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. 2026 Apr 10;10(4):e70131. doi: 10.1002/pld3.70131

Genome‐Wide Identification of MYB Genes and Analysis of Their Expression Under Cold Stress Conditions in Artocarpus heterophyllus

Xiangwei Ma 1,2,3, Pengjin Zhu 1,2,3,✉, Weiyan Ye 1,2,3, Chenxin Yi 1,2,3, Xiuguan Tang 1,2, Jianjun Liang 1,2, Zhuangmin Wei 1,2, Qiqi Song 1,2, Hailan Zhou 1,2, Shengli Tang 1,2
PMCID: PMC13067916  PMID: 41971348

ABSTRACT

Jackfruit ( Artocarpus heterophyllus ) thrives in subtropical and tropical areas but is vulnerable to winter cold stress. The myeloblastosis (MYB) transcription factor family plays an important role in plant biological and abiotic stress responses; however, their response to regional cold injury in jackfruit remains unexplored. The study aimed to identify and analyze the expression of 298 MYB genes in jackfruit under cold stress conditions. The study used genome‐wide identification, bioinformatics analysis, and qPCR to analyze the expression of 298 MYB genes in jackfruit under cold stress. Evolutionary tree analysis showed that the AhMYB family members were divided into seven subfamilies. Chromosome mapping results showed that 298 AhMYB family members were unevenly distributed on 27 chromosomes. In addition, analysis of conserved motifs and gene structure showed that members of the AhMYB family located in the same subfamily had similar conserved motifs and gene structure. Collinear analysis identified 1439 duplicated fragments involving AhMYB family members within the species. In addition, the cis‐acting elements in the promoters of AhMYB family members are implicated in many aspects of plant growth and development. Transcriptomic analysis of jackfruit under low‐temperature stress showed that the expression patterns of AhMYB family members differed in jackfruit varieties. qPCR analysis further verified this result, confirming that AhMYB family members are involved in the response to low‐temperature stress in jackfruit varieties. These findings provide new insights into the functions of AhMYB family members.

Keywords: Artocarpus heterophyllus , cold stress, expression profile, myeloblastosis (MYB)

Abbreviations

BLAST

Basic Local Alignment Search Tool

CAT

catalase

CDS

coding DNA sequence

ERE

ethylene response elements

FPKM

fragments per kilobase of transcript per million mapped reads

LTR

low‐temperature‐responsive

ROS

reactive oxygen species

SOD

superoxide dismutase

TFs

transcription factors

WGCNA

weighted gene co‐expression network analysis

WT

wild‐type

1. Introduction

Transcription factors (TFs) are commonly found in eukaryotes. They form transcription initiation complexes with RNA polymerase and participate in the process of transcription initiation. As one of the largest families of multifunctional TFs in plants, MYB proteins are widely involved in regulating plant growth and development, hormone signal transduction, and stress response at the gene transcription level. MYB TFs have a conserved MYB domain at the N‐terminal, which typically comprises one to four incomplete repeats (R). Each incomplete repeat sequence is the basic unit of the DNA‐binding domain of MYB proteins, consisting of 50–53 amino acid residues and forming a 3α‐helical structure that is favorable for the effective binding of MYB TFs and target DNA. The C‐terminal of MYB protein is highly variable, enabling MYB TFs to perform diverse biological functions. The MYB TF family is divided into four subfamilies based on the number of repeats: 1R‐MYB, R2R3‐MYB, 3R‐MYB, and 4R‐MYB. Most MYB TFs are R2R3‐MYB type, which is also the most studied type, with wide distribution and numerous functions. 1R‐MYB type TFs are the second largest group in the MYB family, usually containing one or part of the repeat sequence, while the 4R‐MYB type is the smallest group, found only in very few plants. Previous studies have shown that MYB TFs play an important role in resistance to cold stress in higher plants. For example, overexpression of R2R3‐type MYB genes can enhance cold stress tolerance in rice ( Oryza sativa ) (Yang et al. 2012). Additionally, Malus baccata MbMYB4 overexpression in Arabidopsis significantly improved the cold resistance of transgenic plants. Under cold stress, transgenic Arabidopsis thaliana exhibited increased proline and chlorophyll contents, as well as elevated peroxidase and catalase (CAT) activities, whereas malondialdehyde (MDA) content and relative conductivity significantly decreased (Yao et al. 2022). Tang et al. (2023) revealed that the expression levels of 31 R2R3‐MYB genes increased significantly after cold stress, and transcriptome and weighted gene co‐expression network analysis (WGCNA) showed that SaMYB098, SaMYB015, and SaMYB068 played an important role in the cold resistance process of Santalum album . Overexpression of Pyrus betulaefolia PbrMYB5 in tobacco (Nicotiana benthamiana) increased plant tolerance to low‐temperature stress, whereas its silencing increased the sensitivity of Pyrus betulaefolia to low‐temperature stress (Xing et al. 2019). In addition, Glycine max GmMYBJ1 expression has been induced by low‐temperature stress. A. thaliana overexpressing GmMYBJ1 showed increased tolerance to cold stress compared with wild‐type (WT) plants (Su et al. 2014).

Jackfruit ( Artocarpus heterophyllus Lam.) is an evergreen tree belonging to the mulberry family. Also known as wood pineapple, it is native to India and widely distributed in South Asia, Southeast Asia, Oceania, and other places (Ullah and Haque 2008). It thrives in tropical climates and is commonly cultivated in Guangdong, western Yunnan, Guangxi, and Hainan in China (Elevitch and Manner 2006). Jackfruit can be consumed fresh or processed into preserved fruit and jam (Li et al. 2010). In addition, jackfruit seeds and skins have important applications in the food, pharmaceutical and environmental industries, making it an important tropical agroforestry tree (Zeng et al. 2022). Currently, studies have been conducted on the physiology and biochemistry (Nantongo et al. 2022), nutritive chemical composition (Alves Evaristo et al. 2022), and industrial preparation (Maity et al. 2022) and applications (Annzia et al. 2019) of jackfruit. In addition, progress has been made in gene transcription, expression, and germplasm resources characterization. Jackfruit is adapted to hot and moist tropical climates, with temperature being the key factor affecting its growth and development. When temperatures are lower than 5°C–7°C, jackfruit trees are susceptible to flower and fruit drop (Song et al. 2021). Frequent cold damage in winter has become one of the important factors limiting the development of the jackfruit industry. The identification of superior cold‐resistant gene resources, combined with modern molecular breeding methods, can enhance the selection efficiency of superior cold‐resistant varieties and provide important guidance for their improvement and breeding (Du et al. 2023).

MYB genes have been widely reported in higher plants, but the genome‐wide identification of MYB gene family members in jackfruit and their expression patterns under abiotic stress have not been fully elucidated. In this study, 298 AhMYB genes were identified using the reported genome of “S10” jackfruit (Lin et al. 2022). The basic physicochemical properties, phylogenetic relationships, conserved motifs, gene structure, chromosome localization, and collinearity of these genes were analyzed, and their expression patterns in different jackfruits under cold stress were evaluated. The results of this study enhance our understanding of AhMYB and provide insights for breeding novel cold stress‐resistant jackfruit strains.

2. Materials and Methods

2.1. Plant Material

The experimental material included a local Guangxi jackfruit strain (GX) and an introduced Thai jackfruit strain (THA), both preserved in the germplasm resource nursery of the Guangxi Institute of Subtropical Crops, located at 108°20′34.8″ E and 2°53′56.4″ N. The study was conducted under natural conditions (February 19–24, 2022), following a period of low‐temperature stress in Nanning City, where daily minimum and maximum temperatures reached 3°C and 15°C, respectively. The minimum and maximum daily temperatures at the jackfruit crown were 3.5°C and 14°C, respectively. The leaves of both strains were collected (counting the first leaf on each branch from top to bottom) and stored at −80°C for RNA extraction. Three biological replicates were set up for each experiment.

