Regulatory effects of Pl1 on anthocyanin biosynthesis in maize basal leaf sheaths and the underlying molecular...

Maize leaf sheath anthocyanin accumulation is governed by precise transcriptional regulation, yet its key regulator and molecular mechanisms for this tissue-specific pigmentation remain incompletely understood. Through EMS-based mutagenesis and CRISPR/Cas9 validation, we determined that the R2R3-MYB transcription factor Pl1, but not B1, is essential for anthocyanin biosynthesis in leaf sheaths. Two natural loss-of-function Pl1 alleles were screened, each harboring an 18-bp deletion in exon 3 of the mRNA that likely arises from GC-rich sequence-mediated mis-splicing. Genome-wide DNA affinity purification sequencing revealed 8053 high-confidence Pl1 target genes that were significantly enriched in arginine and proline metabolism. Nine flavonoid pathway structural genes were identified as Pl1 targets, including Zm00001d043174 encoding a putative cytochrome P450 superfamily protein, which is co-targeted by the bHLH factor R1. Comparative transcriptomics across four inbred lines revealed tissue-specific suppression of Pl1 in leaves relative to leaf sheaths. Overexpression of Pl1 intensified sheath pigmentation and induced pigmentation in green leaves. Cross-tissue transcriptome comparison identified 2784 concordantly differentially expressed genes (DEGs), including 218 transcriptional regulator genes. Pull-down coupled with LC-MS analysis of Pl1 promoter-binding proteins in both tissues identified 61 transcriptional effectors, five of which overlapped with the concordant DEGs. These encode AT-hook proteins, a DNA methyltransferase, a knotted homeobox protein, and a nascent polypeptide-associated complex subunit and are proposed as candidate upstream modulators for sheath-specific pigmentation. This study establishes Pl1 as the master regulator of sheath pigmentation, delineates its downstream target network and candidate upstream factors, thereby advancing the mechanistic understanding of spatially restricted anthocyanin accumulation in maize.