Comprehensive analysis of plastid gene expression during fruit development and ripening of kiwifruit

Key message Global survey of plastid gene expression during fruit ripening in kiwifruit provides cis-elements for the future engineering of the plastid genome of kiwifruit. A limitation in the application of plastid biotechnology for molecular farming is the low-level expression of transgenes in non-green plastids compared with photosynthetically active chloroplasts. Unlike other fruits, not all chloroplasts are transformed into chromoplasts during ripening of red-fleshed kiwifruit (Actinidia chinensis cv. Hongyang) fruits, which may make kiwifruit an ideal horticultural plant for recombinant protein production by plastid engineering. To identify cis-elements potentially triggering high-level transgene expression in edible tissues of the 'Hongyang' kiwifruit, here we report a comprehensive analysis of kiwifruit plastid gene transcription in green leaves and fruits at three different developmental stages. While transcripts of a few photosynthesis-related genes and most genetic system genes were substantially upregulated in green fruits compared with leaves, nearly all plastid genes were significantly downregulated at the RNA level during fruit development. Expression of a few genes remained unchanged, including psbA, the gene encoding the D1 polypeptide of photosystem II. However, PsbA protein accumulation decreased continuously during chloroplast-to-chromoplast differentiation. Analysis of post-transcriptional steps in mRNA maturation, including intron splicing and RNA editing, revealed that splicing and editing may contribute to regulation of plastid gene expression. Altogether, 40 RNA editing sites were verified, and 5 of them were newly discovered. Taken together, this study has generated a valuable resource for the analysis of plastid gene expression and provides cis-elements for future efforts to engineer the plastid genome of kiwifruit.

关键信息：对猕猴桃果实成熟过程中质体基因表达的全球调查为未来猕猴桃质体基因组工程提供了顺式作用元件。质体生物技术在分子农业应用中的一个局限是，与具有光合活性的叶绿体相比，转基因在非绿色质体中的表达水平较低。与其他水果不同，红肉猕猴桃（中华猕猴桃‘红阳’品种）果实成熟过程中并非所有叶绿体都转化为有色体，这可能使猕猴桃成为通过质体工程生产重组蛋白的理想园艺植物。为了确定可能触发‘红阳’猕猴桃可食组织中高水平转基因表达的顺式作用元件，我们在此报告了对绿叶和处于三个不同发育阶段的果实中猕猴桃质体基因转录的综合分析。与叶片相比，一些光合作用相关基因和大多数遗传系统基因的转录本在绿色果实中显著上调，而在果实发育过程中，几乎所有质体基因在RNA水平上都显著下调。少数基因的表达保持不变，包括编码光系统II的D1多肽的psbA基因。然而，在叶绿体向有色体分化过程中，PsbA蛋白的积累持续减少。对mRNA成熟的转录后步骤（包括内含子剪接和RNA编辑）的分析表明，剪接和编辑可能有助于质体基因表达的调控。总共验证了40个RNA编辑位点，其中5个是新发现的。总之，这项研究为质体基因表达分析提供了宝贵的资源，并为未来猕猴桃质体基因组工程提供了顺式作用元件。