Mybr97 is the most likely candidate for the gene underlying the A3 locus
Transposon-tagging revealed that interrupting the promoter of Mybr97 was sufficient to confer the a3 intense purple phenotype. The structure of Mybr97 was similar to other R3-MYB repressor genes that act on the anthocyanin pathway in other species. Mybr97 contains a motif similar to the R1/B1 gene family binding motif [DE]Lx2[RK]x3Lx6Lx3R and was demonstrated to bind to the MIR of B1 in vitro (Zimmermann et al. 2004). The pattern of Mybr97 expression follows the same pattern of pigment accumulation in a3 recessive plants. Finally, bulk-segregant RNA-seq analysis showed that Mybr97 was the highest down-regulated gene within the a3 locus. Altogether, Mybr97 is the most likely gene involved with the a3 intense purple phenotype in maize.
Fine-tuning expression of anthocyanin-related transcription factors increases anthocyanin production
A3 is a strong negative regulator of the anthocyanin pathway in maize. Recessive plants display enhanced pigmentation in leaf sheath, husks, and tassels. Anthocyanin content in the husks is up to 100-fold greater than with a functional A3. It is difficult to compare anthocyanin production across the literature since extraction protocols vary widely. In a nearly exhaustive anthocyanin extraction of purple husks from a breeding program, anthocyanin content reached up to 19% of total weight (Li et al. 2008). The population developed in this current study contains the weak B1-b allele, so pigment production was not optimized. A constitutively expressing B1 or including naturally strong B1 alleles with a3 may be the most effective route to enhance pigment in husks, leaf sheaths, and tassels. Negative regulators Myb11, Myb31, Myb42, Sro1, and In1 were expressed in husks, which indicates that reducing the expression of these genes might also help increase pigment yield in husks. Myb11, Myb31, and Myb42 regulate lignin content while Sro1 is a competitive inhibitor of Pl1 (Agarwal et al., 2016; Qin et al., 2021; Vélez-Bermúdez et al., 2015). In1 affects aleurone pigmentation and is not known to be associated with husks, so this is novel regulation for this gene (Burr et al. 1996). Additionally, R1, C1, and Pac1 transcripts were detected in husks (Table S3). These are also not canonically associated with husk pigmentation, so their function is unknown. In a previous study, Pac1 transcript was also found in husks, but it was found that mutant pac1 plants do not show reduced anthocyanin content in vegetative tissues (Selinger and Chandler 1999; Carey et al. 2004). This finding indicates a secondary WD-repeat protein is most likely responsible for gene activation in husks. Pac1 homolog Mp1 was expressed in husks (Table S4) and may complement Pac1 in husks. Increasing anthocyanin content in maize has implications on human health as these pigments are well-characterized antioxidants and have a range of health-promoting benefits (Lao et al. 2017).
Bulk-segregant RNA-seq analysis facilitates the identification of candidate genes
Two methods for pinpointing A3 gene candidates were utilized in this study. Transposon-tagging has long been used to discover genes since this system produces stable knockouts for visible phenotypes. In this study, Ac-im, a transposon that encodes transposase but cannot excise itself, was used to mobilize Ds (Conrad and Brutnell 2005). This is important so that the Ds element is the only mobile element causing phenotypic changes. The transposon-tagging population was developed with the expectation that 4.5% of the plants will have heritable excision events (Conrad and Brutnell 2005). In a pilot experiment utilizing this system, it was estimated that 5000 individuals would need to be generated to insert into an average-sized gene 4 cM away (Ahern et al., 2009). Since Ds prefers closely linked (4 cM or less) sites (Ahern et al. 2009), then it was necessary to produce a large population for a site expected to be 7.7 cM downstream of a1-m3::Ds (Robinett et al. 1995). The readily visible a3 phenotype assisted in finding new insertion events. The second method for narrowing down candidate genes for A3 involved calling SNPs from transcriptomic data. Bulk segregant RNA-seq analysis is an important tool for discovering genes and is beneficial as a form of low-representation sequencing method for complex genomes like maize (Liu et al. 2012). Large coverage for SNPs can be found in gene-coding regions. The two thresholds for determining significance in bulk segregant RNA-seq left conservatively wide confidence intervals. The A3 locus could have been impacted by the inclusion of some A3 dominant individuals in the recessive pools since the 320N allele of the Mybr97 transcript was found in recessive pools. Despite this, this study demonstrated the effectiveness of using bulk segregant RNA-seq analysis to provide a narrow list of candidate genes that traditional linkage mapping methods are unable to do with the same generation time and population size.
