Comparative transcriptomics provide insight into the morphogenesis and evolution of fistular leaves in Allium

As one of the largest genera of the petaloid monocotyledons, Allium (Amaryllidaceae) comprises more than 920 species [1] and includes several economically important crops that are cultivated for consumption or medicinal uses, such as garlic (A. sativum), welsh onion (A. fistulosum), leek (A. porrum), Chinese chives (A. tuberosum), onion (A. cepa), Chinese jiaotou (A. chinense), and shallot (A. ascalonicum). Varied leaf shapes can be observed among Allium species, including flat, columnar, solid, and fistular morphologies. Morphological and cellular studies have found that fistular leaves develop from solid precursors [2]. Developmental investigation of the leaves of A. fistulosum found that the process of fistular leaf formation involved programmed cell death (PCD) [2]. PCD is a spontaneous, programmed, self-destructive cellular process that plays a key role in tissue differentiation, homeostasis, and organ morphogenesis, including that of leaves [3–5]. However, molecular evidence for the involvement of PCD in the formation of fistular leaf cavities is still absent in Allium, owing to the limited availability of genetic resources for Allium spp.

The paucity of genetic resources in Allium is, in part, due to the fact that Allium spp. have the largest genomes among eukaryotes [6, 7], with genome sizes ranging from 6860 to 30,870 Mbp per C [8]. The enormous size of these genomes has been a major obstacle for their characterization and for gene mining in the family as a whole. In the past 10 years, the next generation sequencing technologies have undergone rapid development, and more than 80 plant species have had their complete genome [9]. However, none of genome of Allium species has been characterized, because of their enormous size.

Because transcriptome analysis by next generation sequencing is rapid, inexpensive, and unconstrained by genomic complexity, it has been widely used as a primary tool for gene discovery and expression profiling in hundreds of plant species [10, 11]. Moreover, transcriptome analysis can also be used as an important tool for investigating the domesticated patterns of crops [12, 13], as well as for investigating the mechanisms of development for specific traits [14]. In Allium, the transcriptome of four species, i.e. garlic [15, 16], onion [17], Chinese chive [18], and welsh onion [19], have been sequenced and de novo assembled. Among these, onion and Welsh onion are fistular-leaved species, whereas the other two produce solid leaves. However, despite the large numbers of generated expressed sequence tags, the molecular mechanism for the formation of fistular leave cavities is still uncharacterized.

Therefore, we analyzed the transcriptomes of nine economically important Allium species, including eight vegetable species and one herbal species (A. macrostemon). Thereafter, genes that had undergone significant selection were identified in both fistular- and solid-leaf species, respectively, and the genes with evolutionary divergence between fistular- and solid-leaf species were screened. As a result, the current study presents molecular evidence regarding a potential evolutionary mechanism for the development of fistular leaves.

Although Allium comprises a large number of species and even includes a few species of significant economic importance, the transcriptomes of only four species (i.e. garlic, onion, welsh onion, and Chinese chive) have been analyzed [15–19]. Therefore, in the present study, the transcriptomes of nine Allium species were sequenced and assembled de novo, and large numbers of genes were identified in each species. These results will help us investigate the major genes responsible for important agronomic traits in Allium species, and will also further our understanding of the mechanisms underlying the formation and development of such traits, including leaf morphology.

Leaves are the primary site of plant photosynthesis and respiration. Diverse leaf forms can be observed in angiosperms, and they can be categorized into two major categories: bifacial or unifacial. Development studies have proposed that the diversity of leaf morphology is caused by differential and asymmetrical growth along the adaxial–abaxial, central–marginal, and proximal–distal polarities [32–34]. Allium spp. exhibit a wide variety of leaf shapes, including cylindrical and hollow, flat and solid, and hollow and triangular types, the differences of which can be observed in transverse sections [35]. As in other angiosperms, there is a great possibility that the diversity of leaf appearance in Allium is caused by these three polarities. For example, only the abaxial identity existed in leaf blades results in a unifacial fistular leaf in Welsh onion [33, 34] However, the formation of fistular leaf cavities is still poorly understood. Although that a cellular investigation of the leaves of A. fistulosum found that the process of fistular leaf formation involved PCD [2], molecular evidences for the involvement of PCD in the formation of fistular leaf cavities is still unknown in Allium.

