How spermidine shields citrus from cold damage: Scientists identify key gene regulation circuit
image: Silencing of CiWRKY27 repressed Spd accumulation and compromised cold tolerance. (a–c) Plant phenotype (a), chlorophyll fluorescence imaging (b), and Fv/Fm ratios (c) of the TRV control and VIGS (TRV-CiWRKY27) plants before and after the cold treatment. Scale bars, 1 cm. (d–e) Electrolyte leakage (d) and MDA content (e) of the tested plants before and after the cold treatment. (f–g) The expression levels of CiSPDS1 (f) and Spd contents (g) of the TRV control and VIGS plants before and after the cold treatment. Error bars represent ± SD (n = 3). ANOVA was conducted for statistics analysis, and the asterisks indicate presence of significant difference between each other (**P < 0.01; ***P < 0.001).
Credit: Horticulture Research
Cold injury is a major limitation to citrus production, yet the molecular mechanisms enabling hardy species to survive freezing remain unclear. This study reveals that spermidine, a polyamine metabolite, plays a central role in protecting citrus against cold stress. Researchers found that cold exposure sharply increased spermidine levels, and external application of spermidine significantly enhanced plant survival under freezing conditions. They identified CiSPDS1 as the key biosynthetic gene responsible for spermidine production and demonstrated that manipulating this gene directly affects cold tolerance. The study further uncovered a transcriptional module in which the WRKY factor CiWRKY27 activates CiSPDS1, establishing a regulatory pathway essential for cold resilience.
Cold stress is one of the most damaging abiotic factors affecting plant growth, yield, and geographical distribution. To cope with freezing temperatures, plants activate complex physiological adjustments and reprogram metabolic pathways. Polyamines—particularly spermidine—have been implicated in stabilizing membranes, scavenging reactive oxygen species, and enhancing cellular protection. However, compared with the extensively studied putrescine pathway, the regulatory mechanisms that control spermidine biosynthesis during cold stress remain poorly defined in citrus species. Ichang papeda, one of the cold-hardiest citrus relatives, offers a unique model for uncovering genetic determinants of freezing resilience. Due to these challenges, there is a need to investigate how spermidine biosynthesis is regulated to enhance cold tolerance.
Researchers from Huazhong Agricultural University and collaborating institutions reported (DOI: 10.1093/hr/uhaf065) on 4 March 2025 in Horticulture Research that they have identified a cold-responsive regulatory module in Ichang papeda (Citrus ichangensis). The team demonstrated that the transcription factor CiWRKY27 directly activates the spermidine synthase gene CiSPDS1, triggering a metabolic response that strengthens plant tolerance to freezing. Their findings provide a mechanistic explanation for spermidine-driven cold resilience and offer promising genetic resources for breeding cold-resistant citrus varieties.
Through physiological, molecular, and genetic analyses, the researchers first showed that cold exposure caused a marked rise in endogenous spermidine and spermidine synthase (SPDS) activity in Ichang papeda. Applying spermidine externally significantly reduced leaf damage, electrolyte leakage, malondialdehyde accumulation, and reactive oxygen species generation in both Ichang papeda and cold-sensitive lemon, confirming spermidine’s protective role. Genome-wide analysis identified two SPDS genes, but only CiSPDS1 was strongly upregulated by cold. Overexpressing CiSPDS1 in tobacco and lemon led to higher spermidine levels and markedly enhanced cold tolerance, whereas silencing CiSPDS1 reduced spermidine synthesis and increased cold sensitivity—effects that were reversed by exogenous spermidine supplementation.
To understand upstream regulation, the team discovered two W-box motifs in the CiSPDS1 promoter and identified CiWRKY27 as the cold-induced transcription factor binding directly to these elements. Yeast one-hybrid, EMSA, and luciferase assays confirmed that CiWRKY27 activates CiSPDS1 expression. Silencing CiWRKY27 suppressed CiSPDS1, lowered spermidine accumulation, and caused severe freezing damage. Collectively, the study defines a CiWRKY27–CiSPDS1 transcriptional pathway that regulates spermidine biosynthesis and cold acclimation in citrus.
“The discovery of this regulatory module provides a missing link in understanding how citrus species manage to withstand freezing stress,” said the study’s senior author. “By demonstrating that CiWRKY27 directly activates CiSPDS1 to boost spermidine production, we have uncovered a precise molecular mechanism that explains Ichang papeda’s exceptional cold hardiness. This knowledge not only enriches our understanding of cold-responsive metabolism but also presents a promising genetic target for improving the resilience of commercial citrus crops.”
The identification of the CiWRKY27–CiSPDS1 module offers new opportunities for engineering or breeding citrus varieties capable of surviving low temperatures. Enhancing spermidine production—either through targeted gene editing, marker-assisted selection, or controlled application of spermidine—could help vulnerable cultivars endure cold spells that increasingly threaten global citrus industries. Beyond citrus, the regulatory mechanism may be conserved across plant species, providing a broader framework for improving stress resilience in horticultural and agricultural crops. This research thus opens the door to developing climate-resilient fruit plants in the face of increasing temperature variability.
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References
DOI
Original Source URL
https://doi.org/10.1093/hr/uhaf065
Funding information
This work was supported by the National Key Research and Development Program of China (2022YFD1200503), National Natural Science Foundation of China (32330095), and the Key Research and Development Program of Jiangsu Province (BE2023328).
About Horticulture Research
Horticulture Research is an open access journal of Nanjing Agricultural University and ranked number one in the Horticulture category of the Journal Citation Reports ™ from Clarivate, 2023. The journal is committed to publishing original research articles, reviews, perspectives, comments, correspondence articles and letters to the editor related to all major horticultural plants and disciplines, including biotechnology, breeding, cellular and molecular biology, evolution, genetics, inter-species interactions, physiology, and the origination and domestication of crops.