2.2. Identification and Physicochemical Properties of MYB Gene Family in A. heterophyllus

The “S10” jackfruit genome (Lin et al. 2022) was downloaded from the National Center for Biotechnology Information (NCBI) Sequence Read Archive (Schoch et al. 2020) under accession numbers PRJNA788174 and PRJNA791757. WRKY gene family sequences of A. thaliana were downloaded from TAIR (Swarbreck et al. 2007) (https://www.arabidopsis.org/index.jsp) and used as probe sequences (E < 0.001) for Basic Local Alignment Search Tool (BLAST) analysis. The MYB conserved domain (PF00249) was retrieved from Pfam (http://pfam.xfam.org/) for further annotation of jackfruit MYB gene family members. The physicochemical properties of jackfruit WRKY gene family members were analyzed using the Protein Parameter Calculation function in TBtools (Chen et al. 2020).

2.3. Phylogenetic Tree Analysis

The phylogenetic tree of the jackfruit MYB family was constructed using the neighbor‐joining method in MEGAX (Mega 2018) with 1000 bootstrap replicates. The tree was visually enhanced using iTOL (Letunic and Bork 2021) (https://itol.embl.de).

2.4. Conserved Motif and Gene Structure Analysis

The conserved motifs within the MYB gene family were analyzed using MEME (Letunic and Bork 2021) (http://meme‐suite.org/tools/meme). The jackfruit genome GFF3 file was used for gene structure reference. Visualization was performed using the Gene Structure Display Server function in TBtools with default parameters.

2.5. Chromosomal Mapping and Collinearity Analysis

The chromosomal locations of jackfruit MYB gene family members were visualized using the “Gene Location Visualization from GTF/GFF function” in TBtools based on the jackfruit genome GFF3 file. Collinear gene pairs within the AhMYB family were identified using the “One Step MCScanX” function in TBtools, and the results were visualized using the Advanced Circos feature of TBtools software.

2.6. Analysis of cis‐Acting Elements of MYB Gene Family Promoter in A. heterophyllus

The 2000‐bp upstream nucleotide sequences of jackfruit MYB gene family members were extracted using TBtools. Promoter cis‐acting elements were identified using PlantCARE (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/), and the results were visualized using TBtools.

2.7. Expression Pattern Analysis of MYB Gene Family Under Low‐Temperature Stress in A. heterophyllus

Fragments per kilobase of transcript per million mapped reads (FPKM) values of the AhMYB gene in GX and THA jackfruit were extracted from the published transcriptome data of different varieties under low‐temperature stress, and heat maps were constructed using the “HeatMap” function in TBtools. The total RNA of GX and THA jackfruit was extracted using a HiScript Q RT SuperMix for qPCR (+gDNA wiper) kit (Novizan, Nanjing, China). cDNA was synthesized using a ChamQ SYBR qPCR Master Mix kit (Novizan), diluted tenfold, and used as an amplification template. Quantitative real‐time PCR (qPCR) was conducted using a QuantStudio 5 Real‐time PCR instrument (Thermo Fisher Scientific, Waltham, MA, USA; Table 1), and Tubulin was used as the reference gene for the qPCR of different varieties under low‐temperature stress. The reaction procedure was as follows: predenaturation at 95°C for 30 s, denaturation at 95°C for 10 s, annealing at 60°C for 30 s, and extension at 72°C for 30 s. The process consisted of 50 cycles repeated thrice. The relative expression of the AhMYB gene was calculated using the 2 − ∆∆Ct method.

TABLE 1.

Basic parameters analysis of AhMYB gene family.

