Transcription factors are proteins that bind specific DNA sequences and control how strongly other genes are read, making them the master regulators of how plants react to their environment. The WRKY family, named for the highly conserved WRKY amino acid motif and the zinc-finger structure it accompanies, occupies a central regulatory role in plant responses to abiotic stresses such as drought, salinity and extreme temperature. Despite their importance, the response patterns and molecular mechanisms of Class II WRKY genes involved in low-temperature responses remained poorly understood in apple. The new study set out to close that gap by combining genome-wide identification, gene cloning, Agrobacterium-mediated genetic transformation and physiological index determination, using Malus halliana as the experimental material.

The first stage of the work was a systematic sweep of the apple genome. The researchers identified a total of 71 Class II WRKY genes and classified them into five subgroups, designated IIa through IIe. Among these, subgroup IIc exhibited the most prominent evolutionary expansion, suggesting that members of this lineage have been repeatedly recruited and diversified during apple evolution. Members of the family possessed conserved evolutionary structures, and their promoter regions contained numerous cis-acting elements associated with stress and hormone responses. Cis-acting elements are short DNA motifs located upstream of genes that serve as docking sites for other regulatory proteins, so their abundance in these promoters indicated that the genes are wired to be rapidly switched on when stress hormones or environmental cues arrive.

To find out which of these genes actually responds to cold, the team measured the expression of the candidates under low-temperature treatment using reverse transcription quantitative PCR, or RT-qPCR, a technique that quantifies how many messenger RNA copies a gene produces and therefore how active it is. One gene stood out clearly: MhWRKY71-like, a member of subgroup IIc, was the most strongly induced by low temperature of all the genes examined. The protein encoded by this gene was localized in the nucleus, which is exactly where a transcription factor must reside to bind DNA and regulate target genes. Phylogenetic analysis, which compares protein sequences across species to reconstruct evolutionary relationships, indicated that MhWRKY71-like shared the closest genetic relationship with a WRKY protein from Pyrus x bretschneideri, the Chinese white pear, a fellow member of the Rosaceae family.

With the gene identified, the researchers moved to functional verification, cloning the sense sequence, the antisense sequence and a virus-induced gene silencing fragment of MhWRKY71-like from Malus halliana cDNA and constructing the corresponding expression vectors. They then used Agrobacterium infiltration, a standard laboratory method that co-opts the soil bacterium’s natural DNA-transfer machinery to introduce engineered genes into plant cells, to generate an impressive array of experimental material. This included Arabidopsis thaliana lines overexpressing MhWRKY71-like, apple callus lines with either overexpression or antisense suppression of the gene, and plants of Malus hupehensis var. pingyiensis carrying either root overexpression or VIGS-mediated silencing. Virus-induced gene silencing, or VIGS, exploits a plant viral vector to trigger the plant’s own antiviral RNA interference pathway, thereby specifically degrading the messenger RNA of the target gene and shutting it down without permanently altering the genome.

The results of the low-temperature stress treatments were striking. Overexpression of MhWRKY71-like markedly enhanced cold tolerance across all the systems tested. The overexpressing lines showed significant increases in chlorophyll content, meaning their photosynthetic apparatus stayed greener and more functional under chilling conditions. They also displayed higher activities of the antioxidant enzymes superoxide dismutase, peroxidase and catalase, the trio of enzymes that plants deploy to neutralize reactive oxygen species. Cold stress, like many abiotic stresses, causes a surge of reactive oxygen species, or ROS, chemically reactive molecules containing oxygen that damage proteins, membranes and DNA when they accumulate unchecked. In the overexpression lines, ROS accumulation decreased obviously, and the plants were better able to keep this oxidative threat under control.

Two other physiological markers told the same story from a different angle. Relative electrical conductivity, or REC, measures how much electrolytes leak out of plant cells, and it rises when membranes lose their integrity under stress. Malondialdehyde, or MDA, is a breakdown product of lipid peroxidation and serves as a classic chemical fingerprint of membrane damage. In the MhWRKY71-like overexpression lines, both REC and MDA content decreased obviously, indicating that the gene helps maintain membrane integrity when temperatures drop. In contrast, the lines in which the gene was suppressed through antisense inhibition or transient VIGS silencing displayed aggravated cold sensitivity, with the protective physiological responses weakened rather than strengthened. The symmetry of the outcome, with gain of function improving tolerance and loss of function worsening it, is a hallmark of a genuinely causal regulatory relationship.

Taken together, the evidence supports a clear mechanistic model. MhWRKY71-like is a nuclear-localized, cold-inducible transcription factor belonging to the IIc subgroup of the WRKY family. When low temperature strikes, the gene is switched on, and the protein it encodes appears to orchestrate a protective program that alleviates oxidative damage by boosting antioxidant enzyme activities, maintains membrane integrity as reflected by reduced electrolyte leakage and lipid peroxidation, and stabilizes chlorophyll so that photosynthesis can continue. The functional verification in Arabidopsis, apple calli and Malus hupehensis through overexpression, antisense inhibition and VIGS silencing systems confirmed that MhWRKY71-like may serve as a positive regulator involved in the apple cold stress response process. Because the gene responded consistently across a herbaceous model plant, undifferentiated apple tissue and a woody apple relative, the regulatory role is likely conserved rather than species-specific.

The practical implications reach well beyond the laboratory bench. Late frost damage during bud burst is a recurring nightmare for apple growers, and breeding varieties with enhanced cold tolerance has long been a goal of fruit tree genetics. A transcription factor that sits at the top of a stress-response cascade is an attractive breeding target because switching on a single master regulator can potentially activate many downstream protective genes at once. The finding that MhWRKY71-like overexpression improved tolerance in Malus hupehensis var. pingyiensis, a species used as a rootstock in apple production, hints at possible applications in rootstock engineering or in marker-assisted selection programs that screen for favorable variants of the gene. The study was supported by the Biotechnology Breeding Project of Gansu Academy of Agricultural Sciences, reflecting the agricultural motivation behind the work.

There are still questions to answer before the discovery translates into hardier orchards. The downstream genes that MhWRKY71-like directly controls have not yet been mapped, and understanding which branches of the cold-response network the factor activates would clarify how it simultaneously stabilizes chlorophyll, membranes and the antioxidant system. It also remains to be seen how the gene performs under field conditions, where cold stress arrives together with fluctuating light, humidity and pathogen pressure rather than as a controlled laboratory treatment. Nevertheless, by identifying 71 Class II WRKY genes across the apple genome, pinpointing the one most strongly induced by cold, and proving its function through gain-of-function and loss-of-function experiments in three different plant systems, the team has provided both a resource for the apple research community and a concrete molecular lead in the effort to protect one of the world’s most valuable fruit crops from the growing threat of unseasonable cold.

Subject of Research: Cold stress response regulation in apple by the WRKY transcription factor MhWRKY71-like

Article Title: Identification and functional analysis of transcription factor MhWRKY71-like from Malus halliana in response to low temperature

Article References: Zhao, W., Sun, W., Zhang, Z., & Wang, Y. (2026). Identification and functional analysis of transcription factor MhWRKY71-like from Malus halliana in response to low temperature. BMC Plant Biology. https://doi.org/10.1186/s12870-026-09938-0

Image Credits: AI Generated

DOI: 10.1186/s12870-026-09938-0

Keywords: Malus halliana, MhWRKY71-like, WRKY transcription factors, low temperature stress, cold tolerance, reactive oxygen species, antioxidant enzymes, virus-induced gene silencing, Agrobacterium-mediated transformation, apple breeding, chlorophyll stability, membrane integrity