Leguminosae

Colville, Louise, Blanco Sáez, Clara M., Lewis, Gwilym P. & Kranner, Ilse, 2015, The distribution of glutathione and homoglutathione in leaf, root and seed tissue of 73 species across the three sub-families of the Leguminosae, Phytochemistry 115 (1), pp. 175-183 : 179-181

2.3. Occurrence of homoglutathione within the Leguminosae family

No hGSH was detected in the Caesalpinioideae subfamily, and in the Mimosoideae subfamily hGSH was detected only in the roots of Parkia biglobosa (Jacq.) G.Don and leaves and roots of Faidherbia albida (Delile) A.Chev. hGSH was found mainly in the Papilionoideae subfamily in species belonging to the tribes Millettieae , Phaseoleae , Desmodieae , Psoraleae, Loteae , Robinieae , Galegeae , Hedysareae and Trifolieae (Table S3). These tribes are found mainly within just four clades: Millettioid sensu lato, Robinioid, Galegeae sensu lato and Vicioid ( Fig. 1 ), although in roots hGSH was also detected in the Mirbelioid sensu lato clade. All of these clades belong to the Old World Clade, which suggests that a gene duplication event that gave rise to hGSH synthetases likely occurred after divergence of the Old World Clade. A whole genome duplication event is postulated to have occurred within the Leguminosae immediately prior to the radiation in the Papilionoideae subfamily that yielded the Mirbelioid, Millettioid and Hologalegina clades ( Shoemaker et al., 2006; Soltis et al., 2009), and this coincides with the appearance of hGSH. However, not all species within the Old World Clade contain hGSH, and even within the same tribe there is variation in the relative abundance of hGSH and GSH. For example, four species within the Phaseoleae tribe were sampled, and hGSH was the most abundant LMW thiol in roots of Galactia striata (Jacq.) Urb. and Leptospron adenanthum (G.Mey.) A.Delgado , was present at similar levels to GSH in roots of Centrosema pubsecens Benth. , whereas it was completely absent from roots of Rhynchosia minima (L.) DC. Similarly, hGSH was absent from roots of Glycyrrhiza echinata L. and Oxytropis sericea Nutt. of tribe Galegeae , but comprised almost half of the total LMW thiols in Astragalus cicer L., and accounted for virtually all of the LMW thiol content in Galega officinalis L. This suggests that the ability to synthesise hGSH may have been lost in some species. Gene duplication leads to redundancy, and a brief period of relaxed selection pressure, during which time duplicate genes accumulate mutations. Duplicate genes that survive the period of relaxed selection pressure are relatively stable ( Schlueter et al., 2004). Most gene duplicates are silenced, but some are preserved especially when they confer a fitness advantage ( Lynch and Conery, 2000). The occurrence of hGSH in a couple of Mimosoid species suggests that hGSH synthesis may have arisen independently prior to divergence of the Papilionoideae subfamily, possibly due to isolated gene duplication events. The ability to synthesise hGSH may have subsequently been lost from other Mimosoid species, due to gene silencing in the absence of a fitness advantage conferred by hGSH. The retention of hGSH synthesis in numerous Papilionoid species suggests that hGSH plays a beneficial role in some species.

The selective advantage conferred by hGSH is not yet clear; it shares antioxidant functions with GSH, and plays similar roles in sulphur metabolism and heavy metal sequestration. There were no apparent relationships between the occurrence of hGSH and geographic origin of the seeds ( Fig. S4 ) or with altitude, or climatic factors e.g. minimum and maximum median temperatures, and annual rainfall ( Table S1 ), which implies that hGSH is not associated with adaptation to stressful environments.

Interestingly, 26 Papilionoideae species used in this study were also included in a survey of canavanine, a non-protein amino acid with structural similarity to arginine, which is a phytoalexin used for herbivore defence ( Bell et al., 1978). The ‘canavanine-accumulating clade’ presented by Wojciechowski et al. (2004) coincides with the clade associated with the occurrence of hGSH in this study. The abilities to synthesise canavanine and hGSH may have arisen around the same time. Similar to the distribution of hGSH observed in this study, canavanine is also fairly randomly distributed within the Papilionoideae , which is probably due to loss of the ability to synthesise canavanine in some species, but persistence in species where it confers a selective advantage ( Bell et al., 1978).