Gene name Gene ID Number of amino acid Molecular weight (kDa) Theoretical pI Instability index Aliphatic index Grand average of hydropathicity
AhMYB1 AHE.Chr01.1108 82 9.25 9.57 73.47 51.22 −1.08
AhMYB2 AHE.Chr01.514 352 39.22 5.82 55.5 80.45 −0.523
AhMYB3 AHE.Chr01.701 376 41.19 6.12 44.42 67.02 −0.595
AhMYB4 AHE.Chr01.752.2 294 32.2 9.65 35.64 88.27 −0.451
AhMYB5 AHE.Chr01.857 324 34.56 8.92 59.65 68.33 −0.442
AhMYB6 AHE.Chr02.352 121 13.96 8.3 55.6 49.17 −1.26
AhMYB7 AHE.Chr02.660 330 35.65 8.75 60.09 70.91 −0.446
AhMYB8 AHE.Chr02.818 407 44.85 8.55 57.32 61.55 −0.889
AhMYB9 AHE.Chr03.1146 314 34.69 9.28 42.5 65.22 −0.474
AhMYB10 AHE.Chr03.278 842 93.74 5.11 53.17 68.8 −0.606
AhMYB11 AHE.Chr03.345 269 29.63 6.59 48.38 62.38 −0.653
AhMYB12 AHE.Chr03.390 275 30.29 9.71 57.27 59.6 −0.709
AhMYB13 AHE.Chr03.440 288 31.63 7.65 49.33 66.56 −0.788
AhMYB14 AHE.Chr03.491 263 30.35 5.48 51.32 78.17 −0.871
AhMYB15 AHE.Chr03.492 321 36.05 8.84 36.55 78.69 −0.672
AhMYB16 AHE.Chr03.563 765 84.29 6.07 52.47 58.48 −0.872
AhMYB17 AHE.Chr03.725 326 37.09 8.99 37.31 73.87 −0.822
AhMYB18 AHE.Chr04.1172 491 53.68 6.71 60.91 61.3 −0.858
AhMYB19 AHE.Chr04.1351 327 35.99 9.38 42.75 64.46 −0.513
AhMYB20 AHE.Chr04.1431 330 36.82 8.67 54.42 65.7 −0.646
AhMYB21 AHE.Chr04.311 966 107.54 5.27 48.82 69.61 −0.621
AhMYB22 AHE.Chr04.420 269 29.57 6.7 40.47 62.75 −0.657
AhMYB23 AHE.Chr04.489 304 30.09 9.58 48.77 65.46 −0.61
AhMYB24 AHE.Chr04.526 300 33.02 7.63 56.45 64.9 −0.717
AhMYB25 AHE.Chr04.566 277 32.21 5.57 38.02 75.31 −0.848
AhMYB26 AHE.Chr04.567 337 38.06 8.9 44.2 73.8 −0.739
AhMYB27 AHE.Chr04.885 335 38.03 8.66 37.95 68.09 −0.944
AhMYB28 AHE.Chr04.945 73 8.91 6.11 91.91 77.53 −0.556
AhMYB29 AHE.Chr04.947 73 8.91 6.11 91.91 77.53 −0.556
AhMYB30 AHE.Chr05.1006 183 20.84 5.04 62.69 67.7 −0.634
AhMYB31 AHE.Chr05.1086 419 40.03 8.07 46.8 72.63 −0.591
AhMYB32 AHE.Chr05.1460 473 51.49 7.02 61.6 57.36 −0.633
AhMYB33 AHE.Chr05.1769 326 36.68 6.38 41.42 69.75 −0.675
AhMYB34 AHE.Chr05.379 233 26.79 9.71 57.55 67.85 −0.793
AhMYB35 AHE.Chr06.1138 454 49.47 6.99 58.89 59.36 −0.668
AhMYB36 AHE.Chr06.1482 312 35.28 5.95 52.17 68.14 −0.724
AhMYB37 AHE.Chr06.1496 366 41.12 7.55 43.15 75.14 −0.486
AhMYB38 AHE.Chr06.370 387 43.33 5.44 47.88 67.65 −0.758
AhMYB39 AHE.Chr06.481 625 68.56 5.41 43.92 79.28 −0.463
AhMYB40 AHE.Chr06.673 257 29.65 8.19 59.93 69.84 −0.796
AhMYB41 AHE.Chr07.117 380 42.77 8.03 51.76 68.5 −0.614
AhMYB42 AHE.Chr07.1256 363 41.27 6.1 43.34 77.11 −0.707
AhMYB43 AHE.Chr07.1342 158 18.05 9.3 76.1 54.43 −1.169
AhMYB44 AHE.Chr07.1457 312 34.55 8.91 52.9 78.59 −0.545
AhMYB45 AHE.Chr07.1463 293 33.22 8.93 40.28 76.55 −0.689
AhMYB46 AHE.Chr07.2079 787 83.82 4.96 49.12 71.26 −0.682
AhMYB47 AHE.Chr07.2321 246 26.42 8.67 42.03 55.57 −0.661
AhMYB48 AHE.Chr07.2410 1092 121.6 5.36 58.27 66.08 −0.589
AhMYB49 AHE.Chr07.425 87 10.01 6.73 55.42 63.91 −0.893
AhMYB50 AHE.Chr07.441 85 9.5 9.05 60.51 74.59 −0.642
AhMYB51 AHE.Chr07.593.1 687 75.77 6.03 37.44 74.18 −0.592
AhMYB52 AHE.Chr07.623 373 42.69 5.08 53.55 69.33 −0.857
AhMYB53 AHE.Chr07.731 310 33.89 9.27 59.27 74.87 −0.555
AhMYB54 AHE.Chr07.863 401 44.24 5.72 54.8 73.49 −0.591
AhMYB55 AHE.Chr07.872 334 37.19 7.08 48.81 68.08 −0.642
AhMYB56 AHE.Chr07.897 311 34.32 9.69 46.89 63.02 −0.584
AhMYB57 AHE.Chr07.972 302 34.35 9.07 45.41 74.93 −0.867
AhMYB58 AHE.Chr07.975 203 23.31 9.56 55.66 72.07 −0.893
AhMYB59 AHE.Chr08.1057 345 39.15 6.2 47.75 81.42 −0.587
AhMYB60 AHE.Chr08.1093 377 43.05 9.24 58.53 57.24 −0.942
AhMYB61 AHE.Chr08.1204 231 26.81 9.39 44.83 99.61 −0.545
AhMYB62 AHE.Chr08.1363 309 34.57 9.29 55.84 71.75 −0.664
AhMYB63 AHE.Chr08.1368 70 7.91 9.64 29.84 66.86 −0.704
AhMYB64 AHE.Chr08.1369 174 19.88 8.63 58.38 78.45 −0.7
AhMYB65 AHE.Chr08.1650 724 77.38 5.18 43.33 68.29 −0.731
AhMYB66 AHE.Chr08.2021 261 28.83 8.98 53.75 57.28 −0.759
AhMYB67 AHE.Chr08.2134 1043 115.14 5.28 60.27 62.21 −0.682
AhMYB68 AHE.Chr08.2364 314 33.99 6.6 49.26 76.66 −0.533
AhMYB69 AHE.Chr08.301 100 11.17 9.26 52.89 76.1 −0.632
AhMYB70 AHE.Chr08.315 94 10.93 7.99 56.37 71.7 −0.902
AhMYB71 AHE.Chr08.532 383 43.59 4.81 45.72 69.79 −0.845
AhMYB72 AHE.Chr08.668 309 33.76 9.13 59.36 75.15 −0.541
AhMYB73 AHE.Chr08.741 393 43.59 6.42 50.84 70.28 −0.694
AhMYB74 AHE.Chr08.752 331 36.93 8.34 48.91 70.73 −0.641
AhMYB75 AHE.Chr08.783 354 39.02 10.14 46.34 61.41 −0.62
AhMYB76 AHE.Chr08.869 308 35.22 8.94 46.69 71.56 −0.894
AhMYB77 AHE.Chr08.874 202 23.42 9.11 61.08 72.92 −0.968
AhMYB78 AHE.Chr09.1449 398 44.32 5.93 58.59 63.47 −0.908
AhMYB79 AHE.Chr09.1579 95 10.6 9.22 67.31 54.42 −0.98
AhMYB80 AHE.Chr09.1585.1 103 11.53 7.92 60.54 70.1 −0.762
AhMYB81 AHE.Chr09.170 528 58.02 4.89 55.67 70.38 −0.746
AhMYB82 AHE.Chr09.1996 342 37.82 6.51 40.21 66.7 −0.813
AhMYB83 AHE.Chr09.2109 304 34.66 6.46 50.52 62.83 −0.852
AhMYB84 AHE.Chr09.2112 270 31.29 8.75 52.25 71.85 −0.797
AhMYB85 AHE.Chr09.361 267 29.91 9.07 48.58 73.48 −0.656
AhMYB86 AHE.Chr09.580 274 31.44 5.88 56.38 66.24 −0.782
AhMYB87 AHE.Chr09.6 522 57.26 4.74 53.1 70.8 −0.743
AhMYB88 AHE.Chr10.106 431 48.91 5.52 40.25 72.16 −0.815
AhMYB89 AHE.Chr10.1436 95 10.63 9.19 66.04 54.42 −0.998
AhMYB90 AHE.Chr10.2043 346 38.52 5.78 45.09 66.47 −0.839
AhMYB91 AHE.Chr10.264 266 29.72 8.61 46.35 70.08 −0.698
AhMYB92 AHE.Chr10.500 242 27.57 8.98 57.53 65.29 −0.73
AhMYB93 AHE.Chr10.508 265 30.45 9.12 55.42 70 −0.74
AhMYB94 AHE.Chr10.572 493 54.25 5.09 64.37 64.26 −0.749
AhMYB95 AHE.Chr10.67 513 56.61 4.91 57.39 66.14 −0.76
AhMYB96 AHE.Chr10.87 431 49.02 5.45 43.55 71.69 −0.822
AhMYB97 AHE.Chr11.1004 307 34.35 9.47 51.61 58.14 −0.723
AhMYB98 AHE.Chr11.1025 473 52.66 5.65 51.71 67.04 −0.782
AhMYB99 AHE.Chr11.1311 495 54.79 7.71 46.81 61.7 −0.76
AhMYB100 AHE.Chr11.184 366 41.18 5.83 48.34 69.75 −0.699
