A single nucleotide polymorphism in conz1 enhances maize adaptation to higher latitudes

Flowering time is a key trait determining the local adaptation of plants. CONSTANS (CO) is a core regulator controlling photoperiodic flowering in Arabidopsis (Shim et al., 2017). Heading date 1 (Hd1), a CO ortholog in rice, promotes heading under short-day (SD) conditions but inhibits heading under long-day (LD) conditions (Yano et al., 2000). Miller et al. (2008) identified constans of Zea mays1 (conz1), a maize CONSTANS homologue, that exhibited distinct diurnal expression patterns under varied photoperiods. However, how conz1 contributes to maize flowering time variation and adaptation remains largely unknown. Using a BC2S3 population derived from a cross between the maize inbred line B73 (Zea mays ssp. mays) and the teosinte inbred line TIL11 (Zea mays ssp. parviglumis), we performed quantitative trait locus (QTL) mapping for photoperiod response measured as the difference in days to anthesis and leaf number between natural SD (NSD, Sanya, 18.4° N, 109.2° E) and natural LD (NLD, Tieling, 42.1° N, 123.6° E). A significant QTL for flowering time and leaf number photoperiod response (FTPR and LNPR) was simultaneously detected at similar location on chromosome 9, designated qPR9 (Figure 1a). Compared with the TIL11 allele, the B73 allele at qPR9 reduced FTPR and LNPR (Figure 1b,c). We further developed a pair of near isogenic lines (NILs) (qPR9-NILB73 and qPR9-NILTIL11) and scored days to anthesis and leaf number under NLD and NSD conditions. qPR9-NILB73 exhibited later flowering and more leaf number than qPR9-NILTIL11 under NSD (Figure 1d,e), while opposite effects were observed under NLD (Figure 1d,e). In other words, qPR9-NILB73 displayed reduced FTPR and LNPR compared with qPR9-NILTIL11, which is consistent with the observation in the maize-teosinte BC2S3 population. Unfortunately, qPR9 is located in a region with severe recombination suppression (Figure 1f), preventing us from fine mapping qPR9. Interestingly, a known flowering time gene, conz1, resided near the peak of qPR9 (Figure 1a,f). Hence, conz1 was considered a strong candidate gene for qPR9. To validate the function of conz1, we knocked out conz1 using CRISPR/Cas9 and obtained a Cas9-free homozygous mutant with 1-bp deletion that caused truncated protein (Figure 1g). Compared with the wild-type (WT), the conz1 knockout line (KO) exhibited delayed flowering under both NLD and NSD conditions (Figure 1h,i). However, the flowering time difference between KO and WT was much larger under NSD (4.4 days) than under NLD conditions (2.6 days) (Figure 1h,i). Moreover, the conz1 KO plants exhibited increased leaf number under NSD compared with WT, but no difference in leaf number was observed under NLD (Figure 1h,i). Interestingly, the conz1 KO plants displayed no difference in days to anthesis but reduced leaf number under artificial LD (ALD) condition with longer day length (16 h) compared with WT (Figure 1h,i). These results indicated that conz1 is regulated by photoperiod. conz1 promoted flowering under NSD, but this promoting effect was weakened under longer photoperiods. As conz1 was underneath of the peak of qPR9 and the conz1 KO plants tended to exhibit similar effect patterns to the qPR9 NILs under varied photoperiods, conz1 was considered the causative gene underlying qPR9. To examine whether conz1 contributed to the natural variation in photoperiod sensitivity, we conducted an association analysis for conz1 in an association panel containing 513 maize inbred lines scored for FTPR and LNPR. In total, 19 variants (15 SNPs and 4 indels) with minor allele frequency (MAF) ≥ 0.05 were identified around conz1. Among these variants, an A > G single nucleotide polymorphism (SNP166), located 166 bp downstream of the start codon of conz1, showed the most significant association with FTPR (P = 2.75E-06) and LNPR (P = 2.14E-06) using a mixed linear model accounting for kinship (Figure 1j). The SNP166A allele exhibited reduced photoperiod sensitivity compared with the SNP166G allele (Figure 1k,l). We then examined whether the NILs of qPR9 differed at SNP166. Interestingly, qPR9-NILB73 carried the SNP166A allele that reduced photoperiod response, while qPR9-NILTIL11 carried the SNP166G allele that enhanced photoperiod response (Figure 1j). Comparison of CONSTANS protein across different species showed that SNP166 is located in the conserved B-box domain, and the G (TIL11 allele) to A (B73 allele) mutation resulted in change from the conserved aspartic acid (D) to