How do holoparasitic plants exploit vitamin K1?

ABSTRACT

Phylloquinone (vitamin K1) is a thylakoid-embedded electron carrier essential for photosynthesis. Paradoxically, we found that phylloquinone biosynthesis is retained in the nonphotosynthetic holoparasite Phelipanche aegyptiaca (Egyptian broomrape). The phylloquinone pathway genes are preferentially expressed during development of the invasive organ, the haustorium, and exhibit strong coexpression with redox-active proteins known to be involved in parasitism. Unlike in photoautotrophic taxa, the late pathway genes of the holoparasite lack the chloroplast-targeting sequence and their proteins are targeted to the plasma membrane instead. Plasma membrane phylloquinone may enable Phelipanche to sense changes in the redox environment during host interactions. The N-truncated isoforms are conserved in several other Orobanchaceae root holoparasites, and interestingly, in a number of closely related photoautotrophic species as well. This suggests an ancient origin of distinct phylloquinone pathways predating the evolution of parasitic plants in the Orobanchaceae. These findings represent exciting opportunities to probe plasma membrane phylloquinone function and diversification in parasitic and nonparasitic plant responses to external redox chemistry in the rhizosphere.

KEYWORDS:

• plasma membrane electron transport
• phylloquinone
• holoparasite
• redox signaling
• haustorium

Holoparasitic plants have evolved a minimalist lifestyle and depend entirely on their hosts to survive and complete their life cycle. This evolutionary shift is marked by greatly reduced seed reserve size, by loss of photosynthetic machinery, and by possession of a specialized invasive organ called haustorium.^1,2 The multicellular haustoria have functions ranging from host penetration to host vascular connection for nutrient and water acquisition.³ While great strides have been made in understanding the sensing and perception of host-derived signals by parasitic seeds for germination and haustorium initiation, the redox machinery underpinning these processes remains poorly studied. We recently described a novel phylloquinone (PhQ, vitamin K1) biosynthetic pathway in Phelipanche aegyptiaca (Egyptian broomrape) and proposed its participation in plasma membrane (PM) redox signaling and haustorium development.⁴

The discovery of the full complement of PhQ biosynthetic genes in a nonphotosynthetic holoparasite may seem counterintuitive at first, given the well-known role of PhQ as a redox cofactor in photosystem I electron transport and oxidative folding of photosystem II subunits.⁵ However, our finding of an alternative, PM-targeted PhQ pathway in broomrapes⁴ is consistent with the long suggested involvement of PhQ in the PM electron transport chain of heterotrophic plant tissues.^6–10 A PM-localized electron transport chain is also central to xenognosis of parasitic plants, the signaling organogenesis process that couples the vegetative-to-parasitic phase transition with host-derived chemical cues.¹¹ According to the electrochemical framework for xenognosis, host-derived haustorium-inducing factors (HIFs) are perceived by PM-bound electron carriers to initiate redox signaling essential for haustorium formation.¹¹ In Striga asiatica, haustorium signaling only commences within a narrow range of redox potentials and shares similar characteristics with organellar redox circuits such as photosynthesis and oxidative phosphorylation.^11,12 This led Keyes and colleagues 11 to speculate on organellar involvement in the redox relay of haustorium signaling. Our finding that holoparasitic P. aegyptiaca repurposed PhQ from defunct photosynthetic electron transport for PM redox systems represents a parsimonious explanation of the model.⁴

Gene network analysis revealed strong coexpression of P. aegyptiaca PhQ genes with components of the PM electron transport chain, including NAD(P)H quinone reductase (PaQR2) and apoplastic peroxidases (PaPRX1 and PaPRX2) known to be associated with parasitism. The apoplastic PRXs have established roles in reactive oxygen species (ROS) generation for haustorium development.^13,14 PaPRXs are also among the most abundantly expressed genes in early stages of broomrape development, especially during haustorium penetration to host roots.^4,15 QR2 expression is highly induced by the common HIF 2,6-dimethoxy1,4-benzoquinones (DMBQ) in several hemiparasites, including Triphysaria versicolor, S. asiatica, S. hermonthica, and Phtheirospermum japonicum,^16–18 and its silencing by RNAi reduced haustorium formation in Phtheirospermum japonicum.¹⁶ Broomrapes do not respond to DMBQ,¹⁹ but PaQR2 was also strongly induced upon exposure to host roots during haustorium initiation.⁴ QR2 is evolutionarily conserved, with PM association^20,21 and menadione (vitamin K3) reduction activities that can be competitively inhibited by dicumarol, a vitamin K antagonist.²² The data suggest that QR2 may have roles both as a penultimate enzyme in PhQ biosynthesis, and in PM redox cycling and ROS detoxification.⁴

