VERNALIZATION2 alters early tiller development in a facultative spring hexaploid bread wheat Dominique Hirsz1,2 , Harry Taylor2,3 , India Lacey2 , Wenxue Wu4 , Adam Gauley2,5 and Laura Dixon1,2 1Leibniz Institute of Plant Genetics and Crop Plant Research (IPK), 06466, Gatersleben, Germany; 2Faculty of Biological Sciences, University of Leeds, Woodhouse Lane, Leeds, LS2 9NL, UK; 3Department of Plant Sciences, University of Cambridge, Downing Street, Cambridge, CB2 3EA, UK; 4State Key Laboratory of Crop Gene Resources and Breeding/Institute of Crop Sciences, Chinese Academy of Agricultural Sciences, Beijing, 100081, China; 5Agri-Food and Biosciences Institute, Newforge Lane, Belfast, BT9 5PX, UK Authors for correspondence: Dominique Hirsz Email: hirsz@ipk-gatersleben.de Laura Dixon Email: dixon@ipk-gatersleben.de Received: 2 May 2025 Accepted: 4 December 2025 New Phytologist (2026) doi: 10.1111/nph.70907 Key words: photoperiod, temperature, Triticum aestivum, vernalization, VRN1, VRN2, ZCCT1, ZCCT2. Summary � An extended period of cold exposure enables the process of vernalization in winter cereals and is important for the synchronised timing of the floral transition. The cereal-specific floral repressor VERNALIZATION2 (VRN2) has an integral role in vernalization, yet this locus remains poorly characterised in facultative spring hexaploid wheat, Triticum aestivum. � Through the generation of defined germplasm combined with bespoke experimental proto- cols, which enable a realistic simulation of annual field-based UK growth conditions, we were able to distinguish gene expression and phenotypic differences at the subgenomic level of VRN2 in hexaploid bread wheat. � Our research in a facultative wheat suggests that the tandemly duplicated genes comprising the VRN2 locus, ZCCT1 and ZCCT2, have gene expression patterns that respond to multiple environmental factors. These genes also show coregulation, forming a regulatory loop between ZCCT-D1 and ZCCT-D2. The function of these genes beyond the classic vernaliza- tion response is explored in a facultative wheat. Here, we identified that VRN-D2 regulates early tiller development, with an accelerated rate of secondary tiller emergence and presence of coleoptile tillers. � The findings identify that the VRN2 loci in bread wheat are formed of multiple genes, which have not only overlapping but also unique regulation and function. Selecting these genes indi- vidually may offer a route to alter wheat plant architecture without directly impacting vernali- zation requirement. Introduction Bread wheat (Triticum aestivum) is the most widely cultivated cereal crop, made possible by allelic variations in several key genes, which have been enriched for during selection and breed- ing. However, bread wheat remains extremely vulnerable to the effects of climate change. Each global temperature increase of 1°C is expected to result in global losses in yield of 6%, with anticipated losses ranging from c. 4–20% for different regions (Asseng et al., 2015; Liu et al., 2016). The ability to improve and modify the adaptability of wheat to changing climate conditions is vital to maintain yield potential and support a rapidly increas- ing global population and food demand (Godfray et al., 2010). One aspect of adaptability that impacts yield potential is the vernalization requirement. This is the requirement for a pro- longed exposure to cold temperatures, coupled with short-day photoperiods, which provides plants with a means for monitoring winter and timing reproductive development to favourable con- ditions. Vernalization is quantitative, so the length of exposure to cold temperatures directly determines the rate of floral transition. Flowering time is accelerated following a longer cold duration up to a point, after which the vernalization requirement is satisfied and cold temperatures have limited or negative impact on flower- ing date (Dixon et al., 2019). In countries with a suitable climate pattern to meet the vernalization requirement, vernalization- requiring (termed ‘winter’ wheat) has a higher yield potential than other wheat (Woods et al., 2016; Jacott & Boden, 2020). Furthermore, it provides a number of other agronomic benefits including establishment and ground cover over winter and com- petition against weeds such as blackgrass (Liang & Richards, 1994; Andrew et al., 2015; Sainju et al., 2022). Wheat varieties that do not require vernalization are termed ‘spring’ varieties, avoiding the pathway through suppression of floral repressors or constitutive expression of floral activators (Trevaskis et al., 2007; Kippes et al., 2016; Dixon et al., 2019). A further group exists, which are facultative wheats; these do not require vernalization to flower, but flowering time is accelerated when they experience vernalization. Changing environmental � 2026 The Author(s). New Phytologist � 2026 New Phytologist Foundation. New Phytologist (2026) 1 www.newphytologist.com This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. Research https://orcid.org/0000-0002-6421-6260 https://orcid.org/0000-0002-6421-6260 https://orcid.org/0009-0002-4469-1459 https://orcid.org/0009-0002-4469-1459 https://orcid.org/0009-0002-4049-3623 https://orcid.org/0009-0002-4049-3623 https://orcid.org/0009-0008-1061-9022 https://orcid.org/0009-0008-1061-9022 https://orcid.org/0009-0004-1520-3721 https://orcid.org/0009-0004-1520-3721 https://orcid.org/0000-0002-8234-3407 https://orcid.org/0000-0002-8234-3407 mailto:hirsz@ipk-gatersleben.de mailto:dixon@ipk-gatersleben.de http://creativecommons.org/licenses/by/4.0/ patterns are altering previously established agricultural practices, with the optimal timing of crop planting becoming less predict- able. For winter crops, the variable winter temperatures are making the timing for the completion of vernalization unpredict- able. This presents two major challenges: too early completion causing a shift in meristem development and a reduction in plant New Phytologist (2026) www.newphytologist.com � 2026 The Author(s). New Phytologist� 2026 New Phytologist Foundation. Research New Phytologist2 biomass, or too late completion delaying flowering and, in extreme cases, not being satisfied before warmer spring condi- tions, and so a substantial reduction in final yield. Increasingly, facultative wheats are being used, which can balance these requirements. Understanding the contributions of the genetic components of the vernalization pathway is important to enable the development of wheat with a variety of vernalization responses. The genetic core of the vernalization pathway in cereals involves VERNALI- SATION1 (VRN1), VRN2 and FLOWERING LOCUS T1 (FT1), also called VRN3. Mutations in the promoter or first intron of VRN1 lead to overexpression, which results in a domi- nant spring wheat phenotype (Dixon et al., 2019). VRN2 is a monocot-specific floral repressor, highly expressed before vernali- zation and gradually repressed following cold exposure by the increasingly expressed VRN1 (Distelfeld et al., 2009). Although VRN2 is generally referred to as a gene, it is a locus made of two tandemly duplicated genes: ZCCT1 and ZCCT2 (Yan et al., 2004). It has a putative C2H2 zinc finger domain and a CCT domain, named after the genes CONSTANS, CONSTANS-like and TIMING OF CAB 1 identified in Arabidopsis thaliana. Nota- bly, VRN2 is part of a known translocation between chromosome 4A and 5A (Ma et al., 2013). Therefore, the VRN2 gene in bread wheat refers to a total of six genes: ZCCT-A1 and ZCCT-A2 on chromosome 5A, ZCCT-B1 and ZCCT-B2 on 4B and ZCCT- D1 and ZCCT-D2 on 4D, all on the distal long arm of the chro- mosomes (Fig. 1a). The distance between these genes also differs between chromosome pairs, raising the possibility that ZCCT genes are under independent regulation and possibly even selec- tion. It also highlights that the individual genes of the VRN2 loci could be nonredundant with respect to the vernalization response. The role of each ZCCT gene varies across different cereal spe- cies, with the diploid Triticum monococcum showing high expres- sion of ZCCT1 and low levels of expression of ZCCT2. In this wheat species, zcct1 mutants exhibit a spring phenotype despite the presence of a functional ZCCT2 gene, leading to the conclu- sion that ZCCT1 is the primary functional copy (Yan et al., 2004). By contrast, the tetraploid wheat species Triticum turgidum shows higher expression of ZCCT2 than ZCCT1 (Dis- telfeld et al., 2009). This species was used to generate a synthetic hexaploid wheat with a triple knockout of VRN2 (Kippes et al., 2016), in which the D-genome was contributed by Aegilops tauschii. In this synthetic hexaploid, ZCCT-B2 was identified as the key copy giving VRN2 its function as a floral repressor (Kippes et al., 2016). This triple-null has a spring wheat phenotype, with significantly varying flowering times dependent on combinations of functional VRN2 genome copies (Kippes et al., 2016). A recent study in barley has also shown that the ZCCT genes can show differential expression in response to ver- nalization (Montardit-Tarda et al., 2025). Understanding the role of each VRN2 gene in natural hexaploid wheat will be of great interest, as this suggests the potential to modulate the verna- lization requirement of wheat through allelic and copy number variation of specific VRN2 genes. Alongside monitoring ambient temperature to direct floral tran- sition, photoperiod monitoring is integral to ensure floral transition is correctly timed. In both barley and rice, VRN2 expression is regu- lated by photoperiod in conjunction with temperature (Trevaskis et al., 2006; Turner et al., 2013). Photoperiod monitoring genes such as PHOTOPERIOD-1 (Ppd-1) are part of a complex network of gene expression, which regulates the central floral integrator, FT1, to ensure floral transition occurs when conditions are ideal (Shaw et al., 2020). Many of the genes with a role in monitoring photoperiod, including Ppd-1 and the CONSTANS (CO) genes, also contain the highly conserved CCT domain (Li et al., 2011; Li & Xu, 2017; Zheng et al., 2017). In VRN2, mutations in the argi- nine amino acids at 16, 35 and 39 in the CCT domain significantly alter protein properties and can render the protein nonfunctional (Distelfeld et al., 2009). We asked how vernalization genes respond in facultative spring wheat under different environmental conditions and whether plant development could be altered to improve winter traits with- out impacting the vernalization response. We identified that the expression of the VRN2 loci is regulated by both temperature and photoperiod in bread wheat, and this expression alters under vari- able conditions. Therefore, specific genes within the VRN2 loci can be targeted for improving aspects of crop performance. In particular, we identify that altered function of ZCCT-D1 and -D2 regulates early tiller outgrowth without impacting final tiller number and flowering time. This was also recently identified in barley and has the potential to improve water, light and nutrient efficiency as well as improve overall soil health and structure (Liang & Richards, 1994; ter Steege et al., 2005; Sainju et al., 2022; Montardit-Tarda et al., 2025). Materials and Methods Plant materials and growth conditions All plants were germinated at room temperature (21°C) on Whatman filter paper with 5 ml ddH2O in a 9-cm Petri dish and Fig. 1 VERNALIZATION2 (VRN2) location and sequence analysis. (a) A schematic showing the direction and locations of each ZCCT1 and ZCCT2 gene on each chromosome, and subgenome A, B and D in Triticum aestivum as measured from the midpoint of each gene. (b) The aligned protein sequences for each homoeologous copy of ZCCT1 and ZCCT2, highlighting the putative C2H2 zinc finger (pink) and CONSTANS, CONSTANS-like and TOC1 (CCT) domain (blue). (c) A consensus sequence indicates the level of conservation across the five ZCCT genes annotated in the v1.2 reference cultivar ‘Chinese Spring’ for the promoter region, 2-kb upstream of the start site. Motifs were identified using ‘PlantPAN 4.0’ (Chow et al., 2024). Nucleotides are indicated based on colour shown in the key and motifs of interest are indicated, with colours indicating whether the motif is conserved across all three homoeologous copies of ZCCT1 (maroon), ZCCT2 (teal) or across all ZCCT1 and ZCCT2 promoter regions (navy). Motifs that are specific to only one ZCCT promoter region are also indicated as indicated in the key. CArG-W8, CArG box motif; CBF, C-REPEAT BINDING FACTOR; LTRE, low temperature responsive element; NF-Y, NUCLEAR FACTOR Y; A, adenine; T, thymine; C, cytosine; G, guanine. D as for C, but for the intron. � 2026 The Author(s). New Phytologist� 2026 New Phytologist Foundation. New Phytologist (2026) www.newphytologist.com New Phytologist Research 3 seedlings transferred to 24-well seed trays (each cell 50 mm 9 48 mm 9 52 mm) containing John Innes Cereal Mix (Backhaus et al., 2022). The Triticum aestivum L. cultivars used were either from laboratory stocks (cv. Cadenza) or obtained from the Germplasm Resource Unit for all TILLING mutants (Rakszegi et al., 2010; Krasileva et al., 2017). Plants used for gen- eration of zcct-d2_m1 and zcct-d1_m1 mutant lines were then grown under glasshouse conditions (16 h : 8 h, 21°C : 15°C, light : dark). All germplasms used and developed in this study are listed in Supporting Information Table S1. The facultative spring wheat cultivar Cadenza was used for the 24-h time-course expression analysis. These plants were grown in Sanyo Plant Growth cabinets under the following temperature conditions with a constant 12-hour photoperiod: 16°C during the light period/10°C during dark, 10°C during light/16°C during dark, and 10°C constant and 16°C constant. Plants were grown in these conditions for 3 wk, and then leaf tip tissue was sampled at 5-h intervals across a 24-h period, with tissue from two individual plants pooled for each of three biological replicates. Growth conditions for