Dual plastid targeting of protoporphyrinogen oxidase 2 in Amaranthaceae promotes herbicide tolerance

Abstract

Plant tetrapyrrole biosynthesis (TPB) takes place in plastids and provides the chlorophyll and heme required for photosynthesis and many redox processes throughout plant development. TPB is strictly regulated, since accumulation of several intermediates causes photodynamic damage and cell death. Protoporphyrinogen oxidase (PPO) catalyzes the last common step before TPB diverges into chlorophyll and heme branches. Land plants possess two PPO isoforms. PPO1 is encoded as a precursor protein with a transit peptide, but in most dicotyledonous plants PPO2 does not possess a cleavable N-terminal extension. Arabidopsis (Arabidopsis thaliana) PPO1 and PPO2 localize in chloroplast thylakoids and envelope membranes, respectively. Interestingly, PPO2 proteins in Amaranthaceae contain an N-terminal extension that mediates their import into chloroplasts. Here, we present multiple lines of evidence for dual targeting of PPO2 to thylakoid and envelope membranes in this clade and demonstrate that PPO2 is not found in mitochondria. Transcript analyses revealed that dual targeting in chloroplasts involves the use of two transcription start sites and initiation of translation at different AUG codons. Among eudicots, the parallel accumulation of PPO1 and PPO2 in thylakoid membranes is specific for the Amaranthaceae and underlies PPO2-based herbicide resistance in Amaranthus species.

The intraplastidal distribution of protoporphyrinogen oxidase (PPO) 2 of Amaranthaceae is unique among dicotyledonous plants and enables resistance against PPO-inhibiting herbicides.

Introduction

Tetrapyrrole biosynthesis (TPB) is a vital metabolic pathway, which in plants requires at least 25 enzymatic reactions and produces chlorophylls, as well as heme, siroheme, and phytochromobilin (Tanaka and Tanaka 2007; Wang et al. 2022). While chlorophyll is the major end-product of TPB in green plant tissues, the demand for other tetrapyrroles varies, depending on plant tissue type and developmental stage (Mochizuki et al. 2010; Terry and Smith 2013; Woodson et al. 2015; Kobayashi and Masuda 2016). Protoporphyrinogen oxidase (PPO) catalyzes the conversion of protoporphyrinogen IX (Protogen) to protoporphyrin IX (Proto), the last common intermediate in the pathway before TPB diverges into separate chlorophyll- and heme-synthesizing branches. In plants, TPB takes place exclusively in plastids (Hedtke et al. 2023). While all catalytic steps up to the conversion of Protogen are performed by soluble proteins in the plastid stroma, PPOs and all subsequent enzymes are associated with chloroplast membranes (Joyard et al. 2009). All eukaryotic PPOs belong to the HemY family of 55 (kDa) oxygenic, FAD-containing enzymes (Koch et al. 2004). Within the plant kingdom, all embryophyte species encode two differing PPO isoforms, and phylogenetic analyses have pointed to a corresponding gene duplication event early in the evolution of land plants (Kobayashi et al. 2014). Consequently, the two isoforms now share only 25% amino acid sequence identity (Lermontova et al. 1997). Plant PPO1 is translocated into plastids via a cleavable chloroplast transit peptide (cTP) located at the N-terminus of the protein and accumulates mainly on thylakoid membranes (Lermontova et al. 1997). In both Arabidopsis (Arabidopsis thaliana) and tobacco (Nicotiana tabacum), insufficient amounts of PPO1 result in severe photodynamic damage and cell death owing to the accumulation of Protogen, which is nonenzymatically oxidized to Proto in the presence of light. Consequently, ppo1 knock-out mutants are seedling-lethal in A. thaliana (Zhang et al. 2014).

In N. tabacum, PPO2 was initially reported to accumulate in mitochondria, and this gave rise to the idea that these organelles possess a separate system for heme synthesis (Lermontova et al. 1997). However, recent analyses of the similarly structured PPO2 of Arabidopsis have refuted this suggestion and demonstrated that the enzyme is exclusively targeted to plastid envelopes (Hedtke et al. 2023). Proteomic data support the localization of PPO2 to the chloroplast envelope in A. thaliana and indicate that its uptake involves a noncanonical import mechanism that does not depend on a cleavable cTP. The distinct localizations of the two PPO isoforms within Arabidopsis plastids result in the spatial separation of TPB within the organelle—prior to the divergence of the chlorophyll- and heme-synthesizing branches—and was suggested to facilitate the co-ordinated provision of tetrapyrroles (Hedtke et al. 2023).

