 |
-0.484 |
|
|
TraesCS1D01G045700 |
TaMIKC11 |
236 |
26582.99 |
6.77 |
48.39 |
88.01 |
-0.656 |
|
|
TraesCS1D01G262700 |
TaMIKC12 |
250 |
27647.3 |
9.5 |
50.26 |
83.72 |
-0.481 |
|
|
TraesCS1D01G264500 |
TaMIKC13 |
218 |
25232.25 |
9.72 |
61.29 |
79.63 |
-0.678 |
|
|
TraesCS1D01G045600 |
TaMIKC14 |
234 |
26816.34 |
7.07 |
59 |
92.05 |
-0.701 |
|
|
TraesCS1D01G203400 |
TaMIKC15 |
231 |
25871.71 |
9.13 |
45.82 |
91.13 |
-0.57 |
|
|
TraesCS1D01G045500 |
TaMIKC16 |
243 |
27870.47 |
6.77 |
51.74 |
84.65 |
-0.721 |
|
|
TraesCS2A01G311100 |
TaMIKC17 |
202 |
23393.05 |
6.98 |
58.05 |
87.82 |
-0.489 |
|
|
TraesCS2A01G337900 |
TaMIKC18 |
240 |
27514.24 |
8.46 |
45.49 |
90.96 |
-0.675 |
|
|
TraesCS2B01G200800 |
TaMIKC19 |
267 |
29999.59 |
8.52 |
63.13 |
78.54 |
-0.858 |
|
|
TraesCS2B01G328000 |
TaMIKC20 |
202 |
23511.14 |
6.54 |
58.81 |
87.33 |
-0.518 |
|
|
TraesCS2B01G281000 |
TaMIKC21 |
274 |
31307.45 |
9.14 |
62.44 |
69.89 |
-0.886 |
|
|
TraesCS2B01G344000 |
TaMIKC22 |
240 |
27487.07 |
7.8 |
45.07 |
88.96 |
-0.719 |
|
|
TraesCS2B01G440900 |
TaMIKC23 |
253 |
29317.76 |
7.69 |
59.29 |
83.2 |
-0.543 |
|
|
TraesCS2D01G262700 |
TaMIKC24 |
271 |
30996.15 |
9.14 |
64.64 |
71.03 |
-0.877 |
|
|
TraesCS2D01G325000 |
TaMIKC25 |
240 |
27496.19 |
8.78 |
46.7 |
88.96 |
-0.705 |
|
|
TraesCS2D01G309300 |
TaMIKC26 |
202 |
23393.05 |
6.98 |
58.05 |
87.82 |
-0.489 |
|
|
TraesCS3A01G090300 |
TaMIKC27 |
225 |
25603.84 |
5.19 |
63.31 |
81.11 |
-0.635 |
|
|
TraesCS3A01G090900 |
TaMIKC28 |
227 |
25941.19 |
5.12 |
63.49 |
77.8 |
-0.658 |
|
|
TraesCS3A01G435000 |
TaMIKC29 |
167 |
18547.58 |
9.71 |
51.66 |
64.97 |
-0.924 |
|
|
TraesCS3A01G284400 |
TaMIKC30 |
213 |
24370.99 |
9.4 |
47.48 |
86.06 |
-0.554 |
|
|
TraesCS3B01G318300 |
TaMIKC31 |
196 |
22316.6 |
8.88 |
42.61 |
84.13 |
-0.545 |
|
|
TraesCS3D01G284200 |
TaMIKC32 |
196 |
22330.63 |
8.88 |
42.18 |
84.13 |
-0.544 |
|
|
TraesCS3D01G102700 |
TaMIKC33 |
229 |
26261.98 |
5.94 |
52.85 |
78.38 |
-0.668 |
|
|
TraesCS3D01G090300 |
TaMIKC34 |
219 |
24797.88 |
5.63 |
51.81 |
83.29 |
-0.701 |
|
|
TraesCS3D01G401700 |
TaMIKC35 |
209 |
24084.53 |
7.14 |
46.94 |
83.97 |
-0.717 |
|
|
TraesCS4A01G028200 |
TaMIKC36 |
232 |
26851.59 |
8.71 |
58.09 |
82.41 |
-0.697 |
|
|
TraesCS4A01G028100 |
TaMIKC37 |
242 |
27893.62 |
7.7 |
59.77 |
81.45 |
-0.728 |
|
|
TraesCS4B01G245700 |
TaMIKC38 |
216 |
24954.32 |
7.61 |
54.59 |
79.86 |
-0.695 |
|
|
TraesCS4B01G277800 |
TaMIKC39 |
247 |
28358.03 |
6.76 |
64.12 |
82.96 |
-0.698 |
|
|
TraesCS4B01G245800 |
TaMIKC40 |
247 |
28273.22 |
8.96 |
49.41 |
84.86 |
-0.617 |
|
|
TraesCS4B01G351500 |
TaMIKC41 |
164 |
18145.07 |
7.9 |
61.99 |
66.77 |
-0.831 |
|
|
TraesCS4B01G346700 |
TaMIKC42 |
222 |
24963.71 |
8.61 |
63.67 |
81.31 |
-0.552 |
|
|
TraesCS4D01G243700 |
TaMIKC43 |
225 |
26092.55 |
8.47 |
58.91 |
74.98 |
-0.813 |
|
|
TraesCS4D01G341700 |
TaMIKC44 |
222 |
24796.49 |
8.83 |
61.9 |
86.58 |
-0.505 |
|
|
TraesCS4D01G245200 |
TaMIKC45 |
248 |
28506.12 |
7.67 |
53.7 |
79.88 |
-0.786 |
|
|
TraesCS4D01G276100 |
TaMIKC46 |
241 |
27860.47 |
6.33 |
64.84 |
81.37 |
-0.773 |
|
|
TraesCS4D01G301100 |
TaMIKC47 |
226 |
25363.68 |
5.64 |
59.82 |
86.37 |
-0.571 |
|
|
TraesCS5A01G391800 |
TaMIKC48 |
238 |
27482.57 |
8.8 |
60.72 |
82.35 |
-0.645 |
|
|
TraesCS5A01G286800 |
TaMIKC49 |
252 |
29042.01 |
