Planta (2014) 239:1091–1100 DOI 10.1007/s00425-014-2038-x ORIGINAL ARTICLE Perennial peanut (Arachis glabrata Benth.) leaves contain hydroxycinnamoyl-CoA:tartaric acid hydroxycinnamoyl transferase activity and accumulate hydroxycinnamoyl-tartaric acid esters Michael L. Sullivan Received: 25 October 2013 / Accepted: 30 January 2014 / Published online: 21 February 2014 © Springer-Verlag Berlin Heidelberg (outside the USA) 2014 Abstract Many plants accumulate hydroxycinnamoyl determinants of donor and acceptor substrate specificity for esters to protect against abiotic and biotic stresses. Caffeoyl this important class of biosynthetic enzymes. An HTT gene esters in particular can be substrates for endogenous could also provide a means by genetic engineering for pro- polyphenol oxidases (PPOs). Recently, we showed that ducing caffeoyl- and other hydroxycinnamoyl-tartaric acid perennial peanut (Arachis glabrata Benth.) leaves con- esters in forage crops that lack them. tain PPO and identified one PPO substrate, caftaric acid (trans-caffeoyl-tartaric acid). Additional compounds were Keywords BAHD acyl transferase · Caftaric acid · believed to be cis- and trans-p-coumaroyl tartaric acid and Hydroxycinnamoyl-CoA:shikimic acid hydroxycinnamoyl cis- and trans-feruloyl-tartaric acid, but lack of standards transferase · Hydroxycinnamoyl-CoA:tartaric acid prevented definitive identifications. Here we characterize hydroxycinnamoyl transferase · Hydroxycinnamoyl-CoA enzymatic activities in peanut leaves to understand how caf- thiolesterase taric acid and related hydroxycinnamoyl esters are made in this species. We show that peanut leaves contain a hydrox- ycinnamoyl-CoA:tartaric acid hydroxycinnamoyl trans- Introduction ferase (HTT) activity capable of transferring p-coumaroyl, caffeoyl, and feruloyl moieties from CoA to tartaric acid Leaves of many plant species accumulate hydroxycinnamic 1 (specific activities of 11 2.8, 8 1.8, 4 0.8 pkat mg− acid derivatives, particularly hydroxycinnamoyl esters. For ± ± ± crude protein, respectively). The HTT activity was used example, arabidopsis (Arabidopsis thaliana [L.] Heynh.) to make cis- and trans-p-coumaroyl- and -feruloyl-tartaric accumulates hydroxycinnamoyl-malate esters (especially acid in vitro. These products allowed definitive identifica- sinapoyl-malate) (Lehfeldt et al. 2000); tomato (Solanum tion of the corresponding cis- and trans-hydroxycinnamoyl lycopersicum L.) (Niggeweg et al. 2004) and coffee (Cof- esters extracted from leaves. We tentatively identified fea canephora L.) (Lallemand et al. 2012) accumulate sinapoyl-tartaric acid as another major phenolic compound hydroxycinnamoyl-quinic acid esters like chlorogenic in peanut leaves that likely participates in secondary reac- acid (caffeoyl-quinic acid); and bean (Phaseolus vulgaris tions with PPO-generated quinones. These results suggest L.) and red clover (Trifolium pratense L.) accumulate hydroxycinnamoyl-tartaric acid esters are made by an acyl- hydroxycinnamoyl-malic acid (especially caffeoyl-malic transferase, possibly a BAHD family member, in perennial acid [phaselic acid]) ([Sullivan and Zeller 2012] and M. peanut. Identification of a gene encoding HTT and fur- Sullivan and F. Booker, unpublished). In vivo, these com- ther characterization of the enzyme will aid in identifying pounds presumably act to protect against abiotic (e.g. UV radiation, ozone, reactive oxygen species) and biotic (insect and pathogen) stresses (Lallemand et al. 2012; Thip- M. L. Sullivan (*) yapong et al. 2004). In particular, caffeic acid derivatives, US Dairy Forage Research Center, US Department upon damage to plant tissues, can be oxidized by endog- of Agriculture, Agricultural Research Service, 1925 Linden Drive, Madison, WI 53705, USA enous polyphenol oxidases (PPOs) to their corresponding e-mail: [email protected] caffeoyl quinones. Although the role of this PPO-mediated 1 3 1092 Planta (2014) 239:1091–1100 oxidation is not fully understood, it may be involved in behaviors, and UV absorption spectra were consistent with defense against insects and pathogens (Thipyapong et al. their being cis- and trans-p-coumaroyl- and -feruloyl-tar- 2004). In addition, it is the secondary reactions of PPO- taric acid esters (Fig. 2 and Table 1, peaks b, c, e, and f). formed quinones with cellular nucleophiles that lead to Unfortunately, lack of authentic standards did not allow the undesirable browning of fresh produce (Vamos-Vig- definitive identification of these.A n additional peak (Fig. 2 yazo 1981). In some cases, the post-harvest reactions of and Table 1, peak h) was presumed to be an o-diphenol PPO-generated quinones are useful or advantageous. For substrate for PPO due to its disappearance when incubated example, in the production