Cultivating vibrant apples: the role of nitrogen signaling in orchestrating anthocyanin biosynthesis for enhanced fruit coloration and quality

Abstract

Plants respond to nitrogen fluctuations through sophisticated metabolic reprogramming, with anthocyanin accumulation serving as a hallmark of stress adaptation. Nitrogen signaling, as a core regulatory pathway in this process, finely regulates anthocyanin biosynthesis through an intricate network of transcription factors, metabolic pathways, and signaling cascades. However, the molecular mechanisms underlying this regulation remain elusive. In an article recently published in New Phytologist, Guo et al. (2025, doi: 10.1111/nph.70040) unveiled a small part of these mechanisms with their outstanding research work. They proposed an ‘ubiquitination–phosphorylation–hormone’ tripartite regulatory framework, revealing how nitrate signaling dynamically coordinates with gibberellin pathways through posttranslational modifications to precisely regulate anthocyanin biosynthesis. Their work systematically deciphers the molecular logic of nutrient–hormone cross talk, offering novel insights into the interplay between nitrate signaling and phytohormone interaction networks. This discovery is of great significance in terms of revealing the molecular mechanisms of plant adaptation to environmental changes, as well as for future applications in agriculture, ecology, and other fields.

It unveils the dynamic integration of post-translational modifications… with hormonal cues, through spatiotemporally orchestrated multi-tiered interactions, thereby providing a conceptual framework for optimizing nitrogen–hormone balance in fruit crop cultivation systems.

Nitrogen, as a central element in plant life processes, not only provides the structural foundation for the synthesis of primary metabolites such as amino acids and nucleic acids but also functions as a metabolic hub by dynamically sensing environmental nitrogen availability. This regulatory process involves multilayered coordination mechanisms. Under nitrogen-deficient conditions, plants adopt a ‘survival-priority’ strategy for resource reallocation. For instance, Arabidopsis seedlings exhibit a 42% reduction in Chl content, specific accumulation of anthocyanins in leaves, and a marked increase in lateral root density (Scheible et al., 2004; Peng et al., 2008). Such adaptive remodeling is achieved through dual metabolic adjustments: Nitrate resupply rapidly induces the trehalose biosynthesis gene AtTPS5 while suppressing trehalose-6-phosphate phosphatases (AtTPPA/B), forming a ‘metabolic valve’ that redirects carbon flux from starch synthesis toward phenylpropanoid pathways (Scheible et al., 2004). Concurrently, downregulation of nitrogen-intensive enzymes such as phenylalanine ammonia-lyase enhances nitrogen recycling efficiency in Nicotiana tabacum xylem by 37% (Fritz et al., 2006).

At the transcriptional level, an antagonistic regulatory network involving MYB-bHLH transcription factor complexes (e.g. PAP1/GL3) and LBD37/38/39 repressors governs secondary metabolism. The MYB-bHLH complexes activate anthocyanin biosynthesis by binding ACGT elements in promoters of genes such as CHS and DFR, while LBD proteins competitively occupy regulatory motifs to maintain dynamic equilibrium (Lea et al., 2007; Rubin et al., 2009). Metabolically, nitrogen availability orchestrates shifts to balance growth and defense. Under nitrogen deprivation, plants prioritize nitrogen conservation by suppressing flavonoid synthesis to alleviate substrate competition in phenylpropanoid pathways, thereby favoring anthocyanin accumulation (Scheible et al., 2004). Simultaneously, lignin deposition in N. tabacum stems increases, reinforcing structural integrity as a mechanical adaptation (Fritz et al., 2006).

Over the past two decades, systematic investigations into the evolution of nitrogen signaling networks governing secondary metabolism have revealed key regulatory components through multidimensional approaches. At the transcriptional level, Arabidopsis establishes a bidirectional regulatory framework through antagonistic interactions between LBD37/38/39 and PAP1/GL3 transcription factors (Lea et al., 2007; Rubin et al., 2009). In Malus domestica, the innovative MdHY5 transcription factor creates a species-specific ‘nitrogen uptake-metabolic conversion’ coupling module by coordinately regulating nitrate transporter genes (MdNRT2.1/2.4) and anthocyanin biosynthetic gene MdMYB10 (An et al., 2017). Posttranslational regulation involves the E3 ubiquitin ligase MdBT2 employing dual targeting mechanisms to maintain nitrogen homeostasis. Under high nitrogen conditions, it promotes ubiquitination-mediated degradation of MdMYB1 while modifying MdMYB88/124 to disrupt their interaction with nitrate transporters, thereby coordinately suppressing both anthocyanin biosynthesis and nitrogen uptake (Wang et al., 2018; Zhang et al., 2021). Epigenetic regulation mechanisms include HDA15-mediated activation of anthocyanin genes through H3K9ac deacetylation (Liao et al., 2022), and sex-specific DNA methylation patterns regulating differential accumulation of secondary metabolites in Populus spp. (Yang et al., 2023). Hormonal cross-talk mechanisms feature DELLA-PAP1 interactions and ethylene signaling cascades as molecular hubs for nitrogen–hormone cross talk (Wang et al., 2015; Zhang et al., 2017). However, these breakthroughs predominantly focused on linear analyses within single regulatory hierarchies, failing to elucidate the spatiotemporal coordination logic between ubiquitination/phosphorylation modifications and hormonal networks, resulting in a fragmented understanding of nitrogen signaling transduction.