8.99 |
56.63 |
76.67 |
-0.809 |
|
|
TraesCS5A01G515500 |
TaMIKC50 |
220 |
24563.19 |
8.82 |
67.09 |
88.27 |
-0.515 |
|
|
TraesCS5A01G117500 |
TaMIKC51 |
254 |
28498.53 |
9.33 |
53.68 |
88.07 |
-0.458 |
|
|
TraesCS5A01G391700 |
TaMIKC52 |
244 |
27838.71 |
9.05 |
64.21 |
77.17 |
-0.803 |
|
|
TraesCS5B01G396700 |
TaMIKC53 |
236 |
27285.3 |
7.71 |
56.57 |
82.63 |
-0.615 |
|
|
TraesCS5B01G002400 |
TaMIKC54 |
232 |
26375.1 |
9.39 |
52.89 |
92.11 |
-0.593 |
|
|
TraesCS5B01G115100 |
TaMIKC55 |
252 |
28389.47 |
9.41 |
55.43 |
91.07 |
-0.451 |
|
|
TraesCS5D01G401700 |
TaMIKC56 |
236 |
27207.09 |
8.38 |
54.84 |
79.75 |
-0.634 |
|
|
TraesCS5D01G002100 |
TaMIKC57 |
225 |
26085.34 |
9.77 |
56.46 |
94 |
-0.48 |
|
|
TraesCS5D01G401500 |
TaMIKC58 |
254 |
28772.9 |
8.93 |
64.57 |
77.6 |
-0.711 |
|
|
TraesCS5D01G294500 |
TaMIKC59 |
252 |
29042.01 |
8.99 |
56.63 |
76.67 |
-0.809 |
|
|
TraesCS5D01G118200 |
TaMIKC60 |
252 |
28254.31 |
9.31 |
54.64 |
90.67 |
-0.425 |
|
|
TraesCS6A01G131100 |
TaMIKC61 |
239 |
27127.91 |
8.83 |
44.35 |
70.59 |
-0.555 |
|
|
TraesCS6A01G313800 |
TaMIKC62 |
228 |
25436.59 |
5.64 |
47.8 |
87.28 |
-0.611 |
|
|
TraesCS6A01G158100 |
TaMIKC63 |
252 |
28255.95 |
6.45 |
61.85 |
80.95 |
-0.51 |
|
|
TraesCS6B01G343900 |
TaMIKC64 |
228 |
25450.62 |
5.64 |
49.35 |
87.72 |
-0.61 |
|
|
TraesCS6B01G159400 |
TaMIKC65 |
231 |
26158.68 |
7.71 |
39.85 |
71.34 |
-0.578 |
|
|
TraesCS6B01G186700 |
TaMIKC66 |
253 |
28571.33 |
6.72 |
62.13 |
80.63 |
-0.529 |
|
|
TraesCS6D01G120900 |
TaMIKC67 |
239 |
27156.93 |
8.88 |
42.95 |
70.21 |
-0.566 |
|
|
TraesCS6D01G293200 |
TaMIKC68 |
228 |
25421.62 |
5.64 |
46.95 |
88.99 |
-0.579 |
|
|
TraesCS6D01G147400 |
TaMIKC69 |
252 |
28255.95 |
6.45 |
61.85 |
80.95 |
-0.51 |
|
|
TraesCS7A01G122100 |
TaMIKC70 |
227 |
26286.8 |
8.57 |
47.52 |
77.8 |
-0.783 |
|
|
TraesCS7A01G475000 |
TaMIKC71 |
225 |
25471.6 |
5.57 |
62.88 |
82 |
-0.599 |
|
|
TraesCS7A01G260600 |
TaMIKC72 |
248 |
28732.66 |
8.74 |
52.09 |
76.25 |
-0.803 |
|
|
TraesCS7A01G111700 |
TaMIKC73 |
238 |
27005.66 |
9.07 |
48.01 |
86.13 |
-0.637 |
|
|
TraesCS7A01G122000 |
TaMIKC74 |
227 |
26380.74 |
8.21 |
57.01 |
75.59 |
-0.844 |
|
|
TraesCS7A01G111800 |
TaMIKC75 |
230 |
26210.78 |
7.91 |
52.49 |
89.52 |
-0.646 |
|
|
TraesCS7A01G383800 |
TaMIKC76 |
237 |
26725.2 |
8.42 |
43.55 |
72.49 |
-0.629 |
|
|
TraesCS7A01G111900 |
TaMIKC77 |
230 |
25945.46 |
7.07 |
52.66 |
89.96 |
-0.588 |
|
|
TraesCS7B01G009900 |
TaMIKC78 |
187 |
21888.47 |
9.65 |
54.56 |
92.19 |
-0.512 |
|
|
TraesCS7B01G009500 |
TaMIKC79 |
230 |
26082.6 |
7.03 |
53.72 |
91.22 |
-0.61 |
|
|
TraesCS7B01G220200 |
TaMIKC80 |
229 |
25796.85 |
6.53 |
38.38 |
96.29 |
-0.232 |
|
|
TraesCS7B01G020800 |
TaMIKC81 |
227 |
26196.61 |
8.75 |
55.19 |
75.64 |
-0.806 |
|
|
TraesCS7B01G158600 |
TaMIKC82 |
246 |
28491.37 |
8.74 |
51.65 |
75.28 |
-0.814 |
|
|
TraesCS7B01G009800 |
TaMIKC83 |
230 |
25917.41 |
7.07 |
51.46 |
89.52 |
-0.59 |
|
|
TraesCS7D01G120500 |
TaMIKC84 |
227 |
26203.75 |
8.9 |
58.33 |
80.79 |
-0.741 |
|
|
TraesCS7D01G261600 |
TaMIKC85 |
248 |
28738.7 |
8.74 |
54.09 |
73.1 |
-0.82 |
|
|
TraesCS7D01G106900 |
TaMIKC86 |
230 |
25980.41 |
7.9 |
55.78 |
88.22 |
-0.62 |
|
|
TraesCS7D01G106600 |
TaMIKC87 |
230 |
26114.71 |
7.77 |
56.4 |
91.22 |
-0.608 |
|
|
TraesCS7D01G120600 |
TaMIKC88 |
227 |