However, since ppo2 knock-out mutants of A. thaliana do not display a visible phenotype, PPO1 appeared to substitute for PPO2 function in Arabidopsis. This differs from recent findings in Palmer amaranth (Amaranthus palmeri), in which a double mutation of PPO2 that reduces activity to 0.12% of wild-type (WT) levels was described as lethal based on genetic analyses (Porri et al. 2022). Intriguingly, PPO2 coding sequences in the Amaranthaceae family of eudicots, which includes weed plants of the genus Amaranthus and crops such as sugar beet (Beta vulgaris), spinach (Spinacia oleraceae), and quinoa (Chenopodium quinoa), share a characteristic amino-terminal extension. Moreover, this N-terminal stretch of about 30 amino acids includes two potential in-frame start codons, which have been reported previously to mediate chloroplast and mitochondrial targeting upon initiation at the first and second AUG triplets, respectively (Watanabe et al. 2001).

Furthermore, this N-terminal structure may be related to numerous reports of PPO2-based herbicide tolerance in diverse Amaranthus weed species (Dayan et al. 2018). These herbicides are based on substrate analogs that act by inhibiting binding of Protogen to PPO enzymes. This leads to rapid accumulation of the substrate, which is nonspecifically oxidized to Proto. In the presence of light, Proto generates singlet oxygen, which ultimately results in cell death, primarily owing to lipid peroxidation. Tolerance to these PPO-inhibiting herbicides has so far been reported for 13 weed species, including four Amaranthus species (Barker et al. 2023). Mutations in PPO2 sequences have been identified as the main resistance mechanism that reverses herbicide-mediated inhibition of PPO enzymes (Porri et al. 2022). Thus, our recent study of organellar targeting of PPO2 in Arabidopsis encouraged us to re-evaluate the localization of this isoform in species of Amaranthaceae. We used spinach as well as Palmer amaranth to assess the distribution and expression of the PPO2 isoform in WT plants, as well as in transgenic Arabidopsis lines overexpressing different spinach or amaranth PPO2 variants.

Results

PPO2 is not associated with mitochondria in Amaranthaceae

As in most dicots, PPO2 genes in Arabidopsis and tobacco lack any obvious transit sequence. However, comparison of the primary structures of PPO2 enzymes in angiosperms reveals the presence of extended N-terminal sequences in the Amaranthaceae, a family of dicotyledonous plants that includes spinach and amaranth. Moreover, these N-terminal sequences include not just one, but two potential translation initiation codons (Fig. 1A). Moreover, PPO2 genes in monocotyledonous plants, such as maize (Zea mays) and rice (Oryza sativa), also encode an extended N-terminal segment (Fig. 1A). Indeed, both maize and rice also share the presence of a second in-frame AUG codon, which would give rise to a shorter N-terminal stretch similar to that recently found in Arabidopsis (Hedtke et al. 2023). Since this last PPO2 protein is exclusively localized in plastid envelopes, the subcellular localization of PPO2 in selected species of Amaranthaceae was re-evaluated.

Figure 1.

PPO2 localization in organellar fractions from wild-type palmer amaranth and spinach. A) Alignment of plant PPO2 sequences. Amino termini of aligned PPO2 sequences of A. thaliana (A.t.), N. tabacum (N.t.), S. oleraceae (S.o.), A. palmeri (A.p.), Z. mays (Z.m.), and Oryza sativa (O.s.) are shown. Conserved amino acid residues are highlighted, methionine residues are boxed in red. B, C) Percoll-purified chloroplasts (Cp) of Palmer amaranth (A. palmeri, B) or spinach (S. oleraceae, C) were separated into thylakoid (Thyl) and envelope (Env) membrane fractions and subjected to SDS-PAGE. Membranes were probed with antibodies directed against amaranth PPO2, the chloroplast envelope protein DnaJ-like protein D12 (DNAJD12) and the thylakoid-localized, light-harvesting complex protein B5 (LHCB5). Samples of total protein (TP, in B), Cp and Thyl represent 20 µg of proteins, Env fractions comprised 2 µg of proteins. Protein molecular weight markers were used in all SDS-PAGE gels to enable the identification of the specific signals. Migration of a 66 kDa marker protein and the large subunit of RuBisCo (RBCL, 52 kDa) are indicated in the upper panel in (B) (open triangles). D) Comparison of PPO enzyme activities in purified Arabidopsis (Col-0) and spinach chloroplasts, and in their respective envelope fractions. Activity values are given in pmol of protoporphyrin IX (Proto) formed per min and µg of protein. Error bars indicate the standard deviations of three assay replicates. E, F) Immunoblot analyses of mitochondria purified from amaranth (E) and spinach (F). Mitochondrial and total protein (TP) samples were probed for the presence of the mitochondrial proteins manganese superoxide dismutase (MnSOD) and voltage-dependent anion channel (VDAC), and the chloroplast-proteins Mg chelatase subunit I (CHLI) and PPO2. Mitochondria were treated with increasing concentrations of proteinase K (final concentrations are given in µM) in order to differentiate internal mitochondrial proteins such as MnSOD from outer-membrane polypeptides and unspecific contaminants.

