Evidence That Chlorinated Auxin Is Restricted to the Fabaceae But Not to the Fabeae

Abstract

Auxin is a pivotal plant hormone, usually occurring in the form of indole-3-acetic acid (IAA). However, in maturing pea (Pisum sativum) seeds, the level of the chlorinated auxin, 4-chloroindole-3-acetic acid (4-Cl-IAA), greatly exceeds that of IAA. A key issue is how plants produce halogenated compounds such as 4-Cl-IAA. To better understand this topic, we investigated the distribution of the chlorinated auxin. We show for the first time, to our knowledge, that 4-Cl-IAA is found in the seeds of Medicago truncatula, Melilotus indicus, and three species of Trifolium. Furthermore, we found no evidence that Pinus spp. synthesize 4-Cl-IAA in seeds, contrary to a previous report. The evidence indicates a single evolutionary origin of 4-Cl-IAA synthesis in the Fabaceae, which may provide an ideal model system to further investigate the action and activity of halogenating enzymes in plants.The chlorinated form of auxin, 4-chloroindole-3-acetic acid (4-Cl-IAA), is a highly active hormone that is thought to play a key role in early pericarp growth (Reinecke et al., 1995Ozga et al., 2009). Exogenous 4-Cl-IAA, for example, has been shown to promote the pericarp elongation of deseeded pea (Pisum sativum) pods . Johnstone et al. (2005) reported that 4-Cl-IAA and bioactive GA (GA 3 or GA 1 ) act synergistically on pericarp growth when applied simultaneously, and a growth regulatory role has been proposed for 4-Cl-IAA through induction of GA biosynthesis and inhibition of ethylene action. In other species, e.g. tomato (Solanum lycopersicum), the nonchlorinated form of auxin, indole-3-acetic acid (IAA), also stimulates fruit growth via GAs (Serrani et al., 2008;Tang et al., 2015). The chlorinated auxin is mainly found in reproductive structures (Katayama et al., 1988), in which its levels often exceed those of the more widespread IAA (Tivendale et al., 2012). The chlorinated form is thought to be restricted to members of the leguminous tribe Fabeae (Reinecke 1999), which includes the genera Vicia, Pisum, Lathyrus, Lens, and Vavilovia (Schaefer et al., 2012). However, there is a curious exception: 4-Cl-IAA has been reported also from Scots pine (Pinus sylvestris; Ernstsen and Sandberg, 1986).We previously published evidence that most 4-Cl-IAA in maturing pea seeds is synthesized from 4-Cltryptophan (4-Cl-Trp) via 4-Cl-indole-3-pyruvic acid (Tivendale et al., 2012(Tivendale et al., , 2014. 4-Cl-Trp has been identified in extracts from pea and broad bean (Vicia faba) seeds (Kettner et al., 1992;Manabe et al., 1999), but whether the precursors of Trp can be chlorinated is unknown.Virtually nothing is known about the enzymes that catalyze halogenation reactions in plants. In bacteria, fungi, and marine algae, there are six types of enzymes responsible for the addition of halogen atoms to organic molecules. These include heme haloperoxidases, vanadium-dependent haloperoxidases, mononuclear nonheme iron halogenases, flavin-dependent halogenases, S-adenosyl-L-Met-dependent chlorinases and fluorinases, and methyl halide transferases (But...

Chlorinated Auxins—How Does Arabidopsis Thaliana Deal with Them?

“…There are different studies about the occurrence and endogenous concentration of 4-Cl-IAA in other plant species. Pless et al [15] found the auxin in fully developed leaves of broad beans in high concentrations, whereas in recent studies only small concentrations could be detected, e.g., in young and ripe seeds of beans, respectively [16]. Chlorinated IAA has also been described in other leguminous plants, such as Medicago truncatula and Trifolium repens [16].…”

The single evolutionary origin of chlorinated auxin provides a phylogenetically informative trait in the Fabaceae

“…Parochetus) tribes we analyzed dry seed extracts from the perennial species C. echinospermum (a wild relative of C. aeritinum), Galega officinalis, Parochetus communis and 2 additional species within the Astragalean clade (Astragalus sinicus and A. propinquus) using base hydrolysis (to hydrolyse auxin conjugates) and UPLC-MS/MS as described previously. 6 Chlorinated auxin was not detected in any of these species (Table 1), although all contained IAA, with the level in P. communis being remarkably high (Table 1).…”

“…Our recent discovery of 4-Cl-IAA in the seeds of Medicago truncatula, Melilotus indicus and 3 Trifolium species has pushed the number of recognized 4-Cl-IAA-producing leguminous species to 14 and extended our detection of this hormone beyond species of the tribe Fabeae, to which this hormone was previously thought to be restricted. 6 It is surprising that the ability of these species to produce chlorinated auxin had remained unrecognised, given that clovers (Trifolium species) have a long cultivation history due to their agricultural significance, especially in temperate regions as forage crops 7 ; and that Medicago truncatula is a model species with a sequenced genome. While the phylogenetic relationships of taxa within the Fabaceae have been somewhat enigmatic, the reconstruction of a phylogeny based on the analysis of the plastid MATK gene resolves many well-supported subclades within the legume family.…”

“…Parochetus) tribes. 6 However, there are a number of genera that are closely related to the Fabeae and Trifolieae, according to modern phylogenic relationships in the Fabaceae. 8,9 The closest of these is the genus Galega which is either sister to Cicer or immediately basal to Cicer (Fig.…”

“…Parochetus) tribes, chlorination capacity evolved once, approximately 25 million years ago. 6 Determination of the precise evolutionary origin of 4-Cl-IAA in our previous study was based on the inability to detect this hormone in a single cultivated species from the Cicerae tribe (Cicer aeritinum), which we took as a sole representative of the clade immediately basal to the common ancestor of the Fabeae and Trifolieae (ex. Parochetus) tribes.…”