26431.88 |
8.58 |
56.36 |
74.76 |
-0.871 |
|
|
TraesCS7D01G315900 |
TaMIKC89 |
232 |
26172.35 |
6.53 |
38.4 |
94.18 |
-0.237 |
|
|
TraesCS7D01G176700 |
TaMIKC90 |
226 |
25217.38 |
5.74 |
52.43 |
81.59 |
-0.57 |
|
2.2 Classification, Conserved Motifs, Structural Domains, Gene Structure, Promoter, and Evolutionary Analysis of MIKC Proteins
The MIKC proteins identified in wheat and Arabidopsis were classified using MEGA11 and Evolgenius software. A phylogenetic tree was constructed using the Neighbor-join (NJ) method to visualize the evolutionary relationships among these proteins (Figure 1). Notably, the MIKC proteins in wheat were categorized into two major groups: MIKCc and MIKC*, with 88 and 2 members, respectively.Based on the classification method used for Arabidopsis MIKC-type proteins, the wheat MIKC-type proteins were further divided into nine subfamilies. Among them, the largest subfamilies were those encoding AG17 and SEP proteins, both containing 21 members. The subfamily encoding PI proteins comprised 20 members. The remaining subfamilies, including SVP, SOC, AG, AG12, and AP1, exhibited more balanced distributions, with most containing five members except the SOC family with six members. The MIKC* subfamily had the smallest number of members, with only two proteins. Analysis of motifs in Figure 2 and Figure 3 revealed that almost all MIKC proteins contained motifs 1, 2, 4, 6, 5, and 3, indicating a high degree of conservation among them. However, slight differences were observed based on family classification, suggesting potential divergence during subsequent evolution due to different functional requirements.Structural domain analysis showed that all MIKC proteins, except for the two MIKC* proteins TaMIKC29 and TaMIKC41, contained both the MEF2 and K-box domains. This finding aligns with the phylogenetic classification and supports the reliability of the classification.Gene structure analysis revealed that the MIKC family proteins had 5-9 exon regions. Except for TaMIKC46, TaMIKC55, TaMIKC56, and TaMIKC57, all other sequences had complete non-coding regions. The results of the structural analysis and the classification results showed some correlation, and the different gene structure may be correlated with the functional differentiation. Promoter analysis showed the presence of numerous light-responsive and hormone-responsive elements in the promoter regions of wheat MIKC genes, including methyl jasmonate, abscisic acid, auxin, gibberellin, and salicylic acid response elements. These response elements were distributed across each gene, suggesting that MIKC genes may be involved in photosynthesis and hormone-related physiological regulation processes in plants. Additionally, some gene promoter regions contained low-temperature response elements, defense stress elements, and seed-specific regulatory elements, indicating that MIKC proteins may also participate in low-temperature stress responses and other adverse stress responses in wheat, thus contributing to plant stress resistance.
2.3 Chromosomal Localization and Collinearity Analysis