of tea, PPO-mediated oxidation with extracts prepared from transgenic alfalfa expressing of phenolic compounds plays a significant role in the so- a PPO gene (Sullivan and Foster 2013). Although caftaric called fermentation process (Subramanian et al. 1999). We and others have also implicated PPO-mediated oxidation e of endogenous o-diphenolic compounds (such as caffeic 30 acid derivatives) in a reduction of post-harvest proteolysis b seen in ensiled forage crops (Lee et al. 2004; Sullivan and 20 a h Hatfield 2006). In particular, red clover leaves contain high c 1 10 f levels (5–15 mmol kg− fresh weight) of the caffeic acid d g mAbsorbance units derivatives, phaselic acid and clovamide (caffeoyl-l-DOPA 0 amide) whose oxidation by PPO results in reduced post- 4 5 6 7 8 9 10 11 12 harvest proteolysis (Sullivan and Zeller 2012). When fed as Time (min) preserved forage, crops with PPO and PPO substrates have the potential to lead to improved nitrogen utilization effi- Fig. 2 Reverse phase HPLC separation of peanut phenolics. Peaks ciency in ruminant animals, which poorly utilize degraded were detected with a photodiode array detector at 310–325 nm. Peak assignments are detailed in Table 1 forage protein (see Sullivan and Zeller (2012) for a discus- sion of this). Unfortunately, some important forage crops such as alfalfa (Medicago sativa L.) lack both PPO and Table 1 Major peaks identified in reverse phase HPLC–PDA-MS o-diphenol PPO substrates (Sullivan 2009b; Sullivan et al. analysis of peanut leaf extract 2008). a b Peak λmax (nm) [M-H]− Identity Recently, we showed that perennial peanut (Arachis glabrata Benth.), a forage crop whose haylage has shown a 327 311 trans-caftaric acid better nitrogen utilization than alfalfa haylage in several b 313 295 trans-p-coumaroyl-tartaric acid animal feeding studies (Foster et al. 2009, 2011; Romero c 308 295 cis-p-coumaroyl-tartaric acid et al. 1987), contains high levels of PPO and PPO substrates d 323 311 (?) caftaric acid isomer in its leaves (Sullivan and Foster 2013). One PPO substrate, e 327 325 trans-feruloyl-tartaric acid 1 present at approximately 1 mmol kg− fresh weight, was f 323 325 cis-feruloyl-tartaric acid definitively identified as caftaric acid (trans-caffeoyl- g 318 295 (?) p-coumaroyl-tartaric acid isomer l-tartaric acid; Fig. 1; Fig. 2 and Table 1, peak a) based on h 328 355 (?) trans-sinapoyl-tartaric acid comparison of its HPLC retention time, molecular weight, a Molecular weight of major negative ion detected and UV absorption spectrum with an authentic caftaric acid b Definitive identification of trans-caftaric acid was previously made standard. In addition to caftaric acid, we observed a series (Sullivan and Foster 2013). Definitive identifications made with data of compounds whose molecular weights, chromatographic from this study are in bold. (?) indicates a tentative identification Fig. 1 Reaction of the proposed R1 R1 OH perennial peanut hydroxy- R2 R2 HO2C cinnamoyl-CoA:tartaric acid OH CO2H hydroxycinnamoyl transferase HTT SCoA + HO2C O (HTT) and structures of reac- R3 CO2H R3 tants and products O HO O hydroxycinnamoyl-CoA L-tartaric acid hydroxycinnamoyl-L-tartaric acid p-coumaroyl: R1=H, R2=OH, R3=H caffeoyl: R1=OH, R2=OH, R3=H feruloyl: R1=OCH3, R2=OH, R3=H sinapoyl: R1=OCH3, R2=OH, R3=OCH3 1 3 Planta (2014) 239:1091–1100 1093 acid and other hydroxycinnamoyl-tartaric acid derivatives Plant material have been described in other species, particularly Vitis species (Singleton et al. 1986) and Echinacia purpurea L. Perennial peanut (Arachis glabrata, Benth., variety not (Perry et al. 2001), to our knowledge, enzymatic activities specified) was grown in a field in Beeville,T X, in partial responsible for their biosynthesis have not been described. shade. The field was planted at least 20 years prior and At least two general pathways whereby hydroxycin- was cut at least once per year. Perennial peanut plants namoyl esters are made have been described. In arabidop- with leaves and some rhizome left intact were harvested sis and other members of the Brassicacea, hydroxycin- on August 20, 2012. The material was wrapped in damp namoyl-malate esters, which serve as UV protectants, are paper towels, placed in a plastic bag and shipped overnight formed by a hydroxycinnamoyl-glucose hydroxycinnamoyl to Madison, WI, for the biochemical analyses described transferase (Grawe et al. 1992; Lehfeldt et al. 2000). In below. Upon receipt in Madison, peanut leaves were what may be a more widespread pathway in many spe- removed from stems, frozen in liquid nitrogen, and stored cies, including arabidopsis, p-coumaroyl-shikimic acid is at 80 °C until needed. − formed by the action of a BAHD family acyltransferase (D’Auria 2006), hydroxycinnamoyl-CoA:shikimic acid