MdHY5: bridging auxin and abscisic acid pathways to bolster cold tolerance in apple

Abstract

Cold stress causes irreversible damage to plants, ranging from chilling and freezing injuries to disruptions in cellular processes. The effects of cold stress include reduced membrane fluidity, protein destabilization, inhibition of enzyme activity, downregulation of gene expression, impaired protein biosynthesis, and ice crystal formation (Shi et al., 2018), which leads to an increase in relative electrolyte leakage. In apples (Malus domestica), cold stress impacts overwintering rootstocks and spring shoot sprouting leading to substantial losses for the global apple industry. For instance, a severe late frost in China in 2018 reduced the production of the ‘Red Fuji’ cultivar by c. 25% (Bai et al., 2019). The risk of apple damage from overwintering and early spring frosts is likely to rise with global climate change and climate instability, as the probability of a brief warm spell being followed by a cold wave increases (Unterberger et al., 2018).

‘Liu et al. demonstrate that HY5 regulates ABA synthesis and IAA dissimilation during cold stress and that these pathways enhance cold tolerance both independently and through crosstalk..’

Liu et al. (2024, doi: 10.1111/nph.20333) recently published findings in New Phytologist revealing that the apple transcription factor, MdHY5, reduces the indole-3-acetic acid (IAA) : abscisic acid (ABA) ratio and promotes anthocyanin accumulation by regulating MdGH3-2/12 (encoding IAA conjugating enzymes) and MdNCED2 (encoding an ABA biosynthesis enzyme). Previous studies suggest that IAA and ABA play opposing roles in cold responses (Zhu et al., 2015) and that cold stress inhibits auxin transport (Shibasaki et al., 2009). The ABA enhances cold tolerance through C-repeat binding factor (CBF)-dependent and CBF-independent pathways (Raza et al., 2023). CBFs act as key integrators of cold tolerance by collecting signals from various pathways and functioning as transcription factors (TFs) that activate cold-regulated gene expression (Jaglo-Ottosen et al., 1998; Shi et al., 2018). CBF-independent pathways also play crucial roles in cold stress responses (Ding et al., 2024).

The bZIP transcription factor, HY5, links light signaling with cold response pathways (Li et al., 2021) and regulates phytohormone signaling (Chen et al., 2008). HY5 modulates multiple pathways contributing to cold tolerance, but the molecular mechanisms underlying its diverse functionality remain poorly understood. Liu et al. demonstrate that HY5 regulates ABA synthesis and IAA dissimilation during cold stress and that these pathways enhance cold tolerance both independently and through crosstalk. Using genetic, molecular, and physiological experiments, the authors uncovered a complex regulatory network. Liu et al. first observed elevated MdHY5 expression under cold stress and reduced cold tolerance in MdHY5 RNAi lines, prompting further investigation into MdHY5's downstream mechanisms. They identified MdGH3-2/12 and MdNCED2, as direct MdHY5 targets. Under ambient conditions, MdHY5 was found to negatively regulate MdGH3-2/12 expression inhibiting IAA conjugation and positively regulating auxin-related functions. By using electrophoretic mobility shift assays, they showed that MdHY5 directly interacts with the promotors of MdGH3-2 and MdGH3-12 differentially under ambient conditions when compared to cold stressed conditions. They used a dual-luciferase to determine the functional implications of this differential binding activity. They found that cold stress substantially reduced the transcriptional repression of MdHY5 on the MdGH3-2 and MdGH3-12 promoters, which led to higher levels of MdGH3-2 and MdGH3-12 and thus less free IAA.

Liu et al. also found that MdHY5 activated MdNCED2 expression, enhancing ABA synthesis and its downstream effects. They found that the increased binding affinity of MdHY5 to the MdNCED2 promoter under cold stress, along with its high expression, promoted ABA synthesis. Therefore, high-MdHY5 expression under cold stress concurrently inhibits responses mediated by auxin and promotes ABA synthesis leading to a reduced IAA : ABA ratio.

MdHY5 has also been linked to anthocyanins biosynthesis (Catalá et al., 2011). Liu et al. observed increased anthocyanin accumulation and elevated ROS scavenging enzyme gene expression in MdGH3-2/12 OE lines. These findings integrate cold stress responses, IAA content, and antioxidant accumulation into a unified pathway. The enhanced expression of CBF1/3 and its downstream target COR47 was observed both in MdGH3-2/12 OE and MdNCED2 OE lines. Both low-IAA and high-ABA levels promote anthocyanins accumulation, enhancing antioxidant activity (Wang et al., 2018; Khaleghnezhad et al., 2019), consistent with HY5's role in regulating cold tolerance through CBF-dependent and CDF-independent mechanisms.

These findings expand the regulatory network of HY5 by linking it to phytohormone accumulation and identifying distinct downstream targets beyond its modulation of CBF and COR in cold stress responses. This discovery connects HY5, auxin/ABA signaling, and cold tolerance for the first time from both a molecular and genetic perspective. Liu et al. propose that MdHY5 induces cold tolerance by shifting hormonal homeostasis, specifically by reducing the IAA : ABA ratio. This hypothesis is supported by their findings that GH3-2/12 overexpression induces ABA accumulation. Since IAA interacts with multiple phytohormones (Ku et al., 2018) and inhibits ABA signaling pathway transduction, HY5-triggered changes in the IAA : ABA ratio are co-regulated by downstream phytohormone crosstalk (Fig. 1).

Fig. 1

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Schematic representation of the cold tolerance regulatory network involving MdHY5. The plus and minus symbols represent changes in expression or accumulation under cold stress in apple seedlings. The violet colored arrows highlight the key findings in the highlighted article and the blue arrows describe the transcriptional regulation of MdHY5 in C-repeat binding factor (CBF)-dependent and CBF-independent pathways. ABA, abscisic acid; IAA, indole-3-acetic acid.

MdHY5 is an essential integrator of plant responses to abiotic stress due to its ability to respond to environmental signals, including light and temperature, and target phytohormone-dependent and independent pathways. Its function as a transcription factor is modulated by transcriptional regulation and binding activity. Agonistic and antagonistic regulations, as demonstrated by the case of MdGH3-2/12 in the findings by Liu et al. highlight the complexity of predicting the downstream effects of MdHY5 under cold stress. Future studies should explore how environmental factors regulate MdHY5 binding specificity and target gene modulation. Further elucidation of HY5's molecular mechanisms, including how it responds to multiple signals and its potential interactions with chaperone molecules and epigenetic markers, will also provide deeper insights into its multifunctionality. The responses of these chaperone molecules or epigenetic markers to different developmental signals and abiotic stresses may uncover new aspects relating to the multiple functions of HY5. Comprehensive genetic, molecular, and computational modeling approaches may be necessary to fully characterize this regulatory network.

Although some questions remain unanswered, the Liu et al. study significantly advances our understanding of apple cold tolerance mechanisms and offers new directions for breeding cold-resilient crops.