Chloroplasts were isolated from leaves of 8-week-old palmer amaranth (Fig. 1B) and spinach leaves (Fig. 1C), and subsequently enriched for envelope and thylakoid membrane fractions. Use of an antiserum raised against amaranth PPO2 (Fig. 1, B and C) revealed two immune-reacting signals of about 55 kDa in the total protein extracts (Fig. 1B) as well as in isolated chloroplasts. The smaller immunoreactive protein (with the faster mobility in SDS-PAGE) was dominant in the thylakoid membranes, while the envelope fraction displayed a specific signal that corresponded to the higher-migrating band. Enrichment of the two chloroplast membrane fractions was confirmed using antibodies directed against the known envelope protein DNAJD12 [DnaJ-like protein D12 (Pulido and Leister 2018)] and the thylakoid polypeptide LHCB5 (light-harvesting complex B5 protein of photosystem II). While it proved difficult to assess the specificity of the smaller immune-reacting band which accumulates in thylakoid membranes (see Discussion below), the presence of PPO2 in chloroplast envelope membranes of Amaranthaceae species was supported by PPO enzyme activity assays (Fig. 1D). PPO activity in chloroplast envelopes from Arabidopsis was recently shown to be specifically attributable to PPO2 (Hedtke et al. 2023). As in Arabidopsis, preparations of spinach plastid envelope membranes contained high levels of PPO activity (Fig. 1D).

To determine whether PPO2 is targeted to the mitochondria in Amaranthaceae, these organelles were purified from both amaranth and spinach (Fig. 1, E and F). Relative to total leaf protein, the mitochondrial fractions obtained were strongly enriched in manganese superoxide dismutase (MnSOD) and voltage-dependent anion channel (VDAC), which are verifiably localized in mitochondria. In contrast to these findings, PPO2-specific signals were strongly depleted in amaranth, and undetectable in spinach. Relative to total leaf samples, the level of PPO2 in mitochondrial fractions from amaranth plants is similar to that of the chloroplast protein CHLI (magnesium chelatase subunit I), which implies that the PPO2 in these samples represents a plastid-derived contaminant. This is further supported by the fact that the PPO2-specific immune signal is as susceptible to proteinase K treatment as are the chloroplast-derived protein CHLI and VDAC, which is localized in the outer mitochondrial envelope. In contrast, the mitochondrial matrix protein MnSOD was still detectable after incubation with 4- to 6-fold higher amounts of proteinase K (Fig. 1, E and F).

Both PPO2 translation initiation sites in Amaranthaceae give rise to chloroplast-localized proteins

To further evaluate the intracellular localization of spinach PPO2, the Arabidopsis ppo1 mutant was employed. In Arabidopsis, knockout of PPO1 is seedling-lethal, since endogenous expression of PPO2 is insufficient to compensate for the loss of PPO1 following de-etiolation (Zhang et al. 2014). In addition, the specific localizations of Arabidopsis PPO1 and PPO2 to different plastid membranes results in only partial complementation of ppo1 when the envelope-localized isoform PPO2 is overexpressed (Hedtke et al. 2023). Strikingly, when Arabidopsis PPO2 was artificially targeted to thylakoid membranes by employing known cTP sequences, full complementation of ppo1 was achieved (Hedtke et al. 2023).

The ability to differentiate phenotypically between distinct subplastid localizations makes the Arabidopsis ppo1 mutant an attractive tool with which to investigate the targeting of Amaranthaceae PPO2 variants. Three different overexpression constructs were generated, which contained either the full-length coding region of PPO2 (for spinach: SoPPO2), the shorter variant resulting from use of the downstream methionine residue (Met2) for initiation (SoPPO2^Δ26aa), or a “long” mutagenized PPO2 form in which only Met1 is available for initiation (SoPPO2^M27G). These variants were then expressed under the control of the constitutive cauliflower mosaic virus (CaMV) 35S promoter (p35S, Fig. 2A). Following transformation of heterozygous Arabidopsis PPO1/ppo1 mutants, numerous transgenic clones were identified using BASTA selection. The presence of SoPPO2 and the T-DNA insertion was examined in plants of the T1 generation, and homozygous ppo1 transformants were identified for both SoPPO2 and SoPPO2^M27G. Phenotypically, these transgenic lines in the homozygous ppo1 background resembled WT plants. In contrast, the lines expressing the SoPPO2^Δ26aa construct were all heterozygous for ppo1 in generation T1. However, among the T2 offspring of these SoPPO2^Δ26aa-expressing lines, about 25% of slow-growing, yellow–green individuals were detected, and their phenotypes resembled those of AtPPO2 overexpressors in the ppo1 background (Hedtke et al. 2023). The degree of complementation achieved with SoPPO2^Δ26aa was dependent on the expression level of the transgene. Only the strongest SoPPO2^Δ26aa overexpression lines gave rise to viable yellow–green plantlets that were able to flower and set seeds.

Figure 2.

Complementation of the A. thaliana ppo1 mutant with PPO2 sequences from spinach. A) Schematic representations of DNA constructs used for expression of PPO2 variants from spinach, (SoPPO2; Amaranthaceae). Arrows indicate the AