Introduction

Oxygenic photosynthesis originated over 2 billion years ago and is the ultimate source of nearly all energy used by living organisms. Almost 90% of plants fix carbon using the ancestral C₃ cycle, but this process is inefficient in hot environments (Sage & Monson, 1999). This is because the key enzyme responsible for the initial fixation of atmospheric CO₂ (Ribulose-1,5-bisphosphate carboxylase/oxygenase, Rubisco) is less able to discriminate CO₂ from O₂ at higher temperatures, and as a result, energy is lost through photorespiration (Farquhar et al., 1982). To reduce photorespiration, plants have evolved C₄ photosynthesis, wherein atmospheric CO₂ is initially assimilated into a 4-carbon organic acid by phosphoenolpyruvate carboxylase (PEPC) in the mesophyll cells, before shuttling the acid to the neighboring bundle sheath cells where it is decarboxylated and the CO₂ recaptured by Rubisco (Hatch, 1971; Edwards & Ku, 1987). This compartmentalization of Rubisco effectively prevents photorespiration. C₄ photosynthesis is a complex trait that relies on both changes to the leaf anatomy and the coordinated regulation of multiple metabolic enzymes (Hatch, 1987). In order to understand the sequence of events that led to C₄ evolution, comprehensive genomic and phenotypic datasets have been generated in many systems, such as Flaveria (Adachi et al., 2023) and Alloteropsis (Pereira et al., 2023). These existing data sets can potentially be mined for quantitative genetics approaches to identify novel genetic factors involved in the evolution of C₄ (Simpson et al., 2021).

By comparing species with different photosynthetic types, the core C₄ enzymes, multiple accessory genes, and loci associated with C₄ leaf anatomy (often termed ‘Kranz’ anatomy) have been identified (Langdale et al., 1987, 1988; Slewinski et al., 2012; Cui et al., 2014). However, decomposing the individual steps during the transition to C₄ is confounded by the fact that variation in photosynthetic type is usually segregated between distinct species that have been independently evolving for millions of years, meaning that they differ in many aspects besides those linked to the photosynthetic pathway (Heyduk et al., 2019). The interspecific segregation of variation in photosynthetic type makes it challenging to apply quantitative genetics methods, such as quantitative trait loci (QTL) mapping and genome-wide association studies (GWAS), since these rely on traits varying within a species, or the ability to hybridize species with divergent phenotypes. GWAS has been used to investigate the variation of C₄ traits within C₄ species, such as photosynthetic performance during chilling in maize (Strigens et al., 2013), and to identify genes associated with stomatal conductance and water use efficiency in sorghum (Ferguson et al., 2021; Pignon et al., 2021). However, to date there has been no QTL region identified for differences in C₄ carbon fixation or Kranz anatomy (Simpson et al., 2021).

In grasses, the proportion of carbon that is fixed through the C₄ cycle can be measured using the stable carbon isotope ratio (δ¹³C) (O'Leary, 1981; Farquhar et al., 1989). Both ¹²C and ¹³C occur naturally in the atmosphere, and in C₃ plants, Rubisco preferentially fixes ¹²C during photosynthesis (O'Leary, 1981). Conversely, in C₄ plants, carbon is initially fixed by CA and PEPC, and this coupled enzyme system discriminates less than Rubisco between the two isotopes (O'Leary, 1981). The rate of CO₂ release in the bundle sheath is coordinated with the rate of CO₂ fixation by Rubisco, which reduces the fractionation effect of this enzyme. δ¹³C is therefore commonly used as a proxy for photosynthetic type and the relative strength of the C₄ cycle (Bender, 1968; Smith & Epstein, 1971; Smith & Brown, 1973; Von Caemmerer, 1992; Cerros-Tlatilpa & Columbus, 2009; Gowik et al., 2011; Lundgren et al., 2015; Stata et al., 2019; Olofsson et al., 2021). While there is intraspecific variation in δ¹³C for C₄ species such as maize and Gynandropsis (Voznesenskaya et al., 2007), we do not know whether this variation arises from differences in anatomy or biochemistry (Simpson et al., 2021). In addition, some of the observed variation in δ¹³C could also be due to environmental effects on water use efficiency (Farquhar & Richards, 1984), particularly if the phenotypic data comes from individuals sampled in the field. However, differences in the δ¹³C between accessions of some species are maintained in a common environment (Lundgren et al., 2016), indicating that the δ¹³C ratio likely has a genetic component. Intraspecific, heritable variation in δ¹³C offers an excellent opportunity for using quantitative genetic approaches to discover C₄ QTLs.

