Evolution of C4 photosynthesis in Neurachne

As a carbon-concentration mechanism, C4 photosynthesis enables plants to be highly productive even in carbon-deficient, hot and dry conditions. From the ancestral C3 pathway, C4 photosynthesis has evolved more than 60 times, and approximately half of C4 origins have occurred in monocots. Uniquely for monocots, the Australian genus Neurachne R.Br. consists of eight closely related species that conduct different photosynthetic types (i.e. C3, C4, and so-called C3-C4 intermediate photosynthesis). Furthermore, the two C4 species of Neurachne represent two independent origins of C4 photosynthesis opening up the possibility to determine if the mechanisms of establishing C4 photosynthesis differ between the species.By means of mRNA sequencing of leaves of seven of the eight Neurachne species (four C3 species, one C3-C4 intermediate species, and two C4 species) we firstly revisit the phylogenetic relationships of Neurachne. To increase our understanding of differential gene expression between closely related species with differing photosynthetic types, we then conduct a comparative transcriptomic analysis between these seven Neurachne species. By using the newly inferred phylogeny of Neurachne and transcriptome sequence information, we finally test whether the key proteins of C4 photosynthesis are under positive selection.Our phylogenetic analysis shows slightly differing relationships in Neurachne compared to previously inferred relationships, but affirm two independent C4 origins within the genus. The gene expression pattern mostly concurs with results observed in comparative transcriptome analyses between C3 and C4 species of other plant lineages. For example, transcriptional investment of genes known to be involved in C4 photosynthesis is very high in leaves of the two C4 species while insignificant in C3 and C3-C4 intermediate species. Interestingly, the number of differentially expressed genes is highest between the two C4 species. Positive selection is absent in most of the C4 proteins when labelling the two branches where C4 is hypothesised to have evolved. However, several amino acids in the primary C4 carboxylase, phosphoenolpyruvate carboxylase, are detected to be under positive selection; one of these in the N terminus—a region underrepresented in all positive selection analyses up to now. This particular amino acid change not only occurs in the two C4 species in Neurachne, but also is present in other C4 grasses.作为一种碳浓缩机制，C4光合作用（C4 photosynthesis）能让植物在缺碳、高温干旱的环境下依然保持高生产力。C4光合作用从祖先的C3途径独立演化超过60次，其中约半数C4起源发生在单子叶植物中。作为单子叶植物中的特例，澳大利亚管颖草属（Neurachne R.Br.）包含8个亲缘关系极近的物种，它们具有不同的光合类型，即C3、C4以及所谓的C3-C4中间型光合作用。尤为特别的是，该属的两个C4物种代表了两次独立的C4光合起源，这为研究不同物种间建立C4光合作用的机制是否存在差异提供了可能。本研究对该属8个物种中的7个（4个C3物种、1个C3-C4中间型物种以及2个C4物种）的叶片进行了mRNA测序（mRNA sequencing），首先重新解析了管颖草属的系统发育关系。为了深入理解具有不同光合类型的近缘物种间的基因表达差异，我们随后对这7个管颖草属物种开展了比较转录组学分析。借助新构建的管颖草属系统发育树与转录组序列信息，我们最终验证了C4光合作用的关键蛋白是否受到正选择作用。系统发育分析显示，管颖草属的亲缘关系与此前推断的结果存在细微差异，但确认了该属内两次独立的C4起源事件。基因表达模式整体与其他植物谱系中C3与C4物种的比较转录组分析结果一致。例如，已知参与C4光合作用的基因在两个C4物种的叶片中转录投入量极高，而在C3及C3-C4中间型物种中则无显著表达。有趣的是，两个C4物种间的差异表达基因数量最多。当以推测发生C4演化的两个分支作为目标分支时，多数C4蛋白未检测到正选择信号。然而，在主要的C4羧化酶——磷酸烯醇式丙酮酸羧化酶（phosphoenolpyruvate carboxylase）中，多个氨基酸位点被检测到处于正选择作用之下；其中一个位于N端区域——该区域在以往所有正选择分析中均被覆盖不足——的氨基酸替换，不仅存在于管颖草属的两个C4物种中，也出现在其他C4禾本科植物中。