As shown in the figure 4, MIKC proteins were distributed across all wheat chromosomes, with the highest distribution on chromosome 7A, with 8 proteins, and the lowest distribution on chromosome 3B, with only one protein. The collinearity analysis results indicated that there were extensive segmental duplications of genes on wheat chromosomes, and within the MIKC family, there were 126 pairs of segmental duplicate genes. However, all of these duplications were interchromosomal, and no tandemly duplicated segments were detected.
2.4 Expression Pattern Analysis
The expression levels of MIKC genes in wheat under different low-temperature conditions were analyzed to investigate the potential involvement of MIKC-type proteins in the wheat's defense against low-temperature stress( figure 5). The results revealed that the expression of most MIKC-type genes did not significantly change when wheat was exposed to low-temperature stress. However, the expression of 14 MIKC genes were altered, with genes such as TaMIKC58, TaMIKC47, TaMIKC62, TaMIKC68, TaMIKC64, TaMIKC13, and TaMIKC17 showing increased expression as temperatures decreased, suggesting their possible role in wheat's resistance to low-temperature stress.The expression of genes like TaMIKC52, TaMIKC50, TaMIKC44, and TaMIKC90 initially increased and then decreased with decreasing temperature. In contrast, the expression of genes TaMIKC30, TaMIKC31, and TaMIKC32 decreased as temperatures dropped, indicating potential negative regulation in wheat's defense against low-temperature stress.To validate the reliability of the transcriptomic data, RT-qPCR analysis was performed on TaMIKC68 and TaMIKC30. The results showed that the expression trends of TaMIKC68 and TaMIKC30 in wheat tillering nodes were consistent with the transcriptomic data, confirming the reliability of the transcriptomic data( figure 6). In the leaves of wheat, the expression trends of these two genes were slightly different compared to the tillering nodes, suggesting that the expression of these genes may vary in different tissues.
MADS transcription factors constitute one of the largest families of transcription factors in plants and are widely involved in various physiological regulation processes, assisting plants in defending against external stressors. MIKC-type transcription factors are one of the two important members of the MADS family and are widely present in plants. To date, MIKC-type genes have been identified in various plants, such as pomegranate(Zhao et al. 2020), peanut(Mou et al. 2022),sweet potato(Shao et al. 2021),ginkgo(Ke et al. 2022), cotton(Ren et al. 2017), and so on, and there have been reports on MIKC-type genes in Poaceae plants, such as rice(Rita et al. 2007), maize(Zou 2021), and rye(Wang et al. 2024). In this study, 90 MIKC genes were identified from wheat, and the physicochemical property analysis revealed that the MIKC proteins in wheat exhibited considerable variation in terms of length, molecular weight, isoelectric point, and other attributes. According to the classification criteria of MIKC proteins from Arabidopsis, the 90 MIKC genes in wheat could be categorized into nine subfamilies. Additionally, the analysis of conserved motifs and domains indicated that there were slight differences in conserved motifs and domains among different subfamilies, suggesting that these proteins might have undergone divergence during the evolutionary process of wheat.