the long-term seasonal gene expression experiment were conducted in a Conviron gen 2000 growth chamber with conditions listed in Table S2. The facultative spring wheat cultivar Cadenza and mutant lines zcct-d2_m1 and zcct-d1_m1 were grown in these conditions. Leaf tissue samples were taken each week an hour after the relative ‘dawn’ and ‘dusk’ for each day depending on when light changes occurred. Tiller emergence was recorded every day for the first 35 d of growth. Flowering time was defined as half-ear emergence from the flag leaf, GS55 on the Waddingtons scale. Spikelet number was counted for the spike from the primary tiller for each plant. Generation of zcct-d2_m1 and zcct-d1_m1mutant lines The two mutant lines were in the hexaploid cv. Cadenza back- ground: Cadenza0810 and Cadenza1436 (with single nucleotide polymorphisms (SNPs) in ZCCT-D2 and ZCCT-D1 respec- tively) (Krasileva et al., 2017) were twice backcrossed. Plants were genotyped using primers detailed in Table S3 at the SNP of inter- est via KASP assay as described in Dixon et al. (2018) to identify homozygous plants. Genomic DNA was extracted using the chloroform:isoamyl alcohol method adapted from Paterson et al. (1993). PCR amplification was conducted to confirm homozygosity at the SNP of interest using Q5 DNA Polymerase (NEB) with reagent quantities and conditions following the manufacturer’s pro- tocol. Primers are listed in Table S3. The PCR was separated using gel electrophoresis and the fragment was extracted using the Mon- arch® DNA Gel Extraction Kit (NEB, Ipswich, MA, USA) accord- ing to the manufacturer’s protocol. Samples were then sequenced by Eurofins Genomics and analysed to confirm the SNP. Analysis of gene expression To measure the expression, plants were sampled as outlined see ‘Plant materials and growth conditions’ in the Materials and Methods section. All plant samples were flash-frozen in liquid nitrogen and stored at �70°C. The tissue was lysed using the TissueLyserLT (Qiagen, Hilden, Germany) using 3-mm steel ball bearings, and RNA was then extracted for all leaf tissue samples using the SpectrumTM Plant Total RNA Kit (Sigma-Aldrich, St. Louis, MI, USA) or Monarch® Total RNA Miniprep kit (NEB, Ipswich, MA, USA) according to the relevant manufacturer’s protocol. Synthesis of cDNA was conducted according to either Dixon et al. (2018) or using the reverse transcriptase UltraScript 2.0 (PCR Biosystems, London, UK) according to the manufac- turer’s protocol. The cDNA was then diluted (1 : 10) and quan- titative real-time reverse transcriptase PCR (RT-qPCR) was conducted using GoTaq® qPCR Master Mix (Promega, Madi- son, WI, USA) according to the manufacturer. The primers used are listed in Table S3, and the thermal cycling conditions per- formed using the CFX96TM Thermal Cycler (Bio-Rad, Watford, Hertfordshire, UK) and protocol described in Dixon et al. (2018). Expression was calculated relative to the housekeep- ing gene TraesCS5A02G015600 (Borrill et al., 2016), using the formula 2DCT where DCT = [expression of Gene of Interest] – [expression of TraesCS5A02G015600]. Promoter and intron motif analysis Gene and 2-kb putative promoter sequences were extracted from Ensembl plants following alignments using BLAST for the ‘10+ Genomes’ hexaploid wheat pangenome cultivars and wheat relatives (Walkowiak et al., 2020). Additional hexaploid wheat sequences were extracted from the pangenome by Jiao et al., 2025. Sequences were aligned against Chinese Spring v.2.1 using MAFFT to identify each individual ZCCT gene. Identification of potential binding motifs was then conducted using the PLACE (Plant Cis-acting Reg- ulatory DNA Elements) database (Higo et al., 1999). Results ZCCT1 and ZCCT2, which form the VRN2 loci, contain different regulatory domains The number of genes forming the VRN2 loci in hexaploid bread wheat was believed to be six, ZCCT-A1 and -A2, -B and -D. However, a blast analysis of the reference genome cv. Chinese Spring (v.2.1) identified only five genes, missing ZCCT-B2. Spe- cific gene search in the region of ZCCT-B2 identified a truncated version of ZCCT-B2 in the reference, containing a 62 amino acid deletion (Fig. 1b). This highlights that the different ZCCT1 and �2 genes could be selected independently despite being phy- sically close on the chromosome (Fig. 1a). Comparison of the coding region of these sequences (Fig. 1b) shows that the putative zinc finger domain has a high level of variation across the differ- ent ZCCT genes compared with the CCT domain, which is known to be well-conserved (Distelfeld et al., 2009; Li et al., 2011). There is also strong conservation from amino acid residues 73–93, which is not part of either domain but is included in the deletion in cv. Chinese Spring in ZCCT-B2. Through analysis of publicly available wheat pangenomes (Walkowiak et al., 2020; Jiao et al., 2025), we identified two New Phytologist (2026) www.newphytologist.com � 2026 The Author(s). New Phytologist� 2026 New Phytologist Foundation. Research New Phytologist4 main allelic variants for ZCCT-A1, -B1 and -A2 (Tables S4 and S5). The ZCCT-D1 and -D2 genes were shown to be fully con- served across all but one of the 39 varieties analysed, and ZCCT- B2 was able to be identified in all genotypes, although it was not annotated for most varieties. Comparison with ancestral wheat sequences shows little variation across the ZCCT genes (Table S6). To begin to investigate the possibility of independent selection of these genes, we classified the regulatory domains within the intron and 2-kb upstream of the putative start site for each of the ZCCT genes (Fig. 1c,d). Here, we could identify not only that the two genes ZCCT-1 and -2 contained unique regulatory domains but also that even within the ZCCT-1 or -2 genes, there is variation between the promoter regions. For example, in the 2-kb upstream of the putative start site, a 0.25-kb region was deleted in ZCCT-B1 and -D1 compared with ZCCT-A1. To identify potential binding motifs, a database of known motifs across multiple vascular plant species was used (Higo et al., 1999). There was variation between genes for key motifs such as the CArG box, a binding site for MADS box transcrip- tion factors such as VRN1 (Deng et al., 2015; Chow et al., 2024), which was present in all three homoeologous copies of ZCCT1 in different locations but not ZCCT2 (Fig. 1c). Com- parisons of the promoter regions for the main allelic variants of each ZCCT gene show some variation (c. 20–50 SNPs), which altered few key motifs (Dataset S1). ZCCT-B1 had the largest difference between alleles, showing low conservation c. 1.6-kb upstream of the putative start site (Dataset S1). The ancestral wheats analysed also showed generally little variation between their promoters and the hexaploid wheat promoter, often similar (c. 20–50 SNPs) to the variation between allelic variants for ZCCT-A1, -B1 and -A2 genes in hexaploid wheat. The analysis of the single intron was conducted in cv. Cadenza to include ZCCT-B2 and