Nitrogen (N) is one of the important macronutrients in plants, and N deficiency induces leaf senescence. However, the molecular mechanism underlying how N deficiency affects leaf senescence is unclear. Here, we report an apple NAC TF, MdNAC4, that participates in N deficiency-induced leaf senescence. The senescence phenotype of apple leaves overexpressing MdNAC4 was enhanced after N deficiency. Consistently, the chlorophyll content of transgenic leaves was significantly lower than that in the WT control leaves, the expression of chlorophyll catabolism-related genes (MdNYC1, MdPAO, and MdSGR1) was significantly higher than that in the WT controls, and the expression of chlorophyll synthesis-related genes (MdHEMA, MdCHLI, and MdCHLM) was significantly lower than that in the WT control leaves. Furthermore, MdNAC4 was found to directly activate the transcription of the chlorophyll catabolism-related genes MdNYC1 and MdPAO. Additionally, MdNAC4 was proven to interact with MdAPRR2 proteins both in vitro and in vivo, and overexpression of MdAPRR2 seemed to delay N deficiency-induced leaf senescence. Correspondingly, the chlorophyll loss of MdAPRR2-overexpressing (MdAPRR2-OE) lines was significantly lower than in WT control plants. Although downregulated, the expression of the chlorophyll synthesis-related genes MdHEMA, MdCHLI, and MdCHLM in the transgenic plants was more than twice that in the WT control plants. Taken together, our results enrich the regulatory network of leaf senescence induced by N deficiency through the interaction between MdNAC4 and MdAPRR2.

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Although it is well established that nitrogen (N) deficiency induces leaf senescence, the molecular mechanism of N deficiency-induced leaf senescence remains largely unknown. Here, we show that an abscisic acid (ABA)-responsive NAC transcription factor (TF) is involved in N deficiency-induced leaf senescence. The overexpression of MdNAC4 led to increased ABA levels in apple calli by directly activating the transcription of the ABA biosynthesis gene MdNCED2. In addition, MdNAC4 overexpression promoted N deficiency-induced leaf senescence. Further investigation showed that MdNAC4 directly bound the promoter of the senescence-associated gene (SAG) MdSAG39 and upregulated its expression. Interestingly, the function of MdNAC4 in promoting N deficiency-induced leaf senescence was enhanced in the presence of ABA. Furthermore, we identified an interaction between the ABA receptor protein MdPYL4 and the MdNAC4 protein. Moreover, MdPYL4 showed a function similar to that of MdNAC4 in ABA-mediated N deficiency-induced leaf senescence. These findings suggest that ABA plays a central role in N deficiency-induced leaf senescence and that MdPYL4 interacts with MdNAC4 to enhance the response of the latter to N deficiency, thus promoting N deficiency-induced leaf senescence. In conclusion, our results provide new insight into how MdNAC4 regulates N deficiency-induced leaf senescence. Graphical Abstract

The regulation of plant gene expression by nitrate is a complex regulatory process. Here, we identified 90 GARP family genes in apples by genome-wide analysis. As a member of the GARP gene family, the expression of MdHHO3 (Malus domestica HYPERSENSITIVITY TO LOW PHOSPHATE-ELICITED PRIMARY ROOT SHORTENING1 HOMOLOG 3) is upregulated under N (nitrogen) supply. The results of DNA-binding site analysis and electrophoretic mobility shift assays (EMSA) showed that MdHHO3 binds to the motif-containing GAATC. Furthermore, MdHHO3 binds to its promoter sequence and inhibits its activity. In addition, the overexpression of MdHHO3 in apple calli resulted in less accumulation of nitrate in 35S:MdHHO3-GFP calli and downregulated the expression of the nitrate transport-related genes but upregulated the expression of the nitrate assimilation-related genes. Similarly, the expression of the nitrate transport-related genes was downregulated and the expression of the nitrate assimilation-related genes was upregulated in MdHHO3 overexpression Arabidopsis and tobacco plants. Interaction experiments showed that MdHHO3 could bind to the promoter MdNRT2.1 (NITRATE TRANSPORTER 2.1) and negatively regulate its expression. Moreover, the exposure of MdHHO3-overexpressing Arabidopsis and tobacco to nitrate deficiency resulted in an early senescence phenotype as compared to the WT plants. These results show that MdHHO3 can not only negatively regulate nitrate accumulation in response to nitrate but also promote early leaf senescence under nitrate deficiency. This information may be useful to further reveal the mechanism of the nitrate response and demonstrates that nitrate deficiency induces leaf senescence in apples.

24Anthocyanins are the key factors controlling the coloration of plant 25 tissues. However, the molecular mechanism underlying the effects of 26 environmental pH on the synthesis of apple anthocyanins is unclear. 27 In this study, we analyzed the anthocyanin contents of apple calli 28 cultured in media at different pHs (5.5, 6.0, and 6.5). The highest 29 anthocyanin content was observed at pH 6.0. Additionally, the 30 moderately acidic conditions up-regulated the expression of 31 MdMYB3 as well as specific anthocyanin biosynthesis structural 32 genes (MdDFR and MdUFGT). Moreover, the anthocyanin content 33 was higher in calli overexpressing MdMYB3 than in the wild-type 34 controls at different pHs. Yeast one-hybrid assay results indicated 35 that MdMYB3 binds to the MdDFR and MdUFGT promoters in 36 vivo. An analysis of the MdDFR and MdUFGT promoters revealed 37 multiple MYB-binding sites. Meanwhile, electrophoretic mobility 38 shift assays confirmed that MdMYB3 binds to the MdDFR and 39 MdUFGT promoters in vitro. Furthermore, GUS promoter activity 40 assays suggested that the MdDFR and MdUFGT promoter activities 41 are enhanced by acidic conditions, and the binding of MdMYB3 42 may further enhance activity. These results implied that an 43 acid-induced apple MYB transcription factor (MdMYB3) promotes 44 anthocyanin accumulation by up-regulating the expression of 45 MdDFR and MdUFGT under moderately acidic conditions. 46 47 65 temperatures, hormones, high irradiance, sugar, and pH [10-14]. 66There is clear evidence that high pH levels enhance the degradation 67 of anthocyanins in carrot [12], while exposure to low pH induces the 68 expression of anthocyanin biosynthesis genes in crabapple leaves, 69 ultimately resulting in increased anthocyanin levels [15]. 70 Previous studies revealed that the accumulation of anthocyanins in 71 the vacuoles of plant tissue cells depends on the pH of the vacuoles 72 in which anthocyanins localize [16, 17]. In morning glory (Ipomoea 73 tricolor) petals, the pH of vacuoles is relatively low when the flower 74 bud opens, causing the petals to be red, but the vacuolar pH 75 increases over time and the petals eventually turn blue [18]. This 76 color change is due to the Na + /H + exchanger encoded by the 77 PURPLE gene, which transports sodium ions into the vacuole and 78 moves protons out of the vacuole to increase the pH [19]. Petunia 79 hybrida flowers normally have a lower pH than I. tricolor flowers, 80 and the wild-type (WT) flowers remain on the reddish (low pH) side 81 of the color spectrum. The PH4 gene encodes a MYB domain and is 82 expressed in the epidermis of petals [20]. A mutation to PH4 results 83 in the lightening of petal colors as well as increases in the pH of 84 petal extracts and, in some genetic backgrounds, the elimination of 85 anthocyanins in flowers [20]. Additionally, mutations to 86 ANTHOCYANIN1 (AN1), AN2, and AN11 lead to a loss of 87 anthocyanin pigments and an increase in the pH of petal extracts 88 [21]. In apple, some MYB TFs control cell pH, an...