Iron (Fe) is a trace element necessary for plant growth. Many land plants have evolved a set of mechanisms associated with the Fe absorption process to deal with the problem of insufficient Fe supply in the soil. During Fe absorption, reactive oxygen species (ROS) can be used as a signal to initiate a response to stress caused by Fe deficiency. However, the molecular mechanisms underlying the involvement of ROS in the Fe deficiency stress response remains unclear. In this study, we have identified a kinase, MxMPK6-2, from Malus xiaojinensis, an apple rootstock that is highly efficient at Fe absorption. MxMPK6-2 has been shown to be responsive to ROS signals during Fe deficiency, and MxMPK6-2 overexpression in apple calli enhanced its tolerance to Fe deficiency. We further screened for proteins in the Fe absorption pathway and identified MxbHLH104, a transcription factor which interacts with MxMPK6-2. MxbHLH104 can be phosphorylated by MxMPK6-2 in vivo, and we confirmed that its phosphorylation increased Fe absorption in apple calli under Fe deficiency, with the presence of ROS promoting this process. Overall, we have demonstrated that MxMPK6-2 is responsive to ROS signaling during Fe deficiency, and is able to control its response by regulating MxbHLH104.

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ATP-binding cassette (ABC) transporters constitute a large, diverse, and ubiquitous superfamily that is involved in a broad range of processes. The completion of genome sequencing provides an opportunity to understand the phylogenetic history of the ABC transporter superfamily among Rosaceae species. This study identified a total of 1323 ABC transporter genes from nine Rosaceae genomes: 191 from Malus domestica, 174 from Pyrus communis, 138 from Prunus persica, 118 from Prunus avium, 141 from Prunus dulcis, 122 from Fragaria vesca, 98 from Rubus occidentalis, 162 from Prunus mume, and 179 from Rosa chinensis. Their chemical characterization, phylogenetic analysis, chromosomal localization, gene structure, gene duplication, and tissue-specific expression were studied. Their subcellular localization, transmembrane structures, and protein motifs were predicted. All the ABC transporter genes were grouped into eight subfamilies on the basis of their phylogenetic relationships and structural features. Furthermore, cis-element and expression analysis of 10 potential phytohormone transporters in MdABCG subfamily genes were also performed. Loss of the W-box in the promoter region of MdABCG28 was found to reduce the gene expression level and was linked to the dwarfing phenotype in apple rootstocks. MdABCG28 overexpression promoted shoot growth of atabcg14 mutants in Arabidopsis.

Understanding the mechanism of iron (Fe)‐deficiency responses is crucial for improving plant Fe bioavailability. Here, we found that the Arabidopsis Rho‐like GTPase 6 mutant (rop6) is less sensitive to Fe‐deficiency responses and has reduced levels of reactive oxygen species (ROS) compared to wild‐type (WT), while AtROP6‐overexpressing seedlings exhibit more sensitivity to Fe‐deficiency responses and has higher levels of ROS compared to WT. Moreover, treatment with H2O2 improves the sensitivity to Fe‐deficiency responses in rop6 mutants. By using the yeast two‐hybrid system, we further demonstrate the direct interaction between AtROP6 and Arabidopsis respiratory burst oxidase homolog D (AtRBOHD), which controls the generation of ROS. Overall, we suggest that AtROP6 is involved in AtRBOHD‐mediated ROS signaling to modulate Fe‐deficiency responses in Arabidopsis thaliana.

Iron (Fe) deficiency limits the yield of fruit trees. When subjected to Fe deficiency, H+ secretion increases in the rhizosphere of dicotyledonous plants and pH decreases. This leads to the acidification of the soil and promotes Fe3+ to Fe2+ conversion, which plants can better uptake. This study investigated the relationship between two inhibitory transcription factors (ethylene response factors MbERF4 and MbERF72) and the H+-ATPase gene MbHA2. Two species of apple woody plants were studied: the Fe-inefficient Malus baccata and the Fe-efficient Malus xiaojinensis. Yeast one-hybrid and electrophoretic mobility shift assays showed that both MbERF4 and MbERF72 bind to the GCC cassette (AGCCGCC) of the MbHA2 promoter. Moreover, yeast two-hybrid and bimolecular fluorescence complementation assays showed that MbERF4 interacts with MbERF72. Furthermore, β-glucuronidase and luciferase reporter assays showed that the MbERF4- and MbERF72-induced repression of MbHA2 expression is synergistic. Virus-induced gene silencing of MbERF4 or MbERF72 increased MbHA2 expression, and thus lowered the rhizosphere pH in M. baccata. Consequently, the high expressions of MbERF4 and MbERF72 induced by Fe deficiency contributed to the Fe sensitivity of M. baccata. Moreover, the low expressions of MxERF4 and MxERF72 contributed to the Fe-deficiency tolerance of M. xiaojinensis via different binding conditions to the HA2 promoter. In summary, this study identified the relationship of two inhibitory transcription factors with the H+-ATPase gene and proposed a model in which ERF4 and ERF72 affect the rhizosphere pH in response to Fe deficiency.