The haustorium induction chemistry of DMBQ-insensitive broomrapes is poorly understood.²³ Cytokinins have been shown to induce haustorium formation, and cytokinin perception was positively correlated with parasite aggressiveness in P. ramose.²⁴ Two cytokinin marker genes PrCKX2 and PrCKX4 encoding cytokinin oxidases were specifically induced upon exposure to cytokinins or host root exudates during haustorium initiation.²⁴ We identified a single ortholog PaCKX2 in the PhQ-centered gene network for P. aegyptiaca.⁴ PaCKX2 exhibited strong coexpression with four of the seven PhQ pathway genes, whereas PhQ gene coexpression was much weaker for CKX2/4 orthologs in DMBQ-responsive hemiparasites S. hermonthica (three of nine PhQ genes) and T. versicolor (one of ten PhQ genes). The patterns are consistent with what we observed for parasitism genes and redox genes involved in transmembrane electron transport.⁴ PaCKX2 thus provides another line of support for the coupling of PhQ biosynthesis, haustorium signaling, and PM redox systems in P. aegyptiaca.

The PhQ biosynthetic pathway is compartmentalized in different organelles (Figure 1). The early (steps 1–4) and intermediate (steps 5–7) pathway steps occur in chloroplasts and peroxisomes, respectively, with the late steps determining the ultimate targeting of PhQ. The chloroplast envelope, plastoglobule, and thylakoid membrane localization of the late pathway enzymes MenA1, NDC1 and MenG1, respectively,^25–27 completes the canonical PhQ biosynthesis in chloroplasts in support of its photosynthetic role (Figure 1a). The alternative pathway configuration of P. aegyptiaca PhQ biosynthesis is mediated by PM enzymes, PaMenA2, PaQR2, and PaMenG2⁴ (Figure 1a). PaMenA2 and PaMenG2 have shorter N-termini lacking the chloroplast transit signal when compared to their plastidial counterparts. A search of the One Thousand Plant Transcriptomes (1KP) database²⁸ revealed two other Orobanchaceae holoparasites, Aphyllon (syn. Orobanche) fasciculata and Conopholis americana which harbor the alternative MenA2 and MenG2 but not the canonical MenA1 and MenG1 (Figure 1b). We also identified distinct genes encoding putative plastidial (MenA1/G1) and PM (MenA2/G2) isoforms in the draft genome assembly of hemiparasite Phtheirospermum japonicum²⁹ and in the 1KP transcriptomes of nonparasitic Lindenbergia philippensis, Rehmannia glutinosa, and Kigelia africana(Figure 1b,d).⁴ Additional parasitic and nonparasitic taxa – all belonging to Lamiales – contain both MenG1 and MenG2 (T. versicolor, S. hermonthica, and Verbascum thapsus) (Figure 1b, e). These data are consistent with the notion of a PM PhQ pathway that predates the origin of the parasitic plants (Figure 1b). Gene duplication of MenA and MenG in the common ancestor of Lamiales likely gave rise to the two distinct isoforms, followed by differential gene losses in photosynthetic and nonphotosynthetic lineages⁴ (Figure 1).

Figure 1. Evolution of phylloquinone biosynthetic pathways

A, Phylloquinone (PhQ) biosynthetic pathway. B-F, A simplified angiosperm phylogeny with representative branches(B)and the corresponding phylloquinone (PhQ) pathway configurations [C-F) adapted from 4. Example genera in B are color-coded for photosynthetic nonparasitic taxa (green], photosynthetic hemiparasites (light green) and nonphotosynthetic holoparasites (purple). Pathway steps in C-F correspond to A, with late pathway enzymes MenA and MenG abbreviated as A1/A2 and G1/G2, respectively. Circle colors denote different subcellular compartments: green, plastid; orange, peroxisome (px); purple, plasma membrane (PM). Illustrative pathway configurations from top to bottom: exclusively plastidial late steps in most taxa (C), dual plastidial and PM localization (D) following lamiale-specific duplication of MenA and MenG (denoted by a red asterisk in the phylogeny), retention of the plastidial pathway in some Orobanchaceae hemiparasites (E) or exclusively PM targeting in holoparasites (F). ICS, isochorismate synthase; MenD, 2-succinyl-5-enolpyruvyl-6-hydroxy-3-cyclohexene-1-carboxylate synthase; MenH, 2-succinyl-6-hydroxy-2,4-cyclohexadiene-1-carboxylate synthase; MenC, o-succinylbenzoate synthase; MenE, o-succinylbenzoyl-CoA ligase; MenB, 1,4-dihydroxy-2-naphthoyl-CoA synthase; DHNAT, 1,4-dihydroxy-2-naphthoyl-CoA thioesterase; MenA, 1,4-dihydroxy-2-naphthoate phytyltransferase; NDC1, NADPH dehydrogenase C1; QR2, quinone reductase2; MenG, demethylphylloquinone methyltransferase.