showed conservation was better in the latter half of the intron (c. 0.8–1.5 kb) with large deletions present in ZCCT-A2 and ZCCT-B2 and insertions present in ZCCT-D1 and ZCCT-A2, as well as several deletions in all three homoeolo- gous copies of ZCCT2 compared with ZCCT1 (Fig. 1d). This highlighted that the two ZCCT genes have diverged in regulatory domains and strongly suggested ZCCT1 and ZCCT2 are not functionally redundant and may be differentially regulated in response to environmental conditions. Core vernalization genes’ expression alters depending on temperature and photoperiod conditions in facultative wheat The domain analysis suggested that the VRN2 genes could be under different regulation and so we hypothesised that some of the VRN2 genes may offer a route to coordinate environmental responses and development without always impacting the vernali- zation response. It has already been established that expression of the VRN2 genes is responsive to changes in photoperiod (Dub- covsky et al., 2006), so to investigate the effect of temperature, we first characterised how vernalization genes behave in the facul- tative spring wheat cv. Cadenza via RT-qPCR analysis on 3-wk- old leaf tissue across a series of simulated environmental scenar- ios, all with the same day-neutral (DN; 12 h : 12 h, light : dark) photoperiods. Alleles of the key vernalization genes for cv. Cadenza are shown in Table S7. We measured VRN1 as this is the central regulator of vernali- zation, and its expression is known to increase under lower ambi- ent temperatures (Yan et al., 2003; Dixon et al., 2019). We observed a stable level of VRN1 expression across the constant Fig. 2 Expression of VERNALIZATION1 (VRN1) under day-neutral (DN) conditions with variable temperatures. A ribbon plot showing expression of VRN1 for all three subgenome copies (A, B, D) in the facultative spring Triticum aestivum cv. Cadenza across a 24-h time course. Leaf tissue was sampled after 3 wk of growth in 12-h DN conditions under different temperatures. Expression was normalised against TraesCS5A02G015600 and the average of n = 3 biological replicates is shown for each time point. Variation is shown as � SE mean (SEM) and is indicated by the shaded region around each point. The light period is indicated by yellow and dark period by grey background shading. ZT0 is the same sample as ZT24 except for D. (a) Expression of VRN1 under constant 16°C conditions. (b) As for a but for constant 10°C conditions. (c) As for a but for 16°C light/10°C dark conditions. (d) as for a but for 10°C light/16°C dark conditions. � 2026 The Author(s). New Phytologist� 2026 New Phytologist Foundation. New Phytologist (2026) www.newphytologist.com New Phytologist Research 5 10°C time course (Fig. 2a). We then compared this with VRN1 expression at constant 16°C as this temperature is associated with early season conditions in Northern Europe. Expression showed diurnal control with a peak before dusk (Fig. 2b) and generally higher expression than under constant 10°C. The diurnal expres- sion is reminiscent of that observed in T. monococcum in the field, with a peak during the day independent of VRN2 expression (Nishiura et al., 2018). The expression patterns of constant 10°C and 16°C suggested that the constant 10°C conditions were actually impacting the day expression pattern of VRN1; to investigate this, we conducted the 24-h time course under 16°C by day and 10°C at night DN conditions. Here, we again observed a low, constant level of VRN1 expression with a slight hint of a predusk increase in expression (Fig. 2c). This indicated that in the facultative spring wheat, it was actually the night temperature that was driving a diurnal expression of VRN1. To challenge this, we conducted a final 24-h time course during which we altered the day and night temperatures to generate an unrealistic 10°C by day and 16°C at night DN time course. Here, the diurnal rhythmicity returned with a peak during the day (Fig. 2d). This highlights that in the facultative spring wheat, cv. Cadenza, the expression of the main vernalization gene is regulated by temperature, the effect of which is altered depending on the interaction with the light and dark cycle, most likely via the circadian clock. Furthermore, it high- lights a role for warming night temperatures in regulating its expression during the subsequent day. As a main regulation target of VRN1 is the repression of VRN2 (Chen & Dubcovsky, 2012), we anticipated that the temperature-dependent expression patterns observed for VRN1 would also be reflected through the regulation of VRN2 under DN photoperiods. Measuring the two VRN2 genes, ZCCT-1 and -2, as homoeologous groups across the previously described 24-h time courses we observed quite distinct patterns of regulation (Figs 3, S1). Under constant 10°C DN, both ZCCT-1 and -2 expressions were highly reminiscent of the low, constant expression observed for VRN1 (Fig. 3a). However, unlike VRN1 under constant 16°C DN, both ZCCT-1 and -2 still had a low level of expression (Fig. 3b). Only when considered on a higher resolution scale, due to the low actual expression levels, could ZCCT-2 be identified to show a similar diurnal pattern to that observed for VRN1 (c.f. Figs 2b, 3b) whilst ZCCT-1 lacked this rhythmicity. This sup- ported previous reports that the ZCCT genes are only expressed under longer photoperiods (Dubcovsky et al., 2006; Trevaskis et al., 2006). However, when we measured ZCCT-1 and -2 expres- sion under changing light/dark temperature conditions, both were expressed and showed a diurnal pattern in expression, which was synchronous between the genes (Fig. 3c). Notably, depending on the temperature pattern, the peak in expression shifted such that under warm days, the peak was across and just after dusk whilst with warm nights, it was during the light period. Under these con- ditions, the expression of VRN2 is asynchronous with VRN1. Furthermore, through comparing the four 24-h time courses, it identified that temperature is an important regulator in the expres- sion of these genes and that the interaction between the genes may be more complex as the patterns of their expression are altering with variable conditions. We therefore aimed to understand how the ver- nalization genes expression was responding across a growing season. Fig. 3 Expression of ZCCT1 and ZCCT2 under day-neutral (DN) conditions with variable temperatures. A ribbon plot showing expression of ZCCT1 for all three subgenome copies (A, B, D) and ZCCT2 (A, B, D) in pink and teal respectively in the facultative spring Triticum aestivum cv. Cadenza across a 24-h time course. Leaf tissue was sampled after 3 wk of growth in 12-h DN conditions under different temperatures. Expression was normalised against TraesCS5A02G015600 and the average of n = 3 biological replicates is shown for each time point. Variation is shown as � SE mean (SEM) and is indicated by the shaded region around each point. The light period is indicated by