asparagine (N) (Figure 1j,m). These results suggested that SNP166 was very likely the functional variant of conz1 underlying qPR9. The Arabidopsis CO tends to form a multimer through the B-box domain, which enhances the overall binding specificity and affinity of CO to FLOWERING LOCUS T (FT) (Lv et al., 2021). As SNP166 is located in the B-box domain, we tested whether SNP166 affected the formation of dimer or multimer of CONZ1. Yeast two-hybrid assay, β-galactosidase assay and luciferase complementation assay indicated that CONZ1B-box(D) from qPR9-NILTIL11 displayed stronger ability to form dimer or multimer than CONZ1B-box(N) from qPR9-NILB73 (Figure 1n,o). Transactivation analysis in maize protoplasts showed that CONZ1(D) exhibited stronger transcriptional activation activity than CONZ1(N) (Figure 1q). Electrophoretic mobility shift assay showed that CONZ1 could directly bind to the sequences containing ‘CCACA’ motifs in the promoter of the maize florigen gene ZEA CENTRORADIALIS 8 (ZCN8) (Figure 1p). Further dual-luciferase transient expression assay in maize protoplasts showed that CONZ1(D) exhibited stronger promotion of ZCN8 expression than CONZ1(N) (Figure 1r). These results suggested that SNP166 affected the formation of dimer or multimer of CONZ1 and its transcription activation of ZCN8. To examine the evolutionary origin of SNP166, we sequenced the region flanking SNP166 in 19 teosinte lines (Zea mays ssp. parviglumis and Zea mays ssp. mexicana). None of them carried the SNP166A allele, indicating that the SNP166A allele was most likely a de novo mutation that occurred after initial maize domestication (Figure 1s). Analysis of modern maize inbred lines showed that the SNP166A allele showed a very low frequency of 4% in tropical maize, but its frequency rapidly rose to 58% in temperate maize (Figure 1s). To test whether SNP166A was targeted by selection, we analysed the nucleotide diversity of the region surrounding SNP166 in diverse maize and teosinte lines. Notably, the maize lines carrying SNP166A retained only 3.3% of the nucleotide diversity of teosinte. Coalescent simulation analysis (Huang et al., 2018) further verified that the severe loss of genetic diversity in the maize lines carrying SNP166A is significantly deviated from the neutral domestication bottleneck model, indicating strong selection for SNP166A. We further genotyped SNP166 in 377 maize landrace accessions distributed across the Americas (Huang et al., 2018). Interestingly, the SNP166A allele was predominantly distributed in northern United States at high latitudes (Figure 1t), indicating that the SNP166A allele at conz1 played an important role in promoting maize adaptation to northern high latitudes. Previous studies in rice have shown that the function of Hd1 depends on flowering repressors Ghd7 and DTH8 (Zong et al., 2021). Under LD conditions, Hd1 interacts with Ghd7 and DTH8 to form a repressive complex to repress heading, while under SD conditions, Hd1 mainly functions as an activator to promote heading due to reduced accumulation of Ghd7 and DTH8 under SD (Zong et al., 2021). In the absence of functional Ghd7 DTH8 alleles, Hd1 promotes heading regardless of day-length (Zong et al., 2021). Our results potentially indicate that conz1 might function in a similar mechanism, but this speculation requires further studies, especially examining the genetic interaction between conz1 and LD-dependent flowering repressors such as ZmCCT9 and ZmCCT10 (Huang et al., 2018; Hung et al., 2012). Taken together, we demonstrated that SNP166, a nonsynonymous mutation in the conserved B-box domain of CONZ1 conferred the photoperiod flowering QTL qPR9. The SNP166A allele that reduces photoperiod sensitivity occurred after initial maize domestication, and its selection enhanced maize adaptation to higher latitudes of temperate regions. This research was supported by the National Key Research and Development Program of China (2022YFD1201503), the National Natural Science Foundation of China (32025027 and 32101711), Hainan Yazhou Bay Seed Laboratory (B21HJ0111), Sanya Yazhouwan Science and Technology City Administration (SYND-2022-26), the Chinese Universities Scientific Fund (2020TC149 and 2022TC138), and China National Postdoctoral Program for Innovative Talents (BX2021366). The authors declare no conflict of interests. L.W., Y.L., L.G. and F.T. designed the project. L.W., Y.L., L.G., Y.Z., W.Q., W.W. and H.J. performed the experiment and analysed the data. L.W., Y.L., L.G., and F.T. wrote the paper. All the authors read and approved the paper.