The taxonomically restricted occurrence of MenA2 and MenG2 raises questions about the biosynthetic origin of PhQ previously associated with PM electron transport in heterotrophic tissues of non-Lamiales.^6,9,30,31 We found evidence of alternative splicing for both AtMenA and AtMenG in Arabidopsis thaliana,³² with several isoforms predicted to lack the plastid-targeting signal. We suggest that alternative splicing can afford flexible PhQ biosynthesis for PM redox functions in species without MenA2 and MenG2. One such example can be inferred from recent studies with a group of PM-localized leucine-rich-repeat receptor kinases (Hydrogen Peroxide-induced Ca²⁺ Increases [HPCA] or Cannot Respond to DMBQ [CARD]) that function as sensors for ROS as well as DMBQ to induce Ca²⁺ influx in roots.^33,34 This ROS/quinone signaling pathway is involved in pathogen defense of A. thaliana and haustorium formation of Phtheirospermum japonicum, and depends on redox activation of CARD via disulfide bond formation between two cysteine residues in the extracellular sensing domain.^33,34 AtCARD1 exhibits higher substrate affinities for menadione and 1,4-naphthoquinone than DMBQ, and its mutation can be rescued by Phtheirospermum japonicum and S. asiatica CARD orthologs.³³ The work supports a conserved ROS/quinone signaling mechanism predating the origin of parasitism. We suggest that PM-localized PhQ may function as a redox cofactor in disulfide bond formation of CARDs, like plastidial PhQ involvement in disulfide bond formation of photosystem II subunits.⁴

Besides pathogen defense, PhQ may participate in PM-mediated ROS sensing, signaling and processing in response to environmental stimuli, such as ozone, heavy metals, and water and temperature stresses.³⁵ Supporting evidence can indeed be gleaned from the PhQ-centered gene network modeling of parasitic plants.⁴ In eudicot roots, ferric reductase oxidases (FRO) of the flavocytochrome b family mediate electron transfer across the PM associated with iron and copper reduction for uptake from soils.^36,37 The PhQ-coexpressed PaFRO1 is orthologous to A. thaliana AtFRO4/AtFRO5 tandem duplicates that are essential for Cu uptake in root tips.³⁶ The A. thaliana QR2 (AtFQR1) exhibits root-biased expression and is highly responsive to auxin and allelochemicals.^38,39 These data point to root PM redox systems associated with biotic and abiotic interactions as potential avenues for research into PM PhQ function in nonparasitic plants.

In summary, adaptation of an extant PhQ pathway for PM redox signaling associated with haustorium development adds to documented evolutionary innovation of parasitic plants. Our work 4offers a repertoire of candidate genes for hypothesis-driven research to ascertain their roles in transmembrane redox processes associated with parasitism. A better understanding of haustorium signaling is of significance to both basic and applied research for effective control of noxious weeds that cause devastating crop losses around the world. Conservation of the alternative PhQ biosynthetic pathway in nonparasitic angiosperms should motivate future research into the PM function of PhQ in photoautotrophic species.

Acknowledgments

THE authors thank James Westwood and colleagues for parasitic plant transcriptome resources funded by National Science Foundation (OS-1238057), other collaborators who contributed to the original study, and Scott Harding for helpful discussion.

Disclosure statement

No potential conflicts of interest were disclosed.

Additional information

Funding

This work was supported in part by the Georgia Research Alliance-Hank Haynes Forest Biotechnology Endowment (C.-J.T.), University of Georgia Graduate School’s Innovative and Interdisciplinary Research Grant (X.G.), and the Taiwan Government Scholarship to Study Abroad (I.-G.C.).