Identification and Phenotypic Characterization of ZEBRA LEAF16 Encoding a β-Hydroxyacyl-ACP Dehydratase in Rice

Abstract

The chloroplast is a self-independent organelle and contains its own transcription and translation systems. The establishment of genetic systems is vital for normal plant growth and development. We isolated a rice <i>zebra leaf 16</i> (<i>zl16</i>) mutant derived from rice cultivar 9311. The <i>zl16</i> mutant showed chlorotic abnormalities in the transverse sectors of the young leaves of seedlings. The use of transmission electron microscopy (TEM) demonstrated that dramatic defects occurred in variegated <i>zl16</i> leaves during the early development of a chloroplast. Map-based cloning revealed that <i>ZL16</i> encodes a β-hydroxyacyl-ACP dehydratase (HAD) involved in <i>de novo</i> fatty acid synthesis. Compared with the wild type, a missense mutation (Arg164Trp) in the <i>zl16</i> mutant was identified, which significantly reduced enzymatic activity and altered the three-dimensional modeling structure of the putative protein. <i>ZL16</i> was ubiquitously expressed in various plant organs, with a pronounced level in the young leaf. A subcellular localization experiment indicated that ZL16 was targeted in the chloroplast. Furthermore, we analyzed the expression of some nuclear genes involved in chloroplast development, and found they were altered in the <i>zl16</i> mutant. RNA-Seq analysis indicated that some genes related to cell membrane constituents were downregulated in the mutant. An <i>in vivo</i> metabolic assay revealed that the total fatty acid content in the mutant was significantly decreased relative to the wild type. Our results indicate that HAD is essential for the development of chloroplasts by regulating the synthesis of fatty acids in rice.