The grass Alloteropsis semialata has long been used as a model to study C₄ evolution, since it is the only species known to have C₃, C₄, and intermediate genotypes that diverged relatively recently and can be crossed, allowing gene flow among them (reviewed by Pereira et al., 2023). The common ancestor of this species is thought to be an intermediate with some chloroplasts in its bundle sheath and performing a very weak C₄ cycle, with the C₃ being a reversal from this intermediate state as that lineage colonized cooler environments in southern Africa (Dunning et al., 2017). The intermediate populations are found in the grassy ground layer of the Central Zambezian miombo forests that we refer to as ‘C₃+C₄’ because they perform a weak C₄ cycle in addition to directly fixing CO₂ through the C₃ cycle (Lundgren et al., 2016; Dunning et al., 2017). Comparative studies have shown that the transition to a purely C₄ physiology in A. semialata is caused by the overexpression of relatively few core C₄ enzymes (Dunning et al., 2019a) and the acquisition of C₄-like morphological traits, notably the presence of minor veins (Lundgren et al., 2019). The δ¹³C of the C₃+C₄ plants ranges from values characteristic of a weak (or absent) C₄ cycle to values that show that the C₄ cycle accounts for more than half of the carbon acquisition (Von Caemmerer, 1992; Lundgren et al., 2015; Stata et al., 2019; Olofsson et al., 2021). Furthermore, the strengthening of the C₄ cycle in the C₃+C₄ intermediates (measured using δ¹³C) is significantly associated with alterations in a number of leaf anatomical traits related to the preponderance of inner bundle sheath (IBS) tissue, the cellular location of the C₄ cycle in this species (Alenazi et al., 2023), including the distance between consecutive bundle sheaths, the width of IBS cells, and the proportion of bundle sheath tissue in the leaf (Alenazi et al., 2023).

Alloteropsis semialata therefore represents an ideal system to identify the genes correlated with the strengthening of the C₄ cycle. Here, we first conducted a global analysis to identify candidate genes associated with the strength of the C₄ cycle (δ¹³C) using genomic data from 420 individuals representing C₃, C₃+C₄, and C₄ phenotypes. We then focused specifically on the C₃+C₄ intermediates, to identify candidate genes associated with the relative expansion of bundle sheath tissue during the transition from a weak to a strong C₄ cycle. The high level of interspecific variation in A. semialata permits a fine-scale understanding of the genetic basis of C₄ evolution, including the intermediate steps involved in the assembly of this complex trait. This is crucially important to identify the initial changes required for the emergence of this trait, something that may ultimately have applications in the engineering of C₄ photosynthesis in C₃ crops such as rice.

中文翻译：

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鉴定与半叶异形植物 C4 光合活性和叶子解剖结构相关的基因组区域

 介绍

产氧光合作用起源于 20 亿多年前，是生物体使用的几乎所有能量的最终来源。几乎 90% 的植物利用祖先的 C ₃循环来固定碳，但该过程在炎热环境中效率低下（Sage & Monson， 1999 ）。这是因为负责大气 CO ₂初始固定的关键酶（Ribulose-1,5-二磷酸羧化酶/加氧酶，Rubisco）在较高温度下区分 CO ₂和 O ₂的能力较差，因此，能量通过光呼吸丢失（Farquhar等， 1982 ）。为了减少光呼吸，植物进化出了C ₄光合作用，其中大气中的CO ₂最初被叶肉细胞中的磷酸烯醇丙酮酸羧化酶（PEPC）同化为4碳有机酸，然后将酸运送到邻近的束鞘细胞，在此处脱羧以及由Rubisco重新捕获的CO ₂ (Hatch， 1971 ；Edwards & Ku， 1987 )。 Rubisco 的这种分隔有效地阻止了光呼吸。 C ₄光合作用是一个复杂的性状，依赖于叶子解剖结构的变化和多种代谢酶的协调调节（Hatch， 1987 ）。为了了解导致 C ₄进化的事件顺序，许多系统中都生成了全面的基因组和表型数据集，例如Flaveria （Adachi等人， 2023 ）和Alloteropsis （Pereira等人， 2023 ）。 这些现有数据集有可能被挖掘用于定量遗传学方法，以识别参与 C ₄进化的新遗传因素（Simpson等人， 2021 ）。

通过比较具有不同光合作用类型的物种，已鉴定出核心 C ₄酶、多个辅助基因以及与 C ₄叶解剖结构（通常称为“Kranz”解剖结构）相关的基因座（Langdale等人， 1987 年、 1988 年；Slewinski等人） .， 2012 ；崔等人， 2014 ）。然而，分解向 C ₄过渡过程中的各个步骤会受到以下事实的困扰：光合类型的变化通常在已经独立进化了数百万年的不同物种之间分离，这意味着除了与 C 4 相关的方面之外，它们在许多方面都存在差异。光合作用途径（Heyduk等人， 2019 ）。光合类型变异的种间分离使得应用定量遗传学方法具有挑战性，例如数量性状位点（QTL）作图和全基因组关联研究（GWAS），因为这些方法依赖于物种内不同的性状，或者使具有不同表型的物种杂交。 GWAS 已被用于研究 C ₄物种内 C ₄性状的变异，例如玉米冷却过程中的光合性能（Strigens等， 2013 ），并鉴定与高粱气孔导度和水分利用效率相关的基因（Ferguson）等人， 2021 ；Pignon等人， 2021 ）。然而，迄今为止，尚未鉴定出 C ₄碳固定或 Kranz 解剖学差异的 QTL 区域（Simpson等，2014） 。， 2021 ）。