AhMYB101 AHE.Chr11.1847 347 38.86 7.64 46.53 67.69 −0.862
AhMYB102 AHE.Chr11.362 264 30.14 9.47 36.62 71.36 −0.655
AhMYB103 AHE.Chr11.648 793 91.65 7.76 36.63 49.75 −1.523
AhMYB104 AHE.Chr11.657 476 54.01 6.85 64.3 69.58 −0.792
AhMYB105 AHE.Chr11.980 329 35.86 5.84 43.66 69 −0.614
AhMYB106 AHE.Chr12.1020_AHE.Chr12.1021 310 34.57 9.54 55.56 60.06 −0.734
AhMYB107 AHE.Chr12.1287 488 53.49 7.74 50.91 66.19 −0.722
AhMYB108 AHE.Chr12.1296 488 53.49 7.74 50.91 65 −0.735
AhMYB109 AHE.Chr12.15 254 28.97 6.92 40.7 69.13 −0.919
AhMYB110 AHE.Chr12.184 322 36.12 5.15 49.05 67.8 −0.766
AhMYB111 AHE.Chr12.2089 325 36.49 5.11 51.12 74.12 −0.699
AhMYB112 AHE.Chr12.401 966 108.86 5.45 52.47 71.51 −0.934
AhMYB113 AHE.Chr12.404 270 30.48 9.36 44.01 70.19 −0.581
AhMYB114 AHE.Chr12.599 422 47.24 6.46 52.07 54.34 −0.792
AhMYB115 AHE.Chr12.634 543 61.24 5.5 59.05 71.2 −0.658
AhMYB116 AHE.Chr12.827 307 35.01 5.36 57.23 59.06 −0.861
AhMYB117 AHE.Chr13.1074 319 34.21 9.2 67.76 69.12 −0.45
AhMYB118 AHE.Chr13.1123 251 29.01 8.39 47.31 77.65 −0.831
AhMYB119 AHE.Chr13.586 381 42.51 8.26 71.25 55.83 −1.005
AhMYB120 AHE.Chr13.919 401 43.56 6.12 43.74 68.88 −0.505
AhMYB121 AHE.Chr13.931 501 55.12 6.54 54.82 60.5 −0.576
AhMYB122 AHE.Chr13.935 349 39.32 6.23 41.66 70.92 −0.903
AhMYB123 AHE.Chr14.1095 323 36.37 6.26 62.99 71.61 −0.638
AhMYB124 AHE.Chr14.1200 338 38.76 6.8 46.38 73.61 −0.696
AhMYB125 AHE.Chr14.1270 678 73.06 6.89 50.47 84.65 −0.287
AhMYB126 AHE.Chr14.396 329 35.43 9.02 67.76 64.98 −0.559
AhMYB127 AHE.Chr14.560 510 56.28 6.16 53.55 60.8 −0.605
AhMYB128 AHE.Chr14.580 422 45.42 6.27 43.07 65.55 −0.468
AhMYB129 AHE.Chr14.967.1 248 27.88 8.91 38.95 83.02 −0.728
AhMYB130 AHE.Chr15.134 412 45.67 5.63 51.45 61.07 −0.831
AhMYB131 AHE.Chr15.1459 293 31.11 6.84 53.05 58.29 −0.695
AhMYB132 AHE.Chr15.66 350 39.5 6.49 52.09 68.29 −0.678
AhMYB133 AHE.Chr15.822 303 34.41 6.07 46.1 74.26 −0.493
AhMYB134 AHE.Chr15.923 281 32.91 9.13 66.4 69.47 −0.905
AhMYB135 AHE.Chr16.1080 310 33.47 5.81 44.02 85.68 −0.259
AhMYB136 AHE.Chr16.121 673 73.49 5.65 39.59 80.09 −0.441
AhMYB137 AHE.Chr16.131 376 42.19 6.11 48.48 62.74 −0.924
AhMYB138 AHE.Chr16.1463 293 31.24 6.55 52.44 63.58 −0.607
AhMYB139 AHE.Chr16.1629 311 33.89 6.52 66.23 57.4 −0.745
AhMYB140 AHE.Chr16.1647 310 33.84 9.5 37.83 90 −0.342
AhMYB141 AHE.Chr16.1710 394 43.02 5.79 43.54 67.13 −0.593
AhMYB142 AHE.Chr16.779 287 32.59 6.01 52.55 60.49 −0.763
AhMYB143 AHE.Chr16.933 274 32.05 9.13 68.12 73.72 −0.856
AhMYB144 AHE.Chr17.1290 289 33.49 7.15 56.41 67.82 −0.82
AhMYB145 AHE.Chr17.1291 344 38.31 5.76 54.9 71.72 −0.461
AhMYB146 AHE.Chr17.204 376 41.77 9.12 43.96 65.59 −0.789
AhMYB147 AHE.Chr17.390 589 64.73 8.67 58.31 62.43 −0.742
AhMYB148 AHE.Chr17.999 335 36.12 8.23 62.62 56.75 −0.696
AhMYB149 AHE.Chr18.1392 590 64.87 8.31 55.03 65.64 −0.713
AhMYB150 AHE.Chr18.1495 312 35.27 9.13 49.65 59.97 −0.771
AhMYB151 AHE.Chr18.232 70 7.83 5.6 72.19 82.14 −0.597
AhMYB152 AHE.Chr18.373 602 67.07 8.17 51.47 66.76 −0.65
AhMYB153 AHE.Chr18.452 89 10.69 6.61 73.1 62.58 −1.078
AhMYB154 AHE.Chr18.769 326 35.45 6.83 51.81 61.32 −0.624
AhMYB155 AHE.Chr18.8 325 36.57 5.11 51.25 73.82 −0.719
AhMYB156 AHE.Chr18.930 272 30.82 8.79 34.9 72.79 −0.767
AhMYB157 AHE.Chr19.1337 256 29.58 9.15 55.87 69.77 −0.792
AhMYB158 AHE.Chr19.1375 704 80.72 5.36 58.79 70.81 −0.745
AhMYB159 AHE.Chr19.173 398 45.73 5.11 52.28 66.58 −0.954
AhMYB160 AHE.Chr19.29 305 34.47 5.41 64.9 70.62 −0.751
AhMYB161 AHE.Chr19.49 381 42.56 5.95 56.98 62.23 −0.664
AhMYB162 AHE.Chr19.505 418 46.88 6.62 43.5 62.56 −0.592
AhMYB163 AHE.Chr19.556 432 50.04 7.23 46.22 65.83 −0.841
AhMYB164 AHE.Chr19.571 319 36.19 5.93 73.69 65.02 −0.716
AhMYB165 AHE.Chr19.580 134 15.92 9.55 49.46 75.67 −0.806
AhMYB166 AHE.Chr19.664 296 32.74 9.27 50.62 82.43 −0.516
AhMYB167 AHE.Chr19.675 318 34.49 6.56 49.88 73.24 −0.583
AhMYB168 AHE.Chr19.739 318 34.6 6.56 50.62 73.24 −0.596
AhMYB169 AHE.Chr19.862 352 39.09 6.62 48.58 68.27 −0.774
AhMYB170 AHE.Chr19.879 340 37.01 5.95 53.29 64.82 −0.729
AhMYB171 AHE.Chr19.880 442 48.2 6.25 47.3 70.88 −0.651
AhMYB172 AHE.Chr19.881 349 39.29 6.03 51.83 69.37 −0.725
AhMYB173 AHE.Chr20.1240 556 62.27 8.78 52.68 68.54 −0.535
AhMYB174 AHE.Chr20.1252 256 29.45 7.67 53.24 67.46 −0.832
AhMYB175 AHE.Chr20.1270 297 33.67 8.28 63.44 63.37 −0.733
AhMYB176 AHE.Chr20.1303.1 327 35.79 6.97 64.65 62.02 −0.689
AhMYB177 AHE.Chr20.1474 1383 155.31 8.04 54.15 71.56 −0.786
AhMYB178 AHE.Chr20.180 399 45.89 5.23 53.53 67.64 −0.962
AhMYB179 AHE.Chr20.494 418 46.82 6.37 43.67 64.21 −0.594
AhMYB180 AHE.Chr20.555 456 52.5 6.11 48.63 63.27 −0.858
AhMYB181 AHE.Chr20.595 208 23.73 7.03 49.96 64.18 −0.739
AhMYB182 AHE.Chr20.654 370 41.14 6.15 51.44 79.11 −0.484
AhMYB183 AHE.Chr20.671 312 33.84 6.56 50.27 77.15 −0.518
AhMYB184 AHE.Chr20.797 353 39.13 6.46 49.6 68.92 −0.793
AhMYB185 AHE.Chr20.888 426 47.07 6.09 47.5 76.95 −0.574
AhMYB186 AHE.Chr21.1002 489 53.83 5.79 59.09 65.03 −0.817
AhMYB187 AHE.Chr21.1104 134 15.21 6.75 38.42 70.75 −0.504
AhMYB188 AHE.Chr21.1561 237 27.28 9.11 60.54 80.93 −0.645
AhMYB189 AHE.Chr21.181 380 41.93 5.46 64.67 69.82 −0.533
AhMYB190 AHE.Chr21.293 275 31.26 6.09 45.67 63.42 −0.904
AhMYB191 AHE.Chr21.362 464 51.11 6.45 64.82 61.36 −0.673
AhMYB192 AHE.Chr21.420 388 42.91 6.28 48.5 63.66 −0.731
AhMYB193 AHE.Chr21.529 509 57.55 9 42.91 66.27 −0.927
AhMYB194 AHE.Chr21.539 230 26.59 7.32 50.52 68.74 −0.905
AhMYB195 AHE.Chr21.666 229 26.74 8.41 61.03 60.44 −1.179
AhMYB196 AHE.Chr21.807 371 40.49 6.6 51.38 63.15 −0.772
AhMYB197 AHE.Chr21.853 372 42.12 9.32 65.58 76.85 −0.657
AhMYB198 AHE.Chr21.943 354 39.78 5.83 54.34 80.76 −0.636
AhMYB199 AHE.Chr21.950 225 25.91 4.82 60.84 41.2 −1.004
AhMYB200 AHE.Chr21.953 286 32.75 5.66 54.6 60.35 −0.826