Hierarchical clustering analysis of the GSH and hGSH content as a percentage of total thiol content in seed, leaf and root tissue resulted in four clusters. The first cluster comprised species which contained mainly GSH in all three tissues, and this was the largest cluster (48 species). The second cluster comprised 8 species, which contained mainly GSH in the seeds and leaves, but mainly hGSH in the roots. The third cluster comprised 7 species, which contained mainly GSH in the seeds, and hGSH in the leaves and roots. The final cluster contained only 2 species, and both had mainly hGSH in all three tissues ( Fig. 4 ). Mapping of the clusters against the Old World Clade phylogeny did not reveal a pattern in hGSH distribution ( Fig. 5 ). However, hGSH is generally restricted to the roots in the more basal clades, suggesting that the ability to synthesise hGSH either arose in the roots and synthesis in the leaves and seeds has developed more recently, or that the ability to synthesise hGSH in roots has been more widely retained, whilst it was lost in leaves and seeds. In all hGSH-containing species hGSH was found in the roots, and none of the species sampled contained hGSH only in the leaves or seeds. This implies that hGSH has a distinct role to GSH, particularly in the roots of some legume species. GSH and hGSH synthetases were shown to be differentially regulated by nitric oxide and hormones in Lotus japonica roots ( Clemente et al., 2012). Furthermore, Pasternak et al. (2014) reported that GSH promoted cell division in Medicago sativa cv. A2, whereas hGSH was associated with differentiated cells, suggesting that GSH and hGSH play different roles in cell cycle regulation. This could explain the higher abundance of GSH in seeds where cell division is required for seedling growth following germination.

El Msehli et al. (2011) found that hGSH and GSH synthetases were differentially expressed in different regions of nodules of Medicago truncatula , and suggested that this could reflect the different roles of the two LMW thiols in nodules. The Papilionoideae subfamily has the largest proportion of nodulated species, and in this study hGSH occurrence was evenly distributed between species that are known to be nodulated and species which are either non-nodulated or not yet tested for nodulation ( Sprent, 2001; Table S1 ), which suggests that hGSH distribution in roots is unrelated to nodulation. This view is supported by Matamoros et al. (1999), who observed that nodules contained higher levels of GSH and hGSH than leaves or roots, but that the relative abundance of hGSH was unrelated to nodulation. Likewise, little difference was found in the patterns of response of hGSH and GSH to hormone treatments in nodulated and non-nodulated plants ( Clemente et al., 2012).

Figures

Fig. 1. The Leguminosae phylogeny showing the major clades which form the Caesalpinioideae, Mimosoideae and Papilionoideae subfamilies. Numbers correspond to the ‘rank’ assigned to the species used in this study (see Table 1). Based on phylogeny from Lewis et al. (2005).

Fig. 4. Hierarchical clustering analysis of glutathione and homoglutathione content in seed, leaf and root tissue of 65 species yielded four clusters when the dendrogram was cut where indicated by the dotted line. The numbers on the y-axis of the dendrogram represent the ‘rank’ assigned to each species (see Table 1). The line graphs show the patterns of glutathione and homoglutathione composition in seed, leaf and root tissue of species belonging to each cluster. Cluster 1: mainly glutathione in all three tissues (48 species); cluster 2: mainly glutathione in seeds and leaves, and mainly homoglutathione in roots (8 species); cluster 3: mainly glutathione in seeds, and mainly homoglutathione in leaves and roots (7 species); cluster 4: mainly homoglutathione in all three tissues (2 species).

Fig. 5. Distribution of glutathione and homoglutathione across the phylogeny of the Old World Clade (Papilionoideae subfamily). Numbers next to the tribe name correspond to the species ‘rank’ (see Table 1). Circles correspond to the clusters shown in Fig. 4 to which the species belongs (S: seed, L: leaf, R: root; green: glutathione, purple: homoglutathione). The asterisk indicates the possible location of a whole genome duplication event, which may have lead to the evolution of homoglutathione synthesis.