The phytohormone auxin (IAA) plays a fundamental role in vegetative and reproductive plant development. Developing seeds accumulate the highest level of IAA of all tissues in a plant, but poor is known on genes/enzymes critical to endosperm development. We analyzed a seed-specific viable maize mutant, defective endosperm 18 (de18), that accumulates 10- to 15- fold less IAA in endosperm and ~40% less dry matter compared to De18. Gene expression analyses of selected putative IAA-biosynthesis genes, showed an high expression of Trp-aminotransferase co-orthologs in both De18 and de18, while only the ZmYucca1 correlated with the reduced IAA levels in the mutant throughout endosperm development. Further, sequence analyses of ZmYuc1 cDNA and genomic clones revealed many changes specific to the mutant, including a 2-bp insertion that generated a premature stop codon and a truncated YUCCA1 protein of 212 amino acids, compared to the 400 amino acids in the De18. Consistent with these results are the recombinant protein data from E. coli where full length cDNA clones of ZmYuc1 of De18 and de18 endosperm showed a normal and greatly reduced sizes of fusion proteins, ~70 kD and ~50 kD, respectively (each included a GST tag of 26.0 kD). The putative ~1.5 kb ZmYuc1 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 that encoded the YUCCA1 protein is the causal basis of impairment in a critical step in IAA biosynthesis, essential to endosperm development in maize. The lack of compensation of the ZmYuc1 function despite the high transcript abundance of the two ZmTar genes suggests that YUCCA1 constituted a rate-limiting step in a single pathway of multiple steps of IAA biosynthesis, consistent with the recent evidences in Arabidopsis.
Bernardi, J., Lanubile, A., Li, Q., Kumar, D., Chourey, P., Marocco, A., DEFECTIVE ENDOSPERM 18-ENCODED YUCCA1 PROTEIN ISESSENTIAL FOR NORMAL AUXIN-DEPENDENT ENDOSPERMDEVELOPMENT IN MAIZE, in Proceedings of the 56th Italian Society of Agricultural Genetics Annual Congress, (Perugia, 17-20 September 2012), Italian Society of Agricultural Genetics Annual Congress, Perugia 2012: 1-1 [http://hdl.handle.net/10807/41324]
DEFECTIVE ENDOSPERM 18-ENCODED YUCCA1 PROTEIN IS ESSENTIAL FOR NORMAL AUXIN-DEPENDENT ENDOSPERM DEVELOPMENT IN MAIZE
Bernardi, Jamila;Lanubile, Alessandra;Marocco, Adriano
2012
Abstract
The phytohormone auxin (IAA) plays a fundamental role in vegetative and reproductive plant development. Developing seeds accumulate the highest level of IAA of all tissues in a plant, but poor is known on genes/enzymes critical to endosperm development. We analyzed a seed-specific viable maize mutant, defective endosperm 18 (de18), that accumulates 10- to 15- fold less IAA in endosperm and ~40% less dry matter compared to De18. Gene expression analyses of selected putative IAA-biosynthesis genes, showed an high expression of Trp-aminotransferase co-orthologs in both De18 and de18, while only the ZmYucca1 correlated with the reduced IAA levels in the mutant throughout endosperm development. Further, sequence analyses of ZmYuc1 cDNA and genomic clones revealed many changes specific to the mutant, including a 2-bp insertion that generated a premature stop codon and a truncated YUCCA1 protein of 212 amino acids, compared to the 400 amino acids in the De18. Consistent with these results are the recombinant protein data from E. coli where full length cDNA clones of ZmYuc1 of De18 and de18 endosperm showed a normal and greatly reduced sizes of fusion proteins, ~70 kD and ~50 kD, respectively (each included a GST tag of 26.0 kD). The putative ~1.5 kb ZmYuc1 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 that encoded the YUCCA1 protein is the causal basis of impairment in a critical step in IAA biosynthesis, essential to endosperm development in maize. The lack of compensation of the ZmYuc1 function despite the high transcript abundance of the two ZmTar genes suggests that YUCCA1 constituted a rate-limiting step in a single pathway of multiple steps of IAA biosynthesis, consistent with the recent evidences in Arabidopsis.
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