AhMYB201 AHE.Chr22.1327 356 39.66 5.27 52.41 84.41 −0.461
AhMYB202 AHE.Chr22.1336 274 31.56 5.01 63.82 53.07 −0.992
AhMYB203 AHE.Chr22.1337 302 34.26 5.44 62.72 63.01 −0.708
AhMYB204 AHE.Chr22.1502 343 38.23 9.53 49.53 69.48 −0.808
AhMYB205 AHE.Chr22.1564 73 8.39 4.66 47.98 53.56 −0.885
AhMYB206 AHE.Chr22.1869 174 20.17 9.69 47.64 68.85 −0.845
AhMYB207 AHE.Chr22.260 301 34.8 5.58 42.97 67.01 −0.891
AhMYB208 AHE.Chr22.338 505 57.19 9.18 39.62 65.23 −0.923
AhMYB209 AHE.Chr22.344 232 26.72 8 45.24 70.69 −0.866
AhMYB210 AHE.Chr22.478 52 6.24 6.29 72.71 99.42 −0.19
AhMYB211 AHE.Chr22.479 284 31.24 4.96 69.2 70.7 −0.413
AhMYB212 AHE.Chr22.558_AHE.Chr22.559 464 51.42 6.62 64.06 60.73 −0.745
AhMYB213 AHE.Chr22.574 508 55.35 7.87 60.64 62.32 −0.691
AhMYB214 AHE.Chr22.607 382 42.11 6.16 55.23 62.62 −0.779
AhMYB215 AHE.Chr22.643 380 41.59 5.96 51.16 62.45 −0.853
AhMYB216 AHE.Chr22.835 325 36.36 4.72 53.46 76.18 −0.601
AhMYB217 AHE.Chr22.991 246 28.49 6.66 56.04 59.07 −1.076
AhMYB218 AHE.Chr23.1017 79 9.14 4.47 62.52 54.43 −0.844
AhMYB219 AHE.Chr23.1208 374 42.33 6.1 43.56 67.78 −0.73
AhMYB220 AHE.Chr23.1220 139 15.57 5.24 52.82 89.21 −0.246
AhMYB221 AHE.Chr23.184 245 27.88 6.37 53.69 69.63 −0.8
AhMYB222 AHE.Chr23.2116 460 51.62 9 55.46 75.83 −0.642
AhMYB223 AHE.Chr23.500 112 12.75 4.57 44.94 48.84 −0.883
AhMYB224 AHE.Chr23.513 206 23.76 9.64 58.86 70.53 −0.923
AhMYB225 AHE.Chr23.515 386 43.24 6.08 54.76 70.75 −0.769
AhMYB226 AHE.Chr23.524.2 966 107.12 6.27 51.91 80.62 −0.412
AhMYB227 AHE.Chr23.631 362 39.97 6.44 58.15 60.94 −0.663
AhMYB228 AHE.Chr23.652 282 30.37 8.13 62.42 49.5 −0.781
AhMYB229 AHE.Chr24.1100 79 9.13 4.61 75.43 51.9 −1.014
AhMYB230 AHE.Chr24.1232 377 42.76 6.34 44.42 66.76 −0.729
AhMYB231 AHE.Chr24.1250 276 31.56 4.98 56.8 65 −0.829
AhMYB232 AHE.Chr24.1552 366 41.29 9.72 49.29 75.66 −0.746
AhMYB233 AHE.Chr24.170 322 37.16 7.67 54.33 73.57 −0.733
AhMYB234 AHE.Chr24.2232 403 45.46 8.29 48.47 71.14 −0.679
AhMYB235 AHE.Chr24.259 319 34.29 6.21 45.35 74.29 −0.445
AhMYB236 AHE.Chr24.516 238 27.48 6.01 55.57 69.2 −0.83
AhMYB237 AHE.Chr24.517 289 32.89 6.84 45.36 67.85 −0.816
AhMYB238 AHE.Chr24.527 378 42.15 6.24 54.47 67.09 −0.726
AhMYB239 AHE.Chr24.538 232 27.05 5.79 57.5 65.17 −1.081
AhMYB240 AHE.Chr24.539 222 25.5 5.78 61.09 66.71 −0.862
AhMYB241 AHE.Chr24.540 289 32.93 6.84 46.85 69.2 −0.823
AhMYB242 AHE.Chr24.548 206 23.85 9.35 54.41 68.2 −0.912
AhMYB243 AHE.Chr24.551 378 42.18 6.21 54.07 66.06 −0.733
AhMYB244 AHE.Chr24.557 662 72.55 5.9 50.79 67.84 −0.644
AhMYB245 AHE.Chr24.593 380 42.31 6.63 61.21 85.68 −0.393
AhMYB246 AHE.Chr24.735 361 38.79 6.54 57.4 61.88 −0.671
AhMYB247 AHE.Chr25.1183 423 47.59 5.97 58.29 63.43 −0.8
AhMYB248 AHE.Chr25.1250 423 47.76 6.13 58.97 62.51 −0.822
AhMYB249 AHE.Chr25.134 299 32.96 8.47 39.8 66.82 −0.567
AhMYB250 AHE.Chr25.215 485 54.26 8.57 52.18 70.56 −0.912
AhMYB251 AHE.Chr25.237 485 54.37 8.57 52.18 69.55 −0.912
AhMYB252 AHE.Chr25.310 272 30.63 6.9 50.24 99.26 −0.246
AhMYB253 AHE.Chr25.38 360 38.71 8.72 37.05 77.47 −0.476
AhMYB254 AHE.Chr25.57 273 31.43 8.82 41.21 69.63 −0.858
AhMYB255 AHE.Chr26.1178.2 489 54.52 7.17 52.58 73.15 −0.716
AhMYB256 AHE.Chr26.140 281 30.72 8.33 43.19 75.62 −0.391
AhMYB257 AHE.Chr26.1460 338 35.45 6.5 54.64 64.23 −0.487
AhMYB258 AHE.Chr26.1510 405 44.33 9.51 58.12 62.4 −0.728
AhMYB259 AHE.Chr26.1539 290 31.42 7.76 49.48 67.62 −0.834
AhMYB260 AHE.Chr26.24 446 48.69 6.65 36.13 67.58 −0.659
AhMYB261 AHE.Chr26.251 340 37.31 8.35 57.89 63.71 −0.644
AhMYB262 AHE.Chr26.285 249 28.94 4.8 66.94 57.23 −0.988
AhMYB263 AHE.Chr26.41 144 16.73 9.84 54.3 87.36 −0.436
AhMYB264 AHE.Chr26.691 411 46.18 6.06 62.85 63.84 −0.775
AhMYB265 AHE.Chr27.1255 1384 155.44 7.84 54.71 71.79 −0.782
AhMYB266 AHE.Chr27.140 82 9.65 7.7 30.12 77.2 −0.776
AhMYB267 AHE.Chr27.142 312 35.49 6.73 50.74 64.68 −0.785
AhMYB268 AHE.Chr27.1474 406 45.02 6.52 36.65 80.99 −0.526
AhMYB269 AHE.Chr27.1540 557 61.114 5.08 41.38 77.04 −0.464
AhMYB270 AHE.Chr27.1568 259 28.74 9.1 65.13 73.94 −0.809
AhMYB271 AHE.Chr27.1785 357 40.93 7.23 50.97 64.45 −0.828
AhMYB272 AHE.Chr27.1793.2 371 41.31 8.53 46.7 68.63 −0.69
AhMYB273 AHE.Chr27.1794 362 40.29 8.53 48.03 68.18 −0.698
AhMYB274 AHE.Chr27.299 97 11.95 6.85 74.26 64.33 −0.982
AhMYB275 AHE.Chr27.571 369 40.46 9.44 49.5 64.15 −0.681
AhMYB276 AHE.Chr27.65 326 35.81 9.38 53.05 64.66 −0.647
AhMYB277 AHE.Chr27.725 409 45.46 7.33 49.42 72.47 −0.86
AhMYB278 AHE.Chr27.739 264 28.44 8.27 50.89 75.11 −0.532
AhMYB279 AHE.Chr27.837 299 34.13 6.32 58.34 63.28 −0.701
AhMYB280 AHE.Chr27.860 405 43.36 5.98 49.32 68.2 −0.439
AhMYB281 AHE.Chr27.862 405 43.36 5.98 49.32 68.2 −0.439
AhMYB282 AHE.Chr28.1 407 45.11 6.46 38.41 76.73 −0.546
AhMYB283 AHE.Chr28.1005 309 35.27 6.32 53.6 63.75 −0.674
AhMYB284 AHE.Chr28.1663 479 52.26 5.8 66.83 69.48 −0.639
AhMYB285 AHE.Chr28.1740.1 371 41.26 8.54 50.64 68.36 −0.68
AhMYB286 AHE.Chr28.1746 366 42.15 6.73 56.42 63.42 −0.891
AhMYB287 AHE.Chr28.1861 405 45.44 5.27 50.19 73.75 −0.637
AhMYB288 AHE.Chr28.1864 245 27.27 9.1 57.7 58.61 −0.81
AhMYB289 AHE.Chr28.2084 549 59.67 5.06 46.54 70.73 −0.504
AhMYB290 AHE.Chr28.313 380 40.87 9.49 44.36 60.82 −0.722
AhMYB291 AHE.Chr28.386 90 10.9 8.2 73.1 59.56 −1.079
AhMYB292 AHE.Chr28.453 273 30.44 9.17 47.98 75.46 −0.438
AhMYB293 AHE.Chr28.596 276 31.08 5.52 43.1 67.54 −0.756
AhMYB294 AHE.Chr28.645 380 40.82 9.59 43.54 59.79 −0.721
AhMYB295 AHE.Chr28.832 423 47.86 7.36 53.99 65.22 −0.982
AhMYB296 AHE.Chr28.848 260 27.79 7.7 48.55 75.15 −0.53
AhMYB297 AHE.Chr28.959 377 40.46 5.77 52.01 71.46 −0.389
AhMYB298 AHE.fragScaff_scaffold_206_pilon.54 373 41.41 6.9 59.87 84.96 −0.364
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2.8. Determination of MDA Content