yellow and dark period by grey background shading. ZT0 is the same sample as ZT24 except for D. (a) Expression of ZCCT1 and ZCCT2 under constant 16°C conditions. (b) As for a but for constant 10°C conditions. (c) As for a but for 16°C light/ 10°C dark conditions. (d) As for a but for 10°C light/16°C dark conditions. New Phytologist (2026) www.newphytologist.com � 2026 The Author(s). New Phytologist� 2026 New Phytologist Foundation. Research New Phytologist6 ZCCT1 expression increases with photoperiod post-vernalization The role of varying day and night temperatures in the regulation of the vernalization genes’ expression suggested that both envir- onmental conditions play an important role in the regulation of these genes. It also indicated that even in a facultative spring wheat the role of cold was important for the synchronisation of gene expression. We therefore wanted to test this across a more realistic time course. To do this, we generated an experimental profile in which we took the approximate annual environmental conditions for London, UK. We calculated the average day- length, temperature during the light-period and temperature dur- ing the dark-period for each calendar month to generate a time course over 12 weeks with each week containing the average environmental condition for one calendar month. We added in one more week of the mid-winter condition to ensure that the plants experienced a true vernalization period in case our experi- mental time course was not sufficient (Fig. 4a). Cv. Cadenza seeds were stratified and then grown in soil from Day 0 of Week 1. Under these conditions, cv. Cadenza flowered following 125 d �1.73 SD, which confirmed that the conditions were sufficient to support floral development. To assess the gene expression, leaf tissue from the newly emer- ging leaf was sampled each week one hour after dawn and dusk. Confirming the observations from the 24-h time course (Figs 2 and 3), gene expression of VRN1 and -2 differed at these two time points. VRN1 showed a steady increase across the time course, as Fig. 4 Gene expression across the long-term seasonal time course at dawn and dusk for the facultative spring wheat cv. Cadenza. (a) A graph outlining the temperature conditions for each week during the day (yellow) and night (grey), as well as the daylength indicated by number of hours of light (red). (b) Bar graphs showing gene expression across the long-term seasonal time course for facultative spring wheat Triticum aestivum cv. Cadenza. Leaf tissue was sampled 1 h after the relative ‘dawn’ and ‘dusk’ time points and n = 3 for each time point. Expression of ZCCT1was normalised against TraesCS5A02G015600 and � SE mean (SEM) is indicated by the error bar. *, P < 0.05; **, P < 0.01; ***, P < 0.001 determined by paired Student’s t-test only between dawn and dusk timepoints. (c) As for b, but for expression of ZCCT2. (d) As for b, but for expression of VRN1. (e) As for b, but for expression of ZCCT-D1. (f) As for b, but for expression of ZCCT-A2. (g) As for b, but for expression of ZCCT-B2. (h) As for b, but for expression of ZCCT-D2. � 2026 The Author(s). New Phytologist� 2026 New Phytologist Foundation. New Phytologist (2026) www.newphytologist.com New Phytologist Research 7 previously reported (Yan et al., 2003; Xie et al., 2019), consistent with its role in floral activation (Fig. 4b). However, of possible equal biological interest is the substantial over threefold increase between Weeks 11 to 12 observed at the dawn time point. This coincides with an increasing day temperature from 8°C to 10°C, night temperature from 4°C to 5°C and photoperiod 12 to 14, as well as representing the end of the vernalization period. The ZCCT genes also showed differences in expression between the dawn and dusk time points (Fig. 4c–h). For both ZCCT-1 and -2, the dusk time point showed a steady decrease in expression as photoperiod and temperature decreased from Weeks 2 to 5. Expression then remained largely repressed across the time course (Fig. 4c–h); this is consistent with its repression as a result of vernalization (Yan et al., 2004). However, like VRN1, the same pattern was not observed at the dawn time point. Here, the genes not only showed a different regulation to dawn but also between the two ZCCT genes. For ZCCT-1, expression also decreased at dawn with the decreasing tempera- ture and photoperiod, but it was not robustly repressed when temperature and photoperiod started to increase again, at Week 10 onwards (Fig. 4c). This is in contrast to ZCCT-2, which was also sequentially repressed across the time course at the dusk time point as temperature and photoperiod decreased, but was hardly expressed at the dawn time point (Fig. 4d). As ZCCT-1 and -2 showed different expression responses across the seasonal time course, we next asked whether these dif- ferences extended to the level of homeologous genes. To assess this, we designed gene specific RT-qPCR primers. Due to the high level of homology between the genes, we were only able to design primers that were specific for each of the ZCCT-2 genes and ZCCT-D1 (Table S3). Between the ZCCT-2 genes, there were distinct expression patterns, with ZCCT-A2 and -D2 (Fig. 4f,h) showing an increase in expression following the winter-simulated period whilst ZCCT-B2 did not (Fig. 4g). This trend was not observed using the generic ZCCT-2 primers, possi- bly because the expression level of ZCCT-A2 and -D2 was very low (10-fold lower). ZCCT-D1 expression followed that of the generic primers, although its expression was lower than the gen- eric primers at the start of the time course (Fig. 4e). For all of the genes, there were differences in expression between the dawn and dusk time points. Early tiller development is accelerated in VRN-D2 TILLING lines The expression of the ZCCT genes and subgenome copies sug- gested that in facultative spring wheat, they can function beyond the vernalization response. Therefore, we aimed to identify whether any of the VRN2 genes controlled important develop- mental traits without significantly altering the facultative spring vernalization response. To do this, we utilised the Cadenza TIL- LING collection (Krasileva et al., 2017) and were able to identify TILLING mutants, which contained a SNP within the coding sequence of two of the ZCCT genes. The line Cadenza0810 con- tained a predicted stop Q144* just before the CCT domain in ZCCT-D2, hereafter zcct-d2_m1, whilst Cadenza1436 contained a T130I, also preceding the CCT domain in ZCCT-1D, hereafter zcct-d1_m1 (Fig. 5a). To reduce the possible impact of background mutations, each line was backcrossed twice and homozygous mutations were selected to generate homozygous BC2F2 lines. These lines were grown under 16°C long day (LD) conditions to replicate condi- tions at the start of autumn, and the plants were carefully pheno- typed (Fig. 5b). Noticeably, a coleoptile tiller developed in the two TILLING mutants but not cv. Cadenza (Fig. 5c). Then, across the first 30 d of