Phylogenetic approaches are important for discovering new anthocyanin-related genes
The anthocyanin pathway is important in numerous plant species and has its roots as far back as the bryophytes (Markham 1988). It is reasonable to hypothesize then that regulatory control of the anthocyanin pathway may have also been phylogenetically conserved among angiosperms. Indeed, anthocyanin R3-MYB repressor genes appear to be functionally conserved among angiosperms. R3-MYB repressors have been characterized in Arabidopsis, chrysanthemum (Chrysanthemum morifolium), eggplant (Solanum melongena), gentian (Gentiana trifolia), grape hyacinth (Muscari spp.), Iochroma loxense, Lychee (Litchi chinensis), monkeyflower (Mimulus lewisii), petunia (Petunia × hybrida), orchid (Phalaenopsis spp.), poplar (Populus spp.), and tomato (Solanum lycopersicum) (Ma and Constabel 2019; Fu et al. 2019; Xiang et al. 2019; Zhang et al. 2020; Zhao et al. 2022). It is reasonable to conclude that Mybr97, which has homology to these transcriptional regulators is also involved with repressing anthocyanin synthesis in maize. Utilizing the wealth of knowledge from other plant species could decipher clues into additional regulators of the anthocyanin pathway in maize. In this study, Nac and WRKY transcriptional regulators with homology to eudicot anthocyanin regulators were upregulated (Table S2). All the canonical biosynthetic genes involved with anthocyanin production were upregulated as was expected (Table 1 and Figure 4). The AOMT in maize has not been characterized to this date, but Omt4 appears to be a good candidate. Work is currently underway to isolate this protein to test its specificity.
Marker-assisted selection was effective for the a3 locus
Molecular marker umc2008 is an appropriate marker for A3. The marker itself is only 30.2 kb away from Mybr97 according to the B73 RefGen_v4 reference genome and has high variability in simple repeat number among common varieties tested (data not shown). However, in the W22 v2 genome, the distance is as far as 1.85 Mb from Mybr97 (Springer et al. 2018). These large-scale structural variabilities may account for the accidental inclusion of dominant A3 plants in the recessive pools of samples. All a3 recessive plants were checked with umc2008 before sampling. Interestingly, some intensely purple plants contained the B73 marker allele and were excluded. Intense anthocyanin accumulation with a dominant A3 allele indicates that there might be alternative factors involved with intensifying anthocyanins in this population besides A3. More evidence for alternative intensification factors was visible in the pericarps of the segregating population. B73 has yellow kernels and 320N contains white kernels. The F1 segregates for yellow and white kernels as expected. However, after selfing, red and purple kernels were common. The source of the cryptic genetic variation shown in this population is not currently understood. It may be due to the interaction of transcription factor alleles from the two parents in the population or recombination swapping promoter elements.
Increased phenylpropanoid pathway gene expression implies increased plant protection
Not only is the involvement of A3 with the anthocyanin pathway important, but also the upregulation of genes in the monolignol pathway has implications on structural stability; insect, disease, and wounding defense; and forage quality in maize. Three PAL genes, along with C4H and 4CL were upregulated in recessive a3 plants (Table 1 and Figure 4). In maize, there appears to be multiple copies of PAL with various tissue-specific regulation (Guillaumie et al. 2007; Morohashi et al. 2012; Yuan et al. 2019). Pal1, Pal2, and Pal6 were important in husks, which is in agreement with a previous study (Yuan et al. 2019). Increased expression of PAL has been also associated with increased insect and disease defense through the increased production of salicylic acid in another study (Yuan et al. 2019). Production of anthocyanins is very costly for the plant, so it is no wonder that many modern maize varieties have a dominant A3 and do not produce appreciable amounts of anthocyanins. Knocking out anthocyanin biosynthetic genes along with A3 might pull vital precursors away from anthocyanin synthesis into, say, lignin, maysin, or salicylic acid synthesis, where they might be more beneficial for crop protection.