Samples (0.5 g) from different jackfruit varieties subjected to low‐temperature stress were homogenized in 1 mL of freezing extraction buffer containing 5.0‐mmol/L phosphate buffer (pH 5.7) and 8% polyvinylpyrrolidone. After centrifugation at 10,000 rpm for 4 min at 10°C, the supernatant was collected for enzyme activity determination. The MDA content was measured at 532 nm using a spectrophotometer, and nonspecific turbidity was corrected by subtracting the absorbance at 600 and 450 nm. Each experiment was replicated thrice.

2.9. Determination of CAT and Superoxide Dismutase (SOD) Activities

Samples (0.5 g) from different jackfruit varieties subjected to low‐temperature stress were homogenized in 1 mL of frozen extraction buffer containing 5.0‐mmol/L phosphate buffer (pH 5.7) and 8% polyvinylpyrrolidone. After centrifugation at 10,000 rpm for 4 min at 10°C, the supernatant was collected for enzyme activity determination. SOD activity was measured at 560 nm using spectrophotometry, and proline content was measured at 450 nm. Each experiment was replicated thrice.

2.10. Statistical Analysis

Gene expression data were analyzed using one‐way analysis of variance followed by Duncan's test to determine significant differences. Data were analyzed using SPSS Statistics 16.0 (SPSS Inc., Chicago, IL, USA) (Student's t test, **p < 0.01, ***p < 0.001, ****p < 0.0001). GraphPad Prism 9 (GraphPad Software, La Jolla, CA, USA) was used for graphical display.

3. Results

3.1. Identification and Phylogenetic Analysis of MYB Genes in A. heterophyllus

The “S10” A. heterophyllus genome was scanned, and 298 MYB genes were identified using BLAST searches with Arabidopsis and rice MYB genes. The MYB genes were annotated according to their position on the chromosome. To preliminarily investigate the function of MYB genes, the basic physicochemical properties of MYB proteins were analyzed using an online tool (Table 1). The number of amino acids (aa) in the MYB family protein sequences ranged from 52 (AhMYB210) to 1384 (AhMYB265), the theoretical isoelectric points (pI) ranged from 4.47 (AhMYB218) to 10.14 (AhMYB75), and the molecular weights ranged from 6.24 to 155.44 kDa. The instability indexes of 22 MYB proteins were less than 40, indicating they were stable, whereas those of the remaining 276 MYB proteins were greater than 40, classifying them as unstable. The phylogenetic relationships divided the AhWRKY gene family into seven subgroups (I–VII), with subgroup I containing the most members (72 AhWRKY genes) and subgroup VII the fewest (19 AhWRKY genes) (Figure 1).

FIGURE 1.

FIGURE 1

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Phylogenetic tree analysis of MYB proteins from Artocarpus heterophyllus . The seven subgroups are represented by different colors: cream yellow for subgroup I, mint green for subgroup II, ice blue for subgroup III, taro for subgroup IV, light blue for subgroup V, gray for subgroup VI, and bright yellow for subgroup VII.