growth, both zcct-d2_m1 and zcct-d1_m1 developed an additional two to three tillers than cv. Cadenza (Fig. 5d). This increase in early tiller development was not main- tained, and final fertile tiller number was similar between all lines (Fig. 5e). The increase in early tiller development was, however, reflected by a slight but significant (P = 0.01) delay in flowering time of 2.5 d �2.74 SD for zcct-d2_m1 and 2.9 d �1.03 SD for zcct-d1_m1 as measured by half spike emergence (Waddingtons GS55; Fig. 5f). This slight delay did not affect the spikelet num- ber (Fig. S2). ZCCT genes co-regulate their expression As we observed an early-stage developmental phenotype, we next asked whether this altered the expression of the vernalization genes. To test this, we employed our condensed season time course, as described for Fig. 4a. We sampled emerging leaf tissue at dusk and measured the expression of vernalization genes in cv. Cadenza, zcct-d2_m1 and zcct-d1_m1 (Fig. 6). Here, we observed that in plants carrying mutant alleles of ZCCT-D1 or -D2, VRN1 expression was higher, and this was particularly apparent at the start of the time course (Weeks 1 and 2) (Fig. 6a). This increase in VRN1 was also reflected by a generally lower expression level of ZCCT-1 and -2 as measured using the generic primers for these genes (Fig. 6b,c). This supports the molecular inter-regulation of these genes, as previously described (Yan et al., 2004; Chen & Dubcovsky, 2012). Interestingly, this gen- eral trend was not maintained when we measured the expression of individual ZCCT-1 and -2 genes. Here, we observed that the function of the ZCCT genes impacted the expression of other ZCCT genes. The expression of ZCCT-A2 was increased in both lines, which carried either less functional ZCCT-D1 or -D2, sug- gesting that usually these genes repress the expression of ZCCT- A2. Similarly, at the start of the time course, ZCCT-D2 expres- sion was much lower in the zcct-d1_m1, suggesting that -D2 is activated by -D1. The reverse was observed in the zcct-d2_m1, which had higher expression of -D1, highlighting a possible mutual regulation mechanism (Fig. 6d,g). Significant changes in gene expression were most apparent at the start of the time course, supporting a role for these genes in early developmental regulation. Additionally, we measured flowering times and observed a flowering delay in the zcct-d2_m1 line, but not for zcct-d1_m1 (Fig. 6h). This indicates that whilst these genes may coregulate the expression of other ZCCT genes, this is likely not redundant as the resultant phenotype is variable. New Phytologist (2026) www.newphytologist.com � 2026 The Author(s). New Phytologist� 2026 New Phytologist Foundation. Research New Phytologist8 Discussion Vernalization genes are responding to multiple environmental factors Vernalization is the requirement for cold to enable the vegetative to floral transition and this definition reflects the absolute need for lower ambient temperatures during this process (Dixon et al., 2019). Additionally, a seasonal response photoperiod plays an important role, in particular regarding the repression of VRN2 expression by short-days (Dubcovsky et al., 2006; Trevaskis et al., 2006). With the increasingly variable and unpredictable conditions of winter, we aimed to further understand how temperature and photoperiod interacted to regulate the expres- sion of the major vernalization genes in hexaploid bread wheat. We were particularly interested in understanding this function in facultative spring wheat as this growth habit offers a mechanism of increased flexibility to regulate plant development under vari- able winters, as well as earlier spring sowing which is of interest in more mediterranean climates. We measured VRN1 and VRN2 expressions under different temperature patterns with DN photo- periods to further our understanding of how temperature influ- ences the expression of these genes. Interestingly, we observed that both VRN1 and VRN2 had altered expression patterns depending on the temperature (Figs 2 and 3). Of particular inter- est is the altered waveform in expression, which occurred in Fig. 5 Tiller growth in VRN2mutants. (a) An infographic outlining the locations of the mutations in the Triticum aestivum TILLING lines used in this study, zcct-d2_m1with a premature termination codon at Q144*, and zcct-d1_m1 with a T130I missense mutation. All germplasm used was at the BC2F2 stage, homozygous for the mutations shown. The relative positions of the C2H2 zinc finger (pink) and CCT (blue) domains are also as illustrated. (b) A labelled image of example wheat plants for the cv. Cadenza control, zcct-d2_m1 and zcct-d1_m1with the primary tiller (PT) and coleoptile tillers indicated, along with the emerging leaves (L1, L2, L3) and secondary tillers (ST1, ST2) numbered in ascending order. (c) Box plots showing the median average time (as indicated by the horizontal line) in days until emergence of a new coleoptile tiller in the cv. Cadenza control (n = 19) compared with the two independent mutant lines; zcct-d2_m1 (n = 2 1) and zcct- d1_m1 (n = 24). The box indicates the interquartile range and whiskers indicate the maximum and minimum values excluding outliers. (d) As for c, but for emergence of secondary tillers, with colours indicating the secondary tiller number (1 = pink, 2 = orange, 3 = yellow, 4 = green, 5 = purple, 6 = blue). (e) Violin plots showing the final tiller number, where ns = not significant and a difference in letter (a or b) between the WT and mutant lines indicates P < 0.05, as determined by a Kruskal–Wallis test followed by a pairwise Wilcoxon rank sum test with Bonferroni correction. The full range is indicated by the furthest vertical points of the violin, whiskers indicate the 25–75th percentile, box indicates the interquartile range and horizontal line indicates the median. The width of the violin indicates frequency. (f) As for e, but for flowering time defined as half-ear emergence (GS55). � 2026 The Author(s). New Phytologist� 2026 New Phytologist Foundation. New Phytologist (2026) www.newphytologist.com New Phytologist Research 9 response to different temperature conditions. This is highly remi- niscent of what is observed in barley (Hordeum vulgare) and T. monococcum (Ford et al., 2016; Nishiura et al., 2018). It was also notable that the circadian regulation is more robust for VRN1 under warmer temperatures (Fig. 2b). Furthermore, VRN2 indi- cated a possible response to temperature entrainment, such that its expression altered significantly when temperature cycles were introduced and that the phase of the expression altered depend- ing on the cycle temperatures. This is an interesting response, which could hint at possible reasons for altered vernalization responses observed depending on quite small temperature changes, or when major day/night temperature differentials are observed. For example, winters that have greater day/night differences in temperature may take longer for plants to