Photosynthesis-related gene expression was affected in recessive a3 plants
The negative association of the a3 phenotype with photosynthesis is concerning in terms of plant productivity. However, if one’s goal is to create an intensely purple plant, then A3 is an ideal candidate. The mechanism for reduced photosynthetic capacity in recessive a3 plants is currently unknown. Anthocyanins have a slight overlap in absorbance spectrum with chlorophyll in the ultraviolet to blue spectrum. However, the maximum absorbance wavelength of anthocyanins are distinct from Chlorophyll a and b (Zscheile 1934; Chatham et al. 2020). It is possible that the high concentration of anthocyanins in recessive a3 plants may mask some photosynthetically active radiation. If Mybr97 is the gene responsible for the a3 locus, reduced photosynthetic gene expression could be the result of associations with other transcription factors with an impact on photosynthesis and shade sensing. Previous studies have implicated Mybr97 in shade avoidance syndrome. Mybr97 was upregulated in the dark, indicating it may have photosynthetic regulatory functions (Shi et al., 2019; Wang et al., 2016). This is similar AtCPC and other CPC-like genes in Arabidopsis that not only affect anthocyanin accumulation, but have a range of effects on trichome development, stomata, and flowering time (Zhu et al. 2009).
Mybr97 is activated independently of the anthocyanin pathway
The activation of Mybr97 in the absence of the MBW complex in B73 and other normal green varieties implies that Mybr97 is involved with a broader activation response mechanism independent of anthocyanins. Mybr97 seems to be involved with many stress response pathways in maize. A previous study also implicated Mybr97 in cold, heat, salt, and UV stress (Makarevitch et al., 2015), just as GO term analysis did here (Table 2). Promoter elements may infer probable mechanisms for the control of Mybr97. Two G-box elements are in the promoter of Mybr97. These elements are known binding sites for maize anthocyanin bHLH proteins and photosynthetic regulator Hy5 (Ang et al. 1998; Kong et al. 2012). The presence of this G-box element may indicate the gene is auto-regulated by anthocyanin bHLH proteins like R1 and B1. In fact, in grape hyacinth, the R3-MYB repressor gene is activated by MabBhlh1, which is the bHLH member of the MBW complex (Zhang et al. 2020). Using the PROMO tool, predicted binding sites for light-associated transcription factors Gt1, Phytochrome interacting factor1, and Thioredoxin m1 were found (Xu et al. 2001; Farre 2003; Gao et al. 2015). Hormonal control of A3 might be another plausible mechanism of activation. Abscisic acid-responsive cis-element TACGTG and auxin responsive elements TGGTTT and TGTCTC are also present in the promoter of A3 (Guilfoyle and Hagen 2007; Song et al. 2018). Future work needs to investigate the role of these binding sites and hormone levels on the Mybr97.
A major role of Mybr97 may be to competitively inhibit the MBW complex
The association of Mybr97 with B1-MIR suggests Mybr97 is a competitive inhibitor of the MBW complex in maize. No DNA-binding experiments were performed, but R3-MYB genes are not predicted to have DNA-binding capabilities because they lack the R2-domain typically found in most MYB proteins (Dubos et al. 2010). It is not currently understood why full-length B1 protein could not bind to Mybr97 (Figure 3). Anthocyanin bHLH proteins are capable of homodimerizing, which may interfere with the association of Mybr97 to B1 (Kong et al. 2012). In addition, the GST-tag may have interfered with binding sites in vitro. Future experiments will be aimed at discovering other targets for Mybr97 protein using alternate affinity tags or using a yeast two-hybrid approach. A previous study showed that AtCPC is able to bind to the MIR of R1 allele Leaf color1 (Lc1) as demonstrated in a yeast-two hybrid and GST-pulldown assay (Tominaga-Wada et al. 2012). Future experiments should test the MIR of this protein and see if it is able to inhibit anthocyanins in either the pericarp or aleurone layers of the grain. Furthermore, protein interaction assay should be performed with photosynthetic bHLH members to see if Mybr97 physically interacts with regulators of photosynthesis.