Impaired auxin biosynthesis in the defective endosperm18 mutant is due to mutational loss of expression in the ZmYuc1 gene encoding endosperm-specific YUCCA1 protein in maize

Abstract

The phytohormone auxin (indole-3-acetic acid [IAA]) plays a fundamental role in vegetative and reproductive plant development. Here, we characterized a seed-specific viable maize (Zea mays) mutant, defective endosperm18 (de18) that is impaired in IAA biosynthesis. de18 endosperm showed large reductions of free IAA levels and is known to have approximately 40% less dry mass, compared with De18. Cellular analyses showed lower total cell number, smaller cell volume, and reduced level of endoreduplication in the mutant endosperm. Gene expression analyses of seed-specific tryptophan-dependent IAA pathway genes, maize Yucca1 (ZmYuc1), and two tryptophan-aminotransferase co-orthologs were performed to understand the molecular basis of the IAA deficiency in the mutant. Temporally, all three genes showed high expression coincident with high IAA levels; however, only ZmYuc1 correlated with the reduced IAA levels in the mutant throughout endosperm development. Furthermore, sequence analyses of ZmYuc1 complementary DNA and genomic clones revealed many changes specific to the mutant, including a 2-bp insertion that generated a premature stop codon and a truncated YUC1 protein of 212 amino acids, compared with the 400 amino acids in the De18. The putative, approximately 1.5-kb, Yuc1 promoter region also showed many rearrangements, including a 151-bp deletion in the mutant. Our concurrent high-density mapping and annotation studies of chromosome 10, contig 395, showed that the De18 locus was tightly linked to the gene ZmYuc1. Collectively, the data suggest that the molecular changes in the ZmYuc1 gene encoding the YUC1 protein are the causal basis of impairment in a critical step in IAA biosynthesis, essential for normal endosperm development in maize.

Figure 1.

Concentrations of free IAA (ng g⁻¹ dry matter) in De18 and de18 endosperms at 8, 12, 20, and 28 DAP, by UPLC-MS/MS. WT, Wild type. [See online article for color version of this figure.]

Figure 2.

A to D, Spatial distribution of cells at different ploidy classes (A and B) and volumes (C and D) on the longitudinal sections of De18 (A and C) and de18 (B and D) endosperms at 8 to 16 DAP. E to H, Number of cells by volume and total number of endosperm cells belonging to different C value classes in De18 (E and G) and de18 (F and H). Endopolyploid cells with C values >6 are represented by a color scale from azure (12C) to red (192C). The cell volume is presented with a color scale ranging from blue (1 × 10⁻³ µm³) to red (5,000 × 10⁻³ µm³).

Figure 3.

qPCR determinations of transcript abundance of various genes in De18 and de18 endosperm at four developmental stages (DAP). Expression levels are shown as number of copies of transcripts per nanogram of total RNA. Each histogram bar is a mean of three biological replicates and each with three technical replications.

Figure 4.

Fine mapping of de18 on chromosome 10 (A), sequence analysis of the ZmYuc1 coding region (B), protein structure of ZmYUC1 (C), and noncoding putative promoter (D). A, Schematic representation of the region containing the Yuc gene on chromosome 10 (not to scale). Contig 393 maps to bin 10.2, while contigs 394, 395, and 397 map to bin 10.3 (each black bar represents a contig). The simple sequence repeat and the SNP markers used for genotyping are in bold; #1306 represents the gene Arf28. The number of recombinants (R) is shown below each marker. B, Yuc1 gene structure in De18; region between arrow and left vertical bar represents mRNA. Exons and untranslated regions/introns are represented as solid and shaded rectangles respectively. Numbers within the boxes denote exon and intron length (derived from B73 MaizeGDB) in base pairs. Downward and upward triangles represent insertion and deletion, respectively, in the de18 mutant. SNPs present in mutant are shown with thin bar. CDS, Coding sequence. C, Locations of conserved motifs present in YUCs protein. Among all motifs, N-terminal GxGPxGLA and middle GxGxxGME motifs are postulated as nucleotide binding motifs FAD and NADPH, respectively. In de18, NADPH binding motif is absent because of a 2-bp insertion in the mRNA at position 532. aa, Amino acids. D, Promoter region of 1491 bp of the Yuc1 gene. Polymorphisms in de18 respect to De18 are shown. Downward triangles indicate insertions, dashed lines deletions, and continuous lines SNPs. [See online article for color version of this figure.]

Figure 5.

Western-blot analysis of E. coli-expressed proteins from full-length cDNA clones of the ZmYuc1 gene from De18 and de18 endosperm and the corresponding empty vector, as shown; each lane represents crude extract of 1µg of total protein. [See online article for color version of this figure.]