3.2. Gene Structure and Conserved Motif Analysis

Analysis of protein motifs, coding DNA sequences (CDSs), untranslated regions (UTRs), and gene structure of AhMYB genes revealed 10 distinct conserved motifs (Figure 2). Almost all AhMYB proteins contained Motif 3, indicating a high degree of conservation. In addition to the common conserved residue Trp (W), other conserved residues included Lys (K), Arg (R), Thr (T), Leu (L), Gly (G), Glu (E), Val (V), Asp (D), and Pro (P). Less than 25% of AhMYB proteins contained only one to two conserved motifs. Motif 10 was present in less than 10% of AhMYB proteins (Data S2: Table S2). Motifs 1, 3, and 10 were located near the N‐terminal, while Motifs 7 and 8 were positioned near the C‐terminal. AhMYB proteins in the same subfamily exhibited similar conserved domains, suggesting that they have similar functions. Analysis of gene structure is helpful to understand the function and regulation mechanism of gene expression, which is of great significance in biological research. The number of introns in the AhMYB genes ranged from 0 to 11, with 7 of 298 AhMYB genes containing no introns and 28 containing only one intron (Data S2: Table S2). AhMYB22 displayed the highest number of introns (18 introns), and approximately 71% of AhMYB genes had no more than two introns, indicating that the number of introns in the AhMYB family was limited.

FIGURE 2.

FIGURE 2

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Gene structure, conserved motifs, and protein structure of the 298 AhMYB proteins.

3.3. Chromosomal Location Analysis

Using the annotated A. heterophyllus genome as a reference, the chromosomal locations of AhMYB genes were identified. The chromosomal distribution of AhMYB genes was mapped using TBtools (Figure 3). The A. heterophyllus genome contains 28 chromosomes, with chromosome 28 being the largest (44.1 Mb) and chromosome 3 being the smallest (14.6 Mb). The 298 AhMYB genes were distributed irregularly across all chromosomes, with the most AhMYB genes on chromosome 8 (19), followed by chromosomes 7 and 24 (18) and 22 (17). In contrast, fewer AhMYB genes were found on chromosomes 13 (six), 1 (six), 15, and 17 (five). The fewest AhMYB genes were found on chromosome 2, with only three AhMYB genes. The dense clusters of AhMYB genes on chromosomes 8 (19), 7, and 24 (18) suggest that these regions could represent evolutionary hot spots for the expansion of the MYB family in A. heterophyllus .

FIGURE 3.

FIGURE 3

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Chromosomal localization of MYB genes in jackfruit.

3.4. Synteny Analysis of MYB Genes in A. heterophyllus

MCScanX was used to analyze homology and determine the collinear gene pairs within MYB genes in A. heterophyllus . A total of 1439 collinear gene pairs were identified among AhMYB genes, with tandem duplication events observed across all 28 chromosomes of A. heterophyllus (Figure 4). In addition, several collinear gene pairs among AhMYB genes on chromosomes 27 and 28 of A. heterophyllus were observed, suggesting that AhMYB genes are functionally conserved during evolution, and tandem duplication events may be the main route of evolution.

FIGURE 4.

FIGURE 4

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Collinearity of MYB genes in jackfruit.

3.5. Prediction of cis‐Acting Elements of MYB Genes in A. heterophyllus

Promoter cis‐acting elements are important components of gene expression and are involved in plant growth and development, as well as hormone and stress responses (Yamaguchi‐Shinozaki and Shinozaki 2005). To further investigate functional differences among AhMYB gene promoters, cis‐acting elements were analyzed using PlantCARE based on the 2‐kb upstream sequences from the initiation codons of 298 AhMYB genes (Figure 5). The identified cis‐acting elements were divided into four groups: light signaling response (22), hormonal response (8), abiotic stresses (9), and development elements (14). Hormone response elements (ABREs), JA response elements (MYC), and ethylene response elements (ERE) accounted for the largest proportion. MYB (CTAACCAAG), anaerobic response (ARE), and stress response (STRE) components comprised the largest proportion of stress response components. In addition, growth and development‐related cis‐acting elements included the commonly observed meristem‐specific expression site (CAT‐box) and seed‐specific regulatory element (RY‐elements). These results suggest that AhMYB genes are involved in light, hormone regulation, and stress response in A. heterophyllus and may operate through various regulatory modes.

FIGURE 5.

FIGURE 5

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Analysis of the cis‐acting elements of MYB gene promoters in jackfruit. The number of elements involved in photoresponse, hormone response, abiotic stress, and growth and development is different. The color shades and numbers in the squares indicate the number of promoters.

3.6. Expression Profile Analyses of MYB Genes in A. heterophyllus Under Cold Stress

Low temperature is a common environmental stress factor that restricts the geographical distribution of plants and affects their growth and development (Yuanyuan et al. 2009). To explore the expression pattern of AhMYB genes in different jackfruit varieties under low‐temperature stress, we used our previously published transcriptome data (Ma et al. 2024) to illustrate their expression patterns. The results showed that 157 AhMYB genes (FPKM < 1) were almost not expressed in the transcriptomic data of A. heterophyllus (Figure 6A). In the expressed genes, 90 genes were highly expressed in the GX variety, and 51 genes were highly expressed in THA. These results suggest that AhMYB genes may play an important role in resistance to cold stress in different jackfruit varieties. It is worth noting that under the low‐temperature stress, AhMYB genes (AhMYB18/28/45/87/96/133/189/219/238/248/251/252/285) were differentially expressed in different strains of jackfruit. The results indicated that the 13 AhDREB genes of GX might have stronger resistance to cold stress than those of THA.

FIGURE 6.

FIGURE 6

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Expression pattern analysis of MYB genes in jackfruit under low‐temperature stress. (A) Heat map analysis of MYB genes in jackfruit varieties under low‐temperature stress. (B) qPCR expression patterns of differentially expressed genes under low‐temperature stress. *p < 0.1, **p < 0.01, ***p < 0.001, and **** p < 0.0001.

To further verify the expression pattern of AhMYB genes under cold stress, we screened 13 AhMYB genes (AhMYB18/28/45/87/96/133/189/219/238/248/251/252/285) that were differentially expressed in the transcriptome and verified them using qPCR (Figure 6B). Genes such as AhMYB18/28/45/133/189/219/248/251/252/285 showed increased expression in the THA variety, whereas AhMYB87/96/238 exhibited lower expression. In addition, cis‐acting element analysis of AhMYB gene promoters revealed the presence of low‐temperature‐responsive (LTR) elements in response to cold stress, suggesting that AhMYB genes may play an important role in cold stress resistance.

3.7. Functional Analysis of GX and THA Under Cold Stress

CAT and SOD are important antioxidant enzymes in plants that play a key role in the response of plants to oxidative stress, maintenance of reactive oxygen species (ROS) balance, and adaptation to adverse conditions (Azarabadi et al. 2017). An increase in MDA content directly indicates that the cell membrane system is under attack by ROS, leading to membrane structural damage. When plants are subjected to abiotic stress, the MDA content in plants usually increases significantly (Ayala et al. 2014). After exposure to low‐temperature stress, distinct water‐soaked spots appeared on THA leaves, indicating that GX leaves were less damaged. To explore the mechanism by which different jackfruit varieties respond to low‐temperature stress, we determined their contents of CAT and SOD after low‐temperature stress (Figure 7). The results showed that CAT and SOD levels in GX were significantly higher than those in THA. Moreover, we determined the MDA content in different jackfruit varieties and found significantly higher levels in THA than those in GX. These results further indicate that GX exhibits greater cold resistance than THA.

FIGURE 7.

FIGURE 7

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Determination of the physiological indicators of different jackfruit varieties. (A) GX and TAH phenotypes after cold stress. (B) SOD, CAT, and MDA levels in GX and TAH after cold stress *p < 0.1, **p < 0.01, ***p < 0.001, and ****p < 0.0001.

