Watching C4 photosynthesis evolve! Does Salsola divaricata, a C2 species on the Canary Islands, bridge the complex evolutionary transition between C3 and C4 photosynthesis?

C4光合作用（PS）是一个复杂的性状，其进化需要叶片解剖和超微结构、途径生物化学和基因调控的深刻变化。c4ps在被子植物中出现了c. 65次，其中一个热点在藜科植物中有c. 15个起源。C4 PS的进化过程仍然是个谜。C4进化的概念模型总是倾向于以甘氨酸穿梭碳浓缩机制（C2 PS）为关键步骤，从C3逐步过渡到C4（最初由Monson等人（1984）提出）；Sage et al. 2012修改)。支持该模型的经验证据有许多局限性，最重要的缺陷是从未考虑到群体水平上的表型可塑性和性状变异。为了验证目前的C4进化模型，该基金建议研究来自加那利群岛的C2物种Salsola divaricata和来自摩洛哥西南部的两个密切相关的C3物种S. verticillata和S. gymnomaschala，其中假设C4光合作用进化的早期阶段似乎存在。本研究采用下一代测序方法对摩洛哥西南部16个萨尔索拉（Salsola divaricata）及其近缘种的遗传分化进行了评估，并结合当地气候和地理参数进行了基因分型，并对模型提出的c4相关性状进行了调查。这些是C2和C4蛋白的定量分析，碳补偿点测量，C2和C4酶在叶片横截面和束鞘超微结构中的表达模式。对选择的偏离性状表达的样本进行转录组分析将进一步揭示基因调控水平上的分化和/或可塑性。Salsola divaricata C4性状的表型变异可能代表了C4进化的独特遗传固定进化状态，因此支持当前的模型，或者它可能是复杂生理综合征的广泛表型可塑性的一个迷人案例，并刺激对当前模型的基本重新评估，以实现包括表型可塑性的概念。了解C4 PS这一高度复杂性状的进化，不仅是解开一个广泛关注的生物学难题，也是目前将C4途径引入水稻等C3作物的研究热点。

C4 photosynthesis (PS) is a complex trait and its evolution requires profound changes of leaf anatomy and ultrastructure, pathway biochemistry and gene regulation within the ancestral C3 lineage. C4 PS arose c. 65 times in the angiosperms with one hotspot of c. 15 origins in the Chenopodiaceae. The processes of how C4 PS evolved are still a mystery. Conceptual models of C4 evolution always favoured a gradual, stepwise transition from C3 to C4 with the glycine shuttle carbon concentrating mechanisms (C2 PS) as the key step (originally proposed by Monson et al. (1984); modified by Sage et al. 2012). The empirical evidence that supports the model has a number of limitations, most importantly the flaw that phenotypic plasticity and variation of traits at the population level has never been taken into account. To test the current model of C4 evolution this grant proposal suggests to study the C2 species Salsola divaricata from the Canary Islands and the two closely related C3 species S. verticillata and S. gymnomaschala from SW Morocco in which hypothetical early phases of C4 photosynthesis evolution appear to be present. The study includes an assessment of the genetic differentiation among 16 populations of Salsola divaricata and its closest relatives from SW Morocco using the next generation sequencing method genotyping by sequencing, local climate and edaphic parameters and a survey of C4-relevant traits as proposed by the model. These are quantitative assays of C2 and C4 proteins, carbon compensation point measurements, expression patterns of C2 and C4 enzymes in leaf cross sections and bundle sheath ultrastructure. A transcriptome analysis of selected samples with deviating trait expression will furthermore reveal differentiation and/or plasticity at the gene regulatory level. The phenotypic variation of C4 traits in Salsola divaricata might represent distinct genetically fixed evolutionary states of C4 evolution and therefore support the current model or it might be a fascinating case of broad phenotypic plasticity of a complex physiological syndrome and stimulate a fundamental re-evaluation of the current model towards a concept that includes phenotypic plasticity. To understand the evolution of the highly complex trait C4 PS is not only to disentangle a fascinating biological problem of broad interest but also a hot topic in current research efforts to introduce the C4 pathway into C3 crops such as rice.