vernalise due to the activation of VRN2 expression at night. This would be compounded by our observation that VRN1 expression is greater during the day. Therefore, under certain environmental condi- tions, VRN1 and VRN2 expressions are asynchronous, suggesting other genetic factors are involved in the regulation of these genes. Vernalization genes show different responses across development To better understand how these genes respond under natural conditions, we conducted a seasonal time course. The possibility of multiple factors regulating the vernalization gene expression Fig. 6 Gene expression across the long-term seasonal time course for the facultative spring wheat cv. Cadenza and mutant lines zcct-d2_m1 and zcct-d1_m1. Bar graphs showing gene expression across the long-term seasonal time course up to Week 8 for facultative spring wheat Triticum aestivum cv. Cadenza (green) and mutant lines zcct-d2_m1 (pink) and zcct-d1_m1 (blue). Leaf tissue was sampled 1 h after the relative ‘dusk’ time point and n = 3 for each time point. Expression was normalised against TraesCS5A02G015600 and �SE mean (SEM) is indicated by the error bar. Significance was determined by a one-way ANOVA followed by a Tukey’s Honestly Significant Difference post hoc test only between wild-type and mutant lines. A difference in letter (a or b) between the WT and mutant lines for each time point indicates P < 0.05, whilst the absence of a letter indicates no significant difference. (a) Expression of ZCCT1. (b) Expression of ZCCT2. (c) Expression of VRN1. (d) Expression of ZCCT-D1. (e) Expression of ZCCT-A2. (f) Expression of ZCCT- B2. (g) Expression of ZCCT-D2. (h) The flowering time for each genotype (cv. Cadenza in green, zcct-d2_m1 in pink, zcct-d1_m1 in blue) following growth in the outlined conditions. Error bars indicate SD; n = 19 for cv. Cadenza, n = 6 for zcct-d2_m1 and n = 10 for zcct-d1_m1. New Phytologist (2026) www.newphytologist.com � 2026 The Author(s). New Phytologist� 2026 New Phytologist Foundation. Research New Phytologist10 patterns appears more apparent in the seasonal time course (Fig. 4). Here, VRN1 expression differs between dawn and dusk time points. At dusk, its expression follows the reported trajectory of increasing during and following vernalization. By contrast, at dawn, expression shows a gradual increase, followed by a decrease and then a sudden increase post-vernalization as spring condi- tions are reached. This strongly suggests that the gene is being repressed at dawn until post-vernalization conditions. Similarly, the VRN2 genes displayed differential expression patterns between dawn and dusk conditions. Notably, for ZCCT-D1 and -A2, expression was not robustly repressed post-vernalization, suggesting that these genes may have additional roles in regulat- ing wheat development after the floral transition has occurred. These expression patterns strongly imply that the VRN2 genes have additional roles beyond the timing of the apex transition and that future work dissecting the mechanistic basis of their function in both the vegetative and later stage development would be very interesting. Notably, VRN1, in conjunction with FUL2 and 3, has an important role in apex patterning and spike- let development (Li et al., 2019) and so additional roles for the VRN2 genes could be anticipated. Early tiller growth is regulated by ZCCT-D1 and -D2 We aimed to characterise the vernalization genes in a facultative spring wheat and understand whether, through considering the VRN2 genes separately, we could identify routes to alter winter-type traits without impacting the floral regulation. To test this hypothesis, we developed two mutant alleles in ZCCT genes and phenotyped them under 16°C LD. We observed that the ZCCT-D1 and -D2 genes were involved in the regulation of lateral branch, or tiller, outgrowth early in plant development (Fig. 5c,d). This increase in tiller outgrowth associated with the presence of HvVRN2 was also recently identified in barley (Montardit-Tarda et al., 2025). It would be interesting to understand whether this phenotype is based on a common mechanism regarding tiller out- growth or increases in tiller bud number. Additionally, it would be interesting to understand whether the observed tiller phenotype impacted subsequent leaf development (Shaaf et al., 2019). The impact of VRN2 genes early in development was further supported when we measured the expression of these genes across our con- densed growing season time course. Here, we observed differences in gene expression in the first few weeks of the time course, which returned to a similar point by Week 5 (Fig. 6). Given that we observed an early development tiller phenotype, we also pheno- typed other growth stages as plant architecture development is often linked (Dixon et al., 2020). The final plant phenotypes were extre- mely mild, with the final tiller number the same between all lines, along with spikelet number (Figs 5e, S1). The absence of major developmental phenotypes at the later growth stages suggests that changes in ZCCT gene expression did not significantly impact over- all plant growth. However, we did observe a small flowering time change, with the mutant alleles flowering c. 2 d later under constant conditions and 5 d later under variable conditions for zcct-d2_m1, which is not the expected result for a floral repressor gene (Figs 5f, 6h). This suggests that the function of the individual ZCCT genes differs and that their role in plant development may be quite com- plex when considered outside the scope of vernalization. It also remains important to further validate phenotypes observed, in parti- cular the regulation of tiller outgrowth, under multilocation field conditions. ZCCT genes show co-regulation Unexpectedly, when we measured the individual gene expression in the mutant backgrounds, we observed that some of the ZCCT genes appeared to regulate other ZCCT gene expression. For example, between ZCCT-D1 and -D2, a regulation loop could be inferred in which a less-functional -D1 caused a reduction in expression of ZCCT-D2, suggesting that normal functional -D1 promotes the expression of -D2 (Fig. 6g). Whereas the expected less functional -D2 led to higher ZCCT-D1 expression, suggest- ing its normal role is to repress the expression of -D1, however, only at the very start of the time course (Fig. 6d). Furthermore, the genetic and expression data also indicate that both ZCCT-D1 and ZCCT-D2 repress the expression of ZCCT-A2 and that this occurred throughout the vernalization period of the time course (Fig. 6e). Therefore, the gene-specific analysis has identified that the different ZCCT genes not only are expressed under the same Fig. 7 Proposed network outlining interactions between ZCCT genes. An infographic outlining a proposed genetic network for the Triticum aestivum ZCCT genes from this study in day-neutral photoperiod conditions. Arrows in grey indicate proposed genetic interactions from the ZCCT genes whilst black