3.8 MT promoted NO3 uptake in an MdHY5-dependent manner
In this study, MT increased the expression of MdHY5 ,MdNRT2.1 , and MdNRT2.4 . MdHY5 activated the transcription of MdNRT2.1 and MdNRT2.4 . Accordingly, we further investigated whether MT promotes MdNRT2.1 and MdNRT2.4expression in an MdHY5 -dependent manner. Transgenic apple plants interfering with MdHY5 (Ri-1 and Ri-2) were generated for hydroponic N-deficiency experiments (Figure 8 a; Figure S3). The results showed that under N-deficiency conditions, the FW and15N-NO3 influx rates of WT with exogenous MT application were significantly higher than those of WT without MT application, but there was no significant difference between the FW and15N-NO3 influx rate of MdHY5 RNAi plants with versus without exogenous MT application (Figure 8 b,c).
Additionally, RT-qPCR results indicated that the expression ofMdHY5 , MdNRT2.1 , and MdNRT2.4 of WT with exogenous MT application was significantly higher than that of WT without MT application (Figure 8 d-f), but there was no significant difference between MdHY5 RNAi plants with versus without exogenous MT application. These results showed that MT-mediated promotion ofMdNRT2.1 and MdNRT2.4 expression in anMdHY5 -dependent manner, ultimately enhancing N absorption.
DISCUSSION
MT, as an emerging biomolecule, has been shown to regulate plant growth and increase resistance to abiotic and biotic stresses (Gao et al. , 2022). Its content is closely related to the key genes involved in its biosynthesis (Zhang et al. , 2022). ASMT encodes an enzyme that catalyzes the synthesis of MT from N-acetylserotonin, which is the final step in MT biosynthesis. The upregulation ofMzASMT1 in the apple plants is consistent with MT production in the 24-h dark/light cycle (Zuo et al. , 2014). Previous research revealed that MdASMT9 transgenic apple plants have elevated MT accumulation (Zhou, Li, et al. , 2022). In this study, we also observed a positive correlation between MdASMT9 gene expression and endogenous MT levels as-induced by N-deficiency stress. To confirm the function of MdASMT9 in regulating low-N resistance, we analyzed MdASMT9- OE apple plants under low-N stress. We found that overexpressing MdASMT9 led to increased plant height, plant biomass, and enhanced low-N tolerance. These findings indicate thatMdASMT9 is involved in enhancing resistance to low-N treatment in apple plants by regulating MT production.
Leaf N primarily contributes to the allocation of the photosynthetic apparatus, which in turn influences photosynthetic capacity (Bassi et al. , 2018). In this study, N-deficiency treatment affected the N concentration in leaves, which then decreased photosynthesis (Hiratsuka et al. , 2015; Wu et al. , 2019). Key functional attributes of photosynthesis, such as GS, play an important role in regulating Pn during plant growth (Tantray et al. , 2020). Previous studies have shown that N-deficiency conditions impair the photosynthetic apparatus and decrease GS (Wu et al. , 2019), ultimately affecting the production of photosynthates and leading to a decrease in Pn. Pn and GS decreased after low-N treatment in our tests, however, MdASMT9 -OE plants exhibited higher Pn and GS levels compared to WT plants.
ChlF is closely linked to photosynthesis processes and serves as an intuitive, efficient indicator of environmental stress (Wu et al. , 2019). Previous studies have shown that N-deficiency conditions lead to photoinhibition, which impairs PSII photochemical efficiency and reduces the activity of reaction centers, ultimately damaging the photosynthetic structure (Jin et al. , 2015; Zhao et al. , 2017). A decrease in FV/Fm indicates that the reaction center of PSII has induced photoinhibition, decreasing light energy use efficiency (Abbasi et al. , 2015). Y(II) reflects the proportion of absorbed light that is used in PSII photochemistry and is considered an important indicator of plant stress (Murchie and Lawson, 2013). Our results demonstrated a decrease inFV/Fm and Y(II) after N-deficiency treatment, while MdASMT9 -OE plants exhibited higherFV/Fm and Y(II). An increase inF0 and Y(NO) indicates progressive damage to the photosynthetic system, ultimately unbalancing the electron transport chain in the chloroplasts, accelerating reactive oxygen species production, and causing oxidative damage (Affenzeller et al. , 2009; Demmig-Adams and Adams, 1992; Fahnenstich et al. , 2008). Conversely, an increase of NPQ and qN protects the photosynthetic system by dissipating excess energy in the form of heat, thereby reducing oxidative damage (Jia et al. , 2019). Here, MdASMT9 -OE lines showed lower F0 and Y(NO) and higher NPQ during N deficiency treatment. These results indicated that overexpressing MdASMT9 helps maintain higher light harvesting and heat transfer capacity, sustains PSII utilization, and improves photosynthetic capacity under N-deficiency conditions.
