Ammonium is one of the prevalent nitrogen sources for growth and development of higher plants. Ammonium acquisition from soil is facilitated by ammonium transporters (AMTs), which are plasma membrane proteins that exclusively transport ammonium/ammonia. However, the functional characteristics and molecular mechanisms of AMTs in apple remain unclear. In this work, 15 putative AMT genes were identified and classified into four clusters (AMT1–AMT4) in apple. According to expression analysis, these AMTs had varying expressions in roots, leaves, stems, flowers and fruits. Some of them were strongly affected by diurnal cycles. AMT genes showed multiple transcript patterns to N regimes and were quite responsive to osmotic stress. In addition, phosphorylation analysis revealed that there were some conserved phosphorylation residues within the C-terminal of AMT proteins. Furthermore, detailed research was conducted on AMT1;2 functioning by heterologous expression in yeast. The present study is expected to provide basic bioinformatic information and expression profiles for the apple AMT family and to lay a basis for exploring the functional roles and regulation mechanisms of AMTs in apple.

Real-time quantitative PCR (RT-qPCR) has become a widely used method for exploring plant gene expression level. The method requires using some stably expressed genes as a reference to accurately normalize the RT-qPCR data. However, under various stresses and hormone treatments, the levels of most reference genes vary. Environmental variations also influence their expression levels. The lack of validated, stably expressed reference genes can mislead the study of gene function in pears. “Huangguan” pears have recently become the focus of research on stress resistance mechanisms, such as high resistance. Therefore, the aim of this study was to select the optimal reference genes in Huangguan pears, and we analyzed the expression of the genes EF1α, ACT, SKD1, YLS8, UBQ, GAPDH, TUB, and WDP in a series of pear leaf sets under various stresses and hormone treatments. Using different statistical algorithms, we found that under various treatments, the WDP gene had more stable expression, ACT was the most stable under MeJA treatment, YLS8 was the most valuable reference gene under ABA hormone and heat stress conditions, and GAPDH showed worst results compared to other housekeeping genes, except under heat stress. These results will supply valuable and updated information for the selection of housekeeping genes in pears under biotic and abiotic stresses in the future.

The absorption and utilization of NH₄⁺ and NO₃⁻ by plant roots is closely related to soil moisture. In this study, we investigated the effect of short-time drought and rewatering on uptake and assimilation of NH₄⁺ and NO₃⁻ in 1-year-old Malus hupehensis plants, as well as transcription changes of ammonium transporters (AMTs) and nitrate transporters (NRTs). In roots, the NH₄⁺ and NO₃⁻ content and nitrate reductase activity decreased under drought and to some extent was restored after rewatering. Expression analysis indicated that most investigated AMTs and NRTs were down-regulated while MhAMT1;2 was significantly up-regulated in drought-stressed roots. Therefore, the function of MhAMT1;2 was further studied through bioinformatics analysis, tissue-specific expression, subcellular localization, and functional complementation in NH₄⁺ uptake-defective yeast and Arabidopsis mutants. Results showed that MhAMT1;2 was mainly expressed in roots and localized to the cell membrane. Moreover, MhAMT1;2 can mediate NH₄⁺ uptake in both yeast and Arabidopsis mutants, and the transport process was affected by external proton concentrations and ATP. The present study will create a basis for exploring the functional roles of plant AMT members and improving N uptake and use efficiency under drought condition in fruit trees.

Fruit color is a key quality indicator for apples and directly influences their market value. The process of fruit ripening encompasses various physiological and biochemical changes, such as the breakdown of chlorophyll and the buildup of anthocyanins and carotenoids. This study investigated the mechanism of chlorophyll degradation in apple peels using ‘Granny Smith’ varieties. The experiments involving the treatment with methyl jasmonate (MeJA) indicated that a concentration of 10 µM MeJA led to a reduction in chlorophyll degradation, while a higher concentration of 1500 µM MeJA enhanced this degradation, which aligned with the variations observed in the expression of genes associated with chlorophyll degradation. The key chlorophyll degradation gene MdSGR1 was cloned and found to be induced by methyl jasmonate. MdSGR1 encodes a 283-amino-acid protein belonging to the stay-green superfamily. The promoter possesses inducible cis-acting elements that respond to methyl jasmonate, low temperature and light, while the protein is localized to chloroplasts. Overexpression and silencing vectors were constructed. Overexpression of MdSGR1 induced chlorosis in tobacco leaves and ‘Granny Smith’ apple peels, decreased chlorophyll content, and upregulated related gene expression. Conversely, silencing MdSGR1 produced opposite effects. Arabidopsis thaliana plants overexpressing MdSGR1 exhibited low chlorophyll content, reduced photosynthetic rate, upregulated expression of genes associated with chlorophyll degradation. The results of yeast one-hybrid and dual-luciferase reporter assays indicated that the MdMYC2 transcription factor interacts with the promoter region of MdSGR1. In conclusion, MdSGR1 is crucial for the degradation of chlorophyll in apple peel, and it is regulated both by the MdMYC2 transcription factor and different concentrations of MeJA. This study preliminarily elucidated the regulatory mechanism of methyl jasmonate on chlorophyll degradation in fruit peel, and these findings provide an important theoretical basis for controlling degreening and color quality in apple fruit.