4. Discussion

4.1. MYB Genes Are Evolutionarily Conserved in A. heterophyllus

MYB, as one of the largest families of TFs in plants, contains a highly conserved DNA‐binding domain, which enables it to specifically bind to downstream target sequences, thereby regulating gene expression levels and affecting plant growth and development and response to stress (Pireyre and Burow 2015). Klempnauer et al. (1982) identified the first MYB TF in avian myeloblastosis virus, which was named v‐MYB. MYB TFs were later identified in eukaryotes such as animals, plants, and fungi (Lipsick 1996). The maize ( Zea mays ) ZmMYBC1 gene, which regulates pigment synthesis, was the earliest identified MYB‐like TF in plants (Paz‐Ares et al. 1987). Subsequently, a large number of MYB TFs have been cloned and identified in plants, where they play an important role in transcriptional regulation. In the current study, 298 nonredundant MYB genes were identified in A. heterophyllus S10. The number of MYB genes detected in A. heterophyllus was lower than that in Stevia rebaudiana (374) (Chen et al. 2024) and Gossypium hirsutum (524) (Salih et al. 2016) and higher than that in S. rebaudiana (127) (Chen et al. 2024) and Pyrus bretschneideri (231) (Li et al. 2016). These results indicate that MYB genes differentially respond to environmental stress, adaptive changes, and genetic diversity throughout evolution. Analysis of the basic physicochemical properties of AhMYBd proteins showed significant differences in the molecular weight of proteins encoded by different genes, consistent with previous studies (Feng et al. 2017). Conserved motif analysis showed that almost all AhMYB genes contained Motif 3, and members of the same subfamily in the phylogenetic tree shared similar Motif 3 patterns and gene structure. In addition, most AhMYB genes contained only two introns, which are consistent with the fact that most MYB genes in higher plants contain only two introns (Abbas et al. 2021). Chromosome localization analysis showed that the 298 AhMYB genes were unevenly distributed across 28 chromosomes, with small gene clusters also observed. Collinear analysis revealed 1439 collinear gene pairs among AhMYB genes. It is speculated that segmental replication and tandem repeat events are the main modes of AhMYB gene expansion in A. heterophyllus , which may enhance the adaptability of the plant to different environmental conditions (Schilling et al. 2020).

4.2. MYB Genes May Be Involved in Cold Stress Resistance in A. heterophyllus

Under abiotic stress, plants initiate a series of stress responses that activate defense mechanisms to cope with environmental changes (Chaudhry and Sidhu 2022). The signal transduction pathway of plant cells in response to cold stress is complex. To date, several MYB TFs have been confirmed to participate in the process of plant response to cold stress. Agarwal et al. (2006) found that under low‐temperature conditions, the Arabidopsis TF AtMYB15 regulates cold resistance by binding to the MYB recognition sequences of CBF (CBF1, CBF2, and CBF3) gene promoters and inhibiting the expression of CBF genes. Abubakar et al. (2022) identified AvMYB48, AvMYB97, AvMYB8, and AvMYB4 as potential stress response genes by analyzing MYB expression in Apocynum venetum at low temperatures. In the phenylpropane metabolic pathway of Dendrobium officinale under freezing stress, DoMYB97, DoMYB39, fructosyltransferase, and zinc finger protein TFs were significantly affected by freezing and freezing recovery (Zhan et al. 2022). The cis‐acting elements of promoters play an important biological role in regulating plant gene expression by interacting with TFs (Otsuki and Yamamoto 2020). In the present study, analysis of the promoter cis‐acting elements of MYB genes revealed that AhMYB genes contain a large number of promoter cis‐acting elements in response to stress, some of which contained LTR elements. Therefore, we combined the transcriptomic data of different jackfruit varieties under low‐temperature stress to explore whether MYB genes play a role in the cold resistance of jackfruit. The results showed that most AhMYB genes were highly expressed in GX. To further verify this result, differentially expressed genes were screened and verified using qPCR. The expression trend of these differentially expressed AhMYB genes was consistent with the transcriptome results. Thus, it is speculated that AhMYB genes may play an important role in the resistance of jackfruit to cold stress, with each member playing distinct roles in different jackfruit varieties. However, their specific roles require further validation. The results of this study provide new insights into the response mechanism of jackfruit to cold stress.

4.3. GX Experiences Reduced Low‐Temperature Stress‐Induced Damage by Regulating the Antioxidant Enzyme Levels

Plants encounter various stresses during growth. Under low‐temperature stress, the metabolic processes of plants produce ROS, which accumulate in large quantities within the plants and cause membrane lipid peroxidation (Airaki et al. 2012). This effect leads to the gradual accumulation of MDA, which can exacerbate the damage to biological membranes within plants and severely hinder or even disrupt various metabolic activities (Hasanuzzaman et al. 2021). SOD and CAT are important antioxidant protective enzymes in plant cells, and their contents are commonly used physiological indicators of stress tolerance (Hasanuzzaman et al. 2011). MDA, as an indicator of lipid peroxidation, is often used to measure the degree of damage to plants. Yan et al. (2010) found that the MDA levels in Forsythia suspensa at low temperatures increased by 66.7% compared with those at normal temperatures. After low‐temperature treatment, SOD, CAT, and MDA levels in two strawberry varieties (cv. Zoji and Toyonaka) were all higher than those in the control (Luo et al. 2011). After long‐term exposure to cold treatment, the SOD enzyme activities of three citrus varieties (Citrus unshiu, Citrus sinensis , and Citrus limon ) all increased significantly (Mohammadian et al. 2012). After being subjected to low‐temperature stress, water‐soaked spots appeared on the leaves of THA. To further explore the mechanism by which different jackfruit varieties respond to low‐temperature stress, we determined their SOD, CAT, and MDA levels. The results showed that SOD and CAT levels in GX were significantly higher than those in THA. However, MDA levels in GX were significantly lower than those in THA. Therefore, it can be inferred that GX may respond to external low‐temperature stress by regulating the activity of its antioxidant enzymes, thereby increasing its stress resistance. This result is consistent with the mechanism by which wheat (Hou et al. 2010) ( Triticum aestivum ) and strawberries (Luo et al. 2011) (Fragaria × ananassa) respond to low‐temperature stress.

Author Contributions

Conceived the project: Pengjin Zhu and Hailan Zhou. Experiments data collection: Xiangwei Ma, Jianjun Liang, and Chenxin Yi. Analysis and interpretation of results: Chenxin Yi, Weiyan Ye, and Zhuangmin Wei. Draft manuscript preparation: Xiuguan Tang, Qiqi Song, and Shengli Tang. All authors reviewed and approved the final manuscript.

Funding

This research was supported by the Special Project for Basic Scientific Research Business of Guangxi Academy of Agricultural Sciences (GNK 2025YP128, GNK 2026YT021, GNK2026YP054), and the vanguard team of the specialty fruit industry GNKM (202504‐03).

Supporting information

Data S1: Supporting information.

PLD3-10-e70131-s001.xlsx (10.7KB, xlsx)

Data S2: Supporting information.

PLD3-10-e70131-s003.xlsx (9.7KB, xlsx)

Data S3: Supporting information.

PLD3-10-e70131-s002.xlsx (10.2KB, 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

Data S1: Supporting information.

PLD3-10-e70131-s001.xlsx (10.7KB, xlsx)

Data S2: Supporting information.

PLD3-10-e70131-s003.xlsx (9.7KB, xlsx)

Data S3: Supporting information.

PLD3-10-e70131-s002.xlsx (10.2KB, xlsx)

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