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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  zebra leaf 16  ( zl16 ) mutant derived from rice cultivar 9311. The  zl16  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  zl16  leaves during the early development of a chloroplast. Map-based cloning revealed that  ZL16  encodes a β-hydroxyacyl-ACP dehydratase (HAD) involved in  de novo  fatty acid synthesis. Compared with the wild type, a missense mutation (Arg164Trp) in the  zl16  mutant was identified, which significantly reduced enzymatic activity and altered the three-dimensional modeling structure of the putative protein.  ZL16  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  zl16  mutant. RNA-Seq analysis indicated that some genes related to cell membrane constituents were downregulated in the mutant. An  in vivo  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.

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