It has been found that MIKC-type genes are widely present in these plants and play important roles. For example, the overexpression of the wheat MIKC-type transcription factor TaMADS32 in Arabidopsis thaliana can enhance the plant's resistance to temperature and drought stress(Xuemei et al. 2022). Studies have found that under drought stress, several MIKC-type genes in rice show significant changes in expression. For instance, the expression of OsMADS26 decreases in drought conditions(Khong et al. 2015), and overexpression plants have been found to have lower yields than wild-type plants. The expression of rice OsMADS23 and OsMADS25 is influenced by external drought conditions, and the expression of rice OsMADS18, OsMADS22, OsMADS26, and OsMADS27 genes significantly changes when exposed to drought treatment(Puig et al. 2013). Additionally, related studies have shown that overexpression of OsMADS25 in rice seedlings can significantly improve the plant's tolerance to low-temperature stress(Yan et al. 2021). Research has also found that MIKC-type transcription factors can enhance plants' ability to resist salt stress; after treating with NaCl, the expression of LcMADS9 in Leymus chinensis significantly increased, suggesting that this gene may be involved in resisting salt stress(Jia et al. 2018). Once induced, transcription factors can activate or repress the expression of downstream functional genes, playing a role in plants' resistance to salt stress. For example, overexpression of the pepper CaMADS gene in Arabidopsis thaliana can increase seed germination rates under high salt conditions and increase root length and fresh weight of the plant(Chen et al. 2019).Through the analysis of the wheat transcriptome under low-temperature conditions, this study found that the expression of 14 MIKC genes altered in response to cold stress. Specifically, the expression levels of TaMIKC58, TaMIKC47, TaMIKC62, TaMIKC68, TaMIKC64, TaMIKC13, and TaMIKC17 increased with decreasing temperature. The expression of genes such as TaMIKC52, TaMIKC50, TaMIKC44, and TaMIKC90 initially rose but then declined, while the expression of TaMIKC30, TaMIKC31, and TaMIKC32 decreased as the temperature dropped. To validate the reliability of the transcriptomic data, qPCR experiments were conducted on the TaMIKC30 and TaMIKC68 genes. The results showed that the expression of TaMIKC30 reached its peak at 5℃ in the tillering node, with minimal differences observed at -10℃ and -25℃, exhibiting a trend of initial decline followed by stabilization. The expression of TaMIKC68 gradually increased with decreasing temperature. In the leaves, the expression of both genes displayed a pattern of initial increase followed by a decrease. Based on these findings, it can be inferred that MIKC proteins in wheat might be involved in the regulatory process of wheat's response to low-temperature stress.
Related reports on the identification and analysis of wheat genes are relatively few, mainly due to the large genome size of hexaploid wheat, which complicates sequencing and related work. After the publication of the whole-genome sequencing data of wheat in 2018, studies on wheat gene identification gradually increased. In this study, transcriptomic sequencing technology was used to obtain wheat transcriptome data under low-temperature conditions, and with the help of bioinformatics analysis software, 90 MIKC-related genes were identified and analyzed in terms of classification, structure, and physicochemical properties. The results showed that both types of MIKC genes exist in wheat, but MIKC* types are less common, which is similar to the identification results of MIKC-type genes in most plants. Phylogenetic analysis revealed that MIKC-type genes within the same family often show certain correlations in terms of conserved motifs and gene structure. In the promoter region of wheat MIKC-type genes, there are a large number of hormone response elements and stress response elements, indicating that MIKC-type genes may play a role in hormone response and stress resistance. This is supported by current research, and transcriptomic analysis and RT-qPCR results showed that the expression of some MIKC-type genes is temperature-related and may be involved in low-temperature regulation. However, the specific pathways through which they function still need to be further confirmed by experimental studies.
Author Contribution
Zhang. Wang. and Chen. Writing - Original DraftCao .Yang .and Shao .Yu . Jin . ResourcesLiu. Writing - Review & Editing
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No competing interests reported.