As an important transcription factor in organisms, the PHD gene family not only plays a key role in regulating plant growth and development, but also plays an important regulatory role when plants face biotic and abiotic stresses [18].
In this study, we identified 244 TaPHD gene members for the first time in the wheat genome (Table S1), and the vast majority of TaPHD proteins were located in the nucleus. In terms of the number of genes, the number of TaPHD genes has increased significantly compared with other species. It is precisely because the origin of wheat involves two polyploidy events, forming the existing allohexaploid bread wheat. However, compared with 59 in rice and 67 in maize in diploid gramineous crops, it is not multiplied, indicating that the PHD gene in wheat has a more exaggerated expansion and evolution. Gene duplication events are important for the rapid expansion and evolution of plant gene families [60]. Studies have shown that about 70–80% of angiosperms experience duplication events [61,62], and in common wheat (Triticum aestivum L.) more than 85% of the sequences are duplicates [63]. Physical localization on chromosomes and covariance analysis (Fig. 2; Fig. 3) reveal the presence of a large number of segmental duplication events during the evolution of TaPHD genes, suggesting that segmental duplication contributes to the amplification of TaPHD genes. The proportion of TaPHD with the 1:1:1 ratio of the three subgenomes A:B:D accounted for 84.8% of the total proportion (Table 2), which was much higher than the 35.8% of the whole genome, indicating that the PHD gene family is extremely conserved in the three subgenomes compared with other genes. In terms of the covariance and evolutionary relationship of wheat PHD genes among species (Fig. 2; Fig. 4), the PHD-finger family followed the divergence pattern between monocotyledonous and dicotyledonous species, with the average divergence time in monocotyledonous also species oats (12.78Mya) < rice (22.09Mya) < maize (60.87Mya), indicating a more similar genetic structure to barley .
Phylogenetic analysis showed that PHD proteins in three species (including Arabidopsis, Oryza stiva, and Triticum aestivum), which are distantly different from each other, can be divided into four large evolutionary branches, and each group of evolutionary branches contains many smaller evolutionary branches. From the results, there are some small branches containing only wheat PHD genes, which is consistent with the previous results that wheat PHD genes have more exaggerated expansion and evolutionary results. When the PHD genes with different chaperone structural domains were subdivided (Table 1), the folds of divergence were also different, such as ING1, ING2, ROS1, EBS and PKL were expanded 3-fold, while SHL1 was expanded 6-fold, and even more, VIN3 and SIZ1 were expanded to an exaggerated 9-fold and 11-fold. It is probably the presence of so many redundant genes that has contributed to the stability of the genome of hexaploid wheat species [64].
Genes perform their functions through transcription and translation, and the expression patterns of genes reflect the function of genes. PHD gene can regulate the growth and development of plants, so its expression in different tissue parts of plants has also attracted much attention. Studies have shown that the expression patterns of the PHD gene family in rice, maize, potato, and cotton have been concentrated in different tissue types [25–27,29]. In rice, the expression levels of OsPHDs were relatively high in pre-emergence inflorescences and pistils, and the number of low-expressed OsPHDs was significantly higher in seed 10 days compared with other periods [25]. In potato, considerable expression differences were shown between individual StPHD genes from different tissues. For example, StPHD27 showed abundant expression in roots, shoots and stamens, but lower expression levels in petals, carpels and leaves [27]. In cotton, GhPHDs genes have the highest expression levels in ovule and fiber tissue, suggesting that GhPHDs may be involved in regulating ovule and fiber development [26]. This study showed that the TaPHDs gene expression in various tissues of wheat showed great differences with the growth period, especially the TaPHDs gene expression was most highly expressed in the stigma and ovary at the flowering stage. This may be because a large number of PHD proteins in the PHD family regulate the reproductive and developmental process of plants. For example, MMD1, MS1, VIM1 and SHL1 in Arabidopsis previously studied all play key roles in the reproductive growth stage [65–67]. TaPHD100, TaPHD108 and TaPHD122 genes have high expression levels in the whole growth period. These three genes are highly orthologous to AtAL6 and AtAL7. In Arabidopsis, AtAL6 and AtAL7 are methylated with histones through the PHD domain. Modification sites H3K4me3 and H3K4me2 bind to regulate the expression of target genes [68]. Therefore, it can be speculated that TaPHD100, TaPHD108 and TaPHD122 play important roles in the regulation of growth and development of wheat histone methylation. Besides, TaPHD222 and TaPHD232 are only highly expressed in shoots and roots, these two genes are highly orthologous to ORC1A/B, in Arabidopsis, ORC1A/B protein binds methyl groups through the PHD domain lysed DNA and appears to function as a transcriptional activator [69]. Therefore, we infer that these two TaPHD genes are essential for the development of roots and shoots. However, how they function during development needs further verification.
The PHD family not only regulates plant growth and development, but also responds to abiotic stresses. Existing research evidence shows that PHD family transcription factors also play an important role in coping with abiotic stress. For example, the PHD genes AL5 and AL6 in Arabidopsis bind to the promoter regions of downstream target genes, thereby inhibiting various signaling pathways to improve the tolerance of plants to abiotic stresses such as low temperature, drought, and high salt [56,57]. AtSIZ1 accumulates higher levels of sumoylated proteins through an ABA-independent pathway in response to abiotic stresses such as drought, low temperature, and heat shock [70]. In rice, the cis-acting elements DRE/CRT in the OsPHD13 and OsPHD52 promoters in response to stress such as low humidity were induced to be up-regulated by as much as 15-fold under low temperature stress. Overexpression of OsPHD1 can significantly improve plant tolerance to stress (drought, high salt and low temperature) [71]. In maize, the expression of subfamily IX TaPHDs responds to salt, drought and ABA stress [29]. Through transcriptome data, we found that 122 TaPHDs had significant responses to low temperature, drought or high temperature. Among them, 45 TaPHDs genes were significantly changed under two or three treatments, indicating that TaPHDs play an active role in plant response to low temperature, drought or high temperature stress. In order to better verify the adaptability of TaPHDs to the above three abiotic stresses, we selected a representative cultivar "Zhengmai 7698" from the Huanghuai wheat area of China and performed qRT-PCR analysis. We found that TaPHD11 and TaPHD19 genes, which are highly homologous to AtALs, were significantly up-regulated only under drought treatment, which is different from the study in Arabidopsis, indicating that ALs seem to have different responses to abiotic stress in monocotyledonous and dicotyledonous plants Mechanisms. At the same time, the subcellular localization experiments also showed that TaPHD11 and TaPHD19 were localized in the nucleus and cell membrane, indicating that they function not only in the nucleus but also in the membrane in wheat. TaPHD69, which is highly homologous to AtSIZ1, can be significantly up-regulated under low temperature, drought, and high temperature. The accumulation of TaPHD69 seems to be beneficial to plants to cope with abiotic stress, which is very similar to the function of AtSIZ1 in Arabidopsis.
TaPHD117 was significantly up-regulated under high temperature and drought treatments, and significantly down-regulated under low temperature treatments, and had distinct expression patterns in response to different treatments. Therefore, whether TaPHD acts as a key gene in roots to cope with abiotic stress needs further verification. Taken together, our results suggest that TaPHDs have potential functions in plant responses to abiotic stresses.