indicates known interactions. Arrows represent gene activation and bars represent gene repression. ZCCT-D1 and ZCCT- D2 are proposed to repress VRN1 and ZCCT-A2 expression, as well as early tiller formation through an unknown mechanism. ZCCT-D1 is proposed to promote ZCCT-D2 expression, which in turn represses ZCCT- D1 in a potential negative feedback loop. � 2026 The Author(s). New Phytologist� 2026 New Phytologist Foundation. New Phytologist (2026) www.newphytologist.com New Phytologist Research 11 regulation (Fig. 6) but also are able to regulate their own expres- sion. This is a common regulatory mechanism within adaptive response genes (Rees et al., 2022; Gauley et al., 2024) but has not been previously reported for cereal vernalization. Here, we show that the core vernalization genes in facultative spring hexaploid wheat cv. Cadenza are regulated differently depending on the temperature conditions experienced and that this regulation causes an asynchronous expression pattern between the main repressor and activator in the vernalization pathway. Further- more, when considering each of the individual ZCCT genes, we identified inter-regulation in expression as well as independent reg- ulation. Through the development of germplasm, which differed between ZCCT genes, we were able to identify variation in early development of tiller growth, which may have applications in the use of facultative spring wheat (Fig. 7). It is a beneficial phenotype due to the increased coverage during early development, which can improve water, light and nutrient efficiency as well as improve com- petition with biotic stressors (Liang & Richards, 1994; ter Steege et al., 2005; Andrew et al., 2015). To understand the potential use of these mutants in a seasonal context, they will need to be trans- ferred to current breeding material and tested for early development ground cover in field trials. In particular, it would be of great inter- est to understand whether the early-stage tiller development linked with a prostrate phenotype, which has been recorded in wheat and barley (Zhou et al., 2018; Marone et al., 2020; Kumar et al., 2025). Exploring the roles of specific ZCCT genes has shown that these genes have further adaptive potential not only in the context of vernalization but also potentially in other developmental roles. This means these genes may also be involved in regulating devel- opment in wheats, which do not have a winter habit, offering the potential to regulate plant development without impacting verna- lization per se. Acknowledgements We thank Dr Rachel Rusholme-Pilcher at Earlham Institute, UK, for the assistance with the cv. Cadenza VRN2 gene chromo- somal locations. We thank Dr Beth Soanes for her assistance in sampling the long-term seasonal time course. The EMS-muta- genised population of bread wheat cv. Cadenza was developed and characterised by Dr. Andy Phillips at Rothamsted (UK) and Dr. Cristobal Uauy at John Innes Centre (UK). This research was supported through funding to L.E.D. via a UKRI FLF MR/ S031677/1, Rank Prize Funds New Lecturer Award and start-up funds from the University of Leeds. A Molecules to Landscapes grant from BBSRC BB/X005925/1 supported H.T. Open Access funding enabled and organized by Projekt DEAL. Competing interests None declared. Author contributions DH and LD conceived, designed and conducted the experiments and analysis. HT, IL and AG designed and conducted experiments. WW supported with bioinformatic analysis. DH and LD wrote the initial manuscript and all authors have con- firmed its accuracy. ORCID Laura Dixon https://orcid.org/0000-0002-8234-3407 Adam Gauley https://orcid.org/0009-0004-1520-3721 Dominique Hirsz https://orcid.org/0000-0002-6421-6260 India Lacey https://orcid.org/0009-0002-4049-3623 Harry Taylor https://orcid.org/0009-0002-4469-1459 Wenxue Wu https://orcid.org/0009-0008-1061-9022 Data availability All data are available at https://github.com/TweeticumGEA. Raw data are also provided in File S2. From this study, new germplasm is available following request. Gene identifiers used in this study are from Ensembl v.59 for Chinese Spring v.1.2 for all except ZCCT- B2, which is from cv. Julius and are as follows: ZCCT-A1 (TraesC- S5A02G541300); ZCCT-B1 (TraesCS4B02G372700); ZCCT-D1 (TraesCS4D02G364500); ZCCT-A2 (TraesCS5A02G541200); ZCCT-B2 (TraesJUL4B03G02424310); ZCCT-D2 (TraesCS4D0 2G364400); VRN1 (TraesCS5A02G391700, TraesCS5B02G3 96600, TraesCS5D02G401500). References Andrew IKS, Storkey J, Sparkes DL. 2015. A review of the potential for competitive cereal cultivars as a tool in integrated weed management.Weed Research 55: 239–248. Asseng S, Ewert F, Martre P, R€otter RP, Lobell D, Cammarano D, Kimball B, Ottman M, Wall G, White J et al. 2015. Rising temperatures reduce global wheat production. Nature Climate Change 5: 143–147. 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New Phytologist (2026) www.newphytologist.com New Phytologist Research 13 Fig. S2 Final spikelet number for cv. Cadenza, zcct-d2_m1 and zcct-d1_m1 in 16°C LD conditions. Table S1 List of germplasm used in this study. Table S2 Weekly growth conditions for long-term gene expres- sion experiment. Table S3 List of primers. Table S4 Ensembl codes/identified regions for each copy of VRN2 in the 10+ genomes cultivars. Table S5 Promoter regions and haplotypes for each copy of VRN2 from wheat pan-genome. Table S6 Orthologous copies of each VRN2 in related grass species. Table S7 VRN1 and VRN2 alleles in Cadenza. Please note: Wiley is not responsible for the content or function- ality of any Supporting Information supplied by the authors. Any queries (other than missing material) should be directed to the New Phytologist Central Office. Disclaimer: The New Phytologist Foundation remains neutral with regard to jurisdictional claims in maps and in any institutional affiliations. New Phytologist (2026) www.newphytologist.com � 2026 The Author(s). New Phytologist� 2026 New Phytologist Foundation. Research New Phytologist14 VERNALIZATION2 alters early tiller development in a facultative spring hexaploid bread wheat Summary Introduction Materials and Methods Plant materials and growth conditions Generation of zcct-d2_m1 and zcct-d1_m1 mutant lines Analysis of gene expression Promoter and intron motif analysis Results ZCCT1 and ZCCT2, which form the VRN2 loci, contain different regulatory domains Core vernalization genes´ expression alters depending on temperature and photoperiod conditions in facultative wheat ZCCT1 expression increases with photoperiod post-vernalization Early tiller development is accelerated in VRN-D2 TILLING lines ZCCT genes co-regulate their expression Discussion Vernalization genes are responding to multiple environmental factors Vernalization genes show different responses across development Early tiller growth is regulated by ZCCT-D1 and -D2 ZCCT genes show co-regulation Acknowledgements Competing interests Author contributions Data availability References Supporting Information