Previous studies have shown that mitochondria play a central role in controlling PCD in plants [30], by releasing molecules, including cytochrome c that drive the destruction of the cell [36]. In animals, cytochrome c triggers PCD by forming a complex that induces the formation of apoptosomes, and those apoptosomes then activate downstream effector caspases, which are key regulators of PCD [30]. Although no caspase homologs have been found in plants, caspase-like proteins have been described in cells undergoing PCD, such as the cysteine endopeptidases, which can be subdivided into two groups: vacuolar processing enzymes and metacaspases [37, 38]. In the present study, we identified 15 positively selected genes from five orthologous groups that were potentially related to the metabolism of cytochrome c and were found to undergo positive selection. Among these 15 genes, 12 were subjected positive selection in fistular-leaved species, except 3 genes from OG13835 that underwent positive selection in the species with solid or small-gapped fistular leaves. In addition, a KDEL-tailed cysteine endopeptidase-encoding gene from the fistular-leaved ASC exhibited rapid evolution, and a metacaspase-4-like protein-encoding gene was found to undergo positive selection in solid-leaved species, but evolved neutrally in fistular-leaved species.

Like the mitochondria, the chloroplasts are also important in triggering PCD, by mediating reactive oxygen species signaling. In plants, photosynthesis is performed in photosystem II of chloroplasts. When photosynthesis is less efficient, or chloroplasts receive excessive levels of light, chloroplasts produce more reactive oxygen species, and then the reactive oxygen species drives cell death finally [30, 39, 40]. In the present study, we identified five chloroplast genes that are potentially involved in photosynthesis in photosystem II and exhibited rapid diversification. Among them, four of the genes were from fistular-leaved species, and one was from a flat, solid-leaved species (i.e., SAT). In addition, two photosystem II protein-encoding genes were also found to have undergone purfying selection in solid-leaved species, whereas they were evolving neutrally in fistular-leaved species.

After the “death signal” is generated by mitochondria and/or chloroplasts, it is transduced by kinase proteins. Previous studies have shown that PCD signaling involves the activation of mitogen-activated protein kinases that induce hypersensitive responses and cell apoptosis [41]. Recently, the phosphatidyl-3 kinases ATM and ATR were reported as the main sensors of DNA damage-induced PCD [42, 43]. In the present study, the ATM and ATR kinase-encoding genes in the fistular-leaved species FIS, CEP, and CHI exhibited evidence of rapid diversification, but not in solid-leaved species, and mitogen-activated protein kinase-encoding gene underwent positive selection in solid-leaved species, whereas it was evolving neutrally in fistular-leaved species.

In addition, two genes, LSD-one-like protein 2 (LOL2) and constitutively activated cell death 1 (CAD1), have also been reported to play important roles in regulating PCD in Arabidopsis [44, 45]. In the present study, the orthologs of these two genes were also found to exhibit different selective patterns among the fistular- and solid-leaved species. These potential PCD-related genes with evolutionary divergence might have roles in regulating the formation of cavity of fistular leaf in Allium species.

In this study, we compared the leaf transcriptomes of six fistular- and three solid-leaf species of Allium. The selective patterns of orthologous genes in nine species were analyzed, and the result showed that many mitochondrial, chloroplast and nuclear genes that were potentially involved in plant PCD and were either subject to positive selection in fistular-leaved species but not in solid-leaved species, or evolved neutrally in fistular-leaved species but purifying selection in solid-leaved species. These potential PCD-related genes might play roles in the formation of the inner cavities of fistular leaves in Allium, and the differing selection patterns in fistular- and solid-leaved species may be responsible for the evolution of fistular leaves.