The groundbreaking study by Guo et al. transcends these limitations by constructing an ‘ubiquitination–phosphorylation–hormone’ tripartite regulatory model. This model reveals that E3 ligase MdBRG3 and kinase MdMPK7 dynamically regulate MdLBD36 stability through antagonistic posttranslational modifications (high-N-induced ubiquitination degradation vs low-N-triggered phosphorylation protection). This regulatory node further integrates with gibberellin (GA) signaling through DELLA protein MdRGL2a, generating cascade amplification effects that enable precise conversion of nitrate concentration gradients into anthocyanin biosynthetic flux in M. domestica (Fig. 1). This study systematically elucidates a multilayered molecular network governing dynamic nitrate signaling in anthocyanin biosynthesis, transcending conventional boundaries between nitrogen metabolism and secondary metabolism research. For the first time in M. domestica, the LBD family transcription factor MdLBD36 was identified as a central hub for nitrate signal transduction. Unlike the classical pathway in Arabidopsis in which LBD37/38/39 repress anthocyanin biosynthesis by inhibiting PAP1/2 (Rubin et al., 2009), MdLBD36 exhibits dual functionality: (1) directly binding to the MdABI5 promoter to activate its expression and (2) forming a functional complex with MdABI5 to synergistically enhance transcriptional activation of downstream anthocyanin biosynthetic genes. This discovery not only establishes the LBD-ABI5 module as a core regulator of nitrogen deficiency-induced anthocyanin accumulation but also highlights its potential role as a node integrating nitrate and abscisic acid signaling, offering novel insights into multisignal cross talk.

Fig. 1

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Model of nitrogen signaling in regulating anthocyanin biosynthesis in Malus domestica. Under low-nitrogen (low N) conditions, transcription of the protein kinase MdMPK7 is upregulated, whereas expression of the E3 ubiquitin ligase MdBRG3 is suppressed. Conversely, under high-nitrogen (high N) conditions, MdMPK7 transcription is inhibited, while MdBRG3 transcription is activated. Mechanistically, MdMPK7 stabilizes the MdLBD36 protein via phosphorylation, enhancing its ability to promote anthocyanin biosynthesis. By contrast, MdBRG3 directly suppresses anthocyanin biosynthesis by mediating ubiquitination-dependent degradation of MdLBD36. Furthermore, the gibberellin (GA) signaling repressor MdRGL2a amplifies MdLBD36-induced anthocyanin biosynthesis by facilitating its transcriptional activation. Blunt arrows represent inhibition. Sharp arrows represent promotion. The orange spheres with the letter U represent ubiquitination. The purple spheres with the letter P represent phosphorylation.

Guo et al.'s discovery has revealed the precise posttranslational regulatory mechanism of MdLBD36 stability. Under high nitrogen conditions, the E3 ubiquitin ligase MdBRG3 promotes ubiquitination-dependent degradation of MdLBD36, thereby inhibiting metabolic flux. When nitrate is limited, the kinase MdMPK7 is activated, and it phosphorylates MdLBD36 to prevent its degradation, forming an ‘antagonistic interaction between ubiquitination and phosphorylation’. This mechanism fills a critical gap in understanding dynamic nitrogen signal transduction and provides a new paradigm for environmental adaptation research. Furthermore, the study integrates GA signaling into the nitrogen regulatory network. The DELLA protein MdRGL2a, a master regulator of GA signaling and anthocyanin biosynthesis (An et al., 2023), interacts with MdLBD36 to enhance its transcriptional activation capacity. This study delineates a nitrate-initiated regulatory cascade that mechanistically links nitrogen perception to hormonal control, resolving the molecular circuitry governing their bidirectional cross talk. It unveils the dynamic integration of posttranslational modifications (ubiquitination and phosphorylation) with hormonal cues through spatiotemporally orchestrated multitiered interactions, thereby providing a conceptual framework for optimizing nitrogen–hormone balance in fruit crop cultivation systems.

Guo et al. establish a three-tier regulatory model based on ubiquitination, phosphorylation, and hormonal signaling, offering a fresh approach to exploring the interplay among plant nutrition, metabolism, and hormones. The research highlights the key role of the LBD-ABI5 module in nitrate signal transduction and, via a signal amplification effect mediated by MdRGL2a, lays the theoretical foundation for the coordinated regulation of nitrogen uptake and secondary metabolism in crops. The findings hold promise for optimizing agricultural practices under low-nitrogen conditions and for enhancing flower coloration in ornamental plants. Moreover, the proposed strategy for regulating the stability of MdLBD36 provides a new genetic target for breeding crops with high nitrogen use efficiency. Future studies should concentrate on elucidating the signaling connections between nitrate receptors and protein modification networks to further refine the overall regulatory mechanism of nitrate signaling, thereby offering innovative molecular design frameworks for improving crop stress tolerance and metabolic engineering.