Genome-wide identification and analysis of the apple H+-ATPase gene family and its expression against iron deficiency stress

Abstract

Background: Iron (Fe) is indispensable to plants, playing a significant role in life activities such as respiration, chlorophyll biosynthesis, and photosynthetic electron transport. The decrease in pH caused by iron deficiency is related to the activation of H⁺-ATPase in the root plasma membrane, although the reaction mechanism of this enzyme is not well understood in apples. The H⁺-ATPase (HA) gene family has been extensively studied in Arabidopsis but is rarely reported in other species.

Results: In this study, 14 HA genes were identified from the apple genome database through whole genome analysis. These apple H⁺-ATPase (MdHAs) genes were classified into four subsets (I, II, IV, V) based on phylogenetic analysis. Bioinformatics analysis revealed that these genes exhibited diversity in gene structure, chromosomal distribution, conserved motifs, and cis-acting elements. The qRT-PCR analysis revealed that iron deficiency stress significantly induced the upregulation of nine MdHA genes (MdHA5-MdHA14). Furthermore, in the roots of iron deficiency-resistant rootstock variety QZ1 compared to the non-resistant variety QZ2, the expression levels of nine genes (including MdHA1, MdHA6-MdHA13) in QZ2 were significantly lower than those in QZ1. This study lays a foundation for further study on the function of HA gene family.

Conclusions: These findings suggest that the HA gene family is involved in the apple's response to iron deficiency. This study not only lays the groundwork for future research but also highlights the role of MdHAs in iron stress tolerance.

Keywords: H + -ATPase gene family; Apple; Genome - wide identification; Iron deficiency stress.

Fig. 1

Chromosomal localization of MdHAs. The left scale displays chromosome lengths (megabases, Mb), while H.⁺-ATPase gene positions are annotated along the right flank of each chromosome. A color gradient illustrates regional gene density, ranging from red (highest) to blue (lowest)

Fig. 2

Synteny investigation of H⁺-ATPase family genes. A Genome-wide syntenic relationships of MdHA genes. Gray connectors indicate conserved genomic blocks within the apple genome, while red linkages highlight specific homologous gene pairs among MdHA members. B Comparative synteny patterns of H⁺-ATPase genes between apple and four model species. Background gray connectors display genomic conservation between Malus domestica and Arabidopsis thaliana/Solanum tuberosum/Oryza sativa. Species-specific collinear associations are illustrated with distinct color codes: ochre connectors depict grape-apple synteny, pink linkages represent orthologous relationships with rice, azure lines correspond to potato homologs, and violet connectors demonstrate conserved gene pairs with Arabidopsis

Fig. 3

Phylogenetic tree of H⁺-ATPase genes in peach, apple, grape, potato, Arabidopsis, sunflower, rice, sorghum, millet, tomato, and pineapple

Fig. 4

A Apple H⁺-ATPase protein subcellular localization. The figures in the bars represent subcellular localization frequencies. B Tertiary structure analysis of H⁺-ATPase gene family in apple

Fig. 5

Structural and functional characterization of the MdHA gene in Malus domestica. A Identification of conserved protein motifs within the MdHA gene family through systematic motif analysis. B Domain architecture determination of apple MdHA proteins using comprehensive domain scanning. C Genomic architecture visualization illustrating exon–intron organization patterns of MdHA genes

Fig. 6

Cis-regulatory element analysis of the MdHA genes. A Cis-acting elements contained by the different MdHA gene family members. B The species of cis-acting elements contained by the different members of the MdHA gene family are Phytohormone respond in blue, light responsive elements in orange, growth and development in yellow and Biotic/abiotic stress in green

Fig. 7

A Synonymous codon frequency distribution. B Optimal count of synonymous codons. C Inter-codon correlations within the HA gene. Red hues denote positive associations, blue hues negative associations, and white signifies no detectable relationship. Strength of correlation is quantified by color intensity and circle diameter (darker/larger = stronger; lighter/smaller = weaker). The correlation data for panel B are based on 14 observations (n = 14)

Fig. 8

A Functional network mapping of HA protein family interactions in Malus domestica. Nodes illustrate encoded proteins, with hollow shapes designating polypeptides lacking resolved structural configurations and solid geometries depicting molecules with experimentally determined or computationally modeled 3D architectures. Edges connecting nodes portray pairwise molecular associations, chromatically coded to distinguish distinct binding modalities. B Spatiotemporal expression profiles of HA homologs across apple organ systems. Tissue specimens annotated with numerical suffixes reflect progressive developmental phases. A heatmap color gradient displays transcriptional dynamics, where warm/cool tones correspond to elevated (red spectrum) and suppressed (blue spectrum) mRNA abundance relative to baseline measurements. The intensity scale represents normalized expression magnitudes calculated against reference housekeeping genes

Fig. 9

Expression analysis of MdHA gene family in apple histculture seedlings under different treatments. CK (MS liquid culture for 0 h), MS 24 h (MS liquid culture for 24 h), MS 48 h (MS liquid culture for 48 h), MS 72 h (MS liquid culture for 72 h), -Fe 24 h (MS iron deficiency liquid culture for 24 h), -Fe 48 h (MS iron deficiency liquid culture for 48 h), -Fe 72 h (MS iron deficiency liquid culture for 72 h). The CK, which was assigned with a value of 1. Error bars represent the mean ± SE from three biological repeats. Different letters denote significant differences, whereas the same lowercase letters indicate no statistical difference (P < 0.05)

Fig. 10

Expression analysis of the MdHA gene family in apple field seedlings of different cultivars. * Represents a significant difference, while no * indicates no statistical difference (P < 0.05)