NO3 is the one of the main available N sources for plants (Crawford and Glass, 1998). In the present study,MdASMT9 -OE plants accumulated more NO3 in leaves and roots after low-N stress than WT. Previous studies have shown that NRT1.1 and NRT2 protein play crucial roles in response to the low NO3 conditions (Okamoto et al. , 2003; You et al. , 2022). AtNRT2.1 andAtNRT2.4 are primarily expressed in roots and are important for NO3 influx (Okamoto et al. , 2003; Orsel et al. , 2002; Wirth et al. , 2007). In apple plants, there are five main NRT2 proteins: MdNRT2.1, MdNRT2.2, MdNRT2.3, MdNRT2.4, and MdNRT2.5 (Tahir 2020). We detected the expression ofMdNRT1.1 , MdNRT2.1 , MdNRT2.2 , MdNRT2.3 ,MdNRT2 .4, and MdNRT2.5 to find that application of MT and overexpression of MdASMT9 enhanced the expression ofMdNRT2.1 and MdNRT2.4 in apple plants.
HY5, a bZIP TF, regulates root development and NO3 absorption (Gangappa and Botto, 2016). Recently studies have revealed that HY5 moves to roots and activates its own expression, which in turn activates NRT2.1expression to promote N uptake while promoting C assimilation and translocation in shoots (Chen et al. , 2016). EMSA and LUC/REN activity provided evidence here that MdHY5 can bind to theMdNRT2.1 and MdNRT2.4 promoters to activate their expression. In apple calli, overexpression of MdHY5 increasedMdNRT2.1 and MdNRT2.4 expression while silencing ofMdHY5 reduced MdNRT2.1 and MdNRT2.4 expression. Previous research showed that exogenous MT and altered biosynthesis of endogenous MT enhance the expression of HY5 to protect plants from oxidative stress (Yao et al. , 2021). Here, MdASMT9overexpression and application of MT enhanced the expression ofMdHY5 under N-deficiency conditions. However, exogenous application of MT had no effect on the accumulation of N or the expression of MdNRT2.1 and MdNRT2.4 in MdHY5 RNAi plants under N-deficiency conditions. These results indicate that MT promotes N absorption in an MdHY5 -dependent manner.
C and N are two of the most important essential nutrients in all living organisms. Their metabolism is crucial to biological systems (Zhang et al. , 2018). C metabolism includes the TCA cycle, glycolysis, fatty acid, sugar, and organic acid metabolism; nucleotide and amino acid metabolism are related to N metabolism (Zhang et al. , 2018). N deficiency impairs nitrate reduction and amino acid assimilation, thereby affecting C and N metabolism (Schlüter et al. , 2012). It is necessary to balance C and N metabolism for plants to avoid metabolic inefficiencies in response to low-N stress (Santos-Filho et al. , 2014). The TCA cycle is a recognized source of C skeletons, supplying biosynthetic precursors and energy (Niehaus, 2021). It also plays a vital role in N assimilation by providing reducing power and the C-skeleton α-ketoglutarate (KG) and oxaloacetate (Oxa) (Zhou, Hu, et al. , 2022). The photosynthetic apparatus also relies on the availability of N to synthesize Chl, other metabolites, and cellular components (Kaachra et al. , 2018; Nunes-Nesi et al. , 2010). Thus, higher photosynthetic capacity and N utilization efficiency activate the TCA cycle and enhance the TCA cycle activity, which provide the thus energy necessary to withstand adverse environmental conditions.
The TCA cycle is closely linked with amino acids synthesis and metabolism. Amino acid metabolism is central to N management, involving the efficiency of N absorption, assimilation, and reactivation. It is finely regulated under N-deficiency treatment (Dellero, 2020). Previous studies have shown that N use efficiency is closely related to amino acid metabolism, and the level of free amino acids is significantly decreased when N supply is insufficient (Dellero, 2020; Sung et al. , 2015). Enhanced NO3uptake contributes to maintenance of the amino acid pool under N deficiency (Jia et al. , 2020). N-deficiency-tolerant wild soybean can enhance amino acids synthesis and the TCA cycle to improve the low-N tolerance (Liu et al. , 2020). MT has also been proven to be related to amino acid metabolism in N deficiency conditions (Wang et al. , 2022). Wang et al. (2021) showed that MT can promote the expression of glutamate synthase (GOGAT) and amino acid transporter genes, highlighting its importance in N use efficiency. MT enhances the activities of glutamate dehydrogenase (GDH) and GOGAT, leading to enhanced amino acids synthesis under N-deficiency conditions (Wang et al. , 2022). In this study, MdASMT9 -OE plants showed higher levels of certain amino acids (Glu, Asp, Arg, His, Lys, and Thr) compared to WT under low-N stress. This relatively higher and more stable amino acid pool may explain why MdASMT9 transgenic apple lines maintained better growth performance compared to WT during N-deficiency treatment.
In summary, a working model of the manner in which MdASMT9mediates MT biosynthesis to enhance low N tolerance was developed. We found that N-deficiency conditions induce the expression ofMdASMT9 and the accumulation of MT. Overexpression ofMdASMT9 enabled apple plants to maintain higher light harvesting and heat transfer capability while alleviating the damage of low-N stress to the photosynthetic system. MdASMT9 overexpression also promoted the15N-NO3 influx rate and N accumulation. MT promoted the expression of MdNRT2.1 andMdNRT2.4 in an MdHY5 -dependent manner, thereby improving N absorption. Furthermore, an increase in photosynthetic capacity and N uptake was found to enhance the TCA cycle, which positively modulated amino acid metabolism to resist N-deficiency stress.