在草类中，通过C ₄循环固定的碳比例可以使用稳定碳同位素比(δ ¹³ C)来测量(O'Leary， 1981 ；Farquhar等人， 1989 )。 ¹² C 和¹³ C 均天然存在于大气中，并且在 C ₃植物中，Rubisco 在光合作用期间优先固定¹² C (O'Leary, 1981 )。相反，在C ₄植物中，碳最初被CA和PEPC固定，并且该偶联酶系统在两种同位素之间的区分小于Rubisco（O'Leary， 1981 ）。束鞘中CO ₂释放的速率与Rubisco固定CO ₂的速率相协调，这降低了该酶的分级作用。因此，δ ¹³ C 通常用作光合类型和 C ₄循环相对强度的代表（Bender， 1968 ；Smith & Epstein， 1971 ；Smith & Brown， 1973 ；Von Caemmerer， 1992 ；Cerros-Tlatilpa & Columbus， 2009 ；Gowik等人， 2011 ；Lundgren等人， 2015 ；Olofsson等人，2021 ） 。虽然玉米和白花菜等 C ₄物种的 δ ¹³ C 存在种内变异（Voznesenskaya等，2016 ）。， 2007 ），我们不知道这种差异是否源于解剖学或生物化学的差异（Simpson等人， 2021 ）。此外，观察到的一些 δ ¹³ C 变化也可能是由于环境对水利用效率的影响（Farquhar & Richards， 1984 ），特别是如果表型数据来自现场采样的个体。然而，一些物种的种质之间的 δ ¹³ C 差异在共同环境中保持不变（Lundgren等人， 2016 ），表明 δ ¹³ C 比率可能具有遗传成分。 δ ¹³ C 的种内遗传变异为使用定量遗传方法发现 C ₄ QTL 提供了绝佳的机会。

草类Alloteropsis semialata长期以来一直被用作研究 C ₄进化的模型，因为它是已知唯一具有 C ₃ 、 C ₄和中间基因型的物种，这些基因型最近才分化并且可以杂交，从而允许基因在它们之间流动。由 Pereira等人审查， 2023 ）。该物种的共同祖先被认为是一种中间状态，其束鞘中有一些叶绿体，并执行非常弱的 C ₄循环，而 C ₃是这种中间状态的逆转，因为该谱系在南部非洲较冷的环境中定居（邓宁等人， 2017 ）。中间种群存在于赞比西亚中部 miombo 森林的草地地面层中，我们将其称为“C ₃ +C ₄ ”，因为它们除了通过 C ₃循环直接固定 CO ₂之外，还执行弱 C ₄循环（Lundgren）等人， 2016 ；邓宁等人， 2017 ）。比较研究表明， A. semialata向纯 C ₄生理学的转变是由于相对较少的核心 C ₄酶的过度表达（Dunning等人， 2019a ）以及 C ₄样形态特征的获得，特别是存在小静脉（Lundgren等人， 2019 ）。 C ₃ +C ₄植物的δ ¹³ C范围从弱（或不存在）C ₄循环的特征值到显示C ₄循环占碳获取的一半以上的值（Von Caemmerer， 1992 ； Lundgren等人， 2015 ；Stata等人， 2019 ；Olofsson等人， 2021 ）。此外，C ₃ +C ₄中间体中 C ₄循环的加强（使用 δ ¹³ C 测量）与许多与内束鞘（IBS）组织的优势相关的叶片解剖特征的改变显着相关。该物种中 C ₄循环的细胞位置 (Alenazi et al ., 2023 )，包括连续束鞘之间的距离、IBS 细胞的宽度以及叶片中束鞘组织的比例 (Alenazi et al ., 2023 ) ）。

因此， Alloteropsis semialata代表了鉴定与 C ₄循环加强相关的基因的理想系统。在这里，我们首先使用代表 C ₃ 、C ₃ +C ₄和 C ₄表型的 420 名个体的基因组数据进行全局分析，以确定与 C ₄循环强度 (δ ¹³ C) 相关的候选基因。然后，我们特别关注 C ₃ +C ₄中间体，以确定与从弱 C 4 循环到强 C ₄循环过渡期间束鞘组织相对扩张相关的候选基因。 A. semialata的高水平种间变异允许对 C ₄进化的遗传基础进行精细的了解，包括组装这一复杂性状所涉及的中间步骤。这对于确定该性状出现所需的初始变化至关重要，最终可能在水稻等 C ₃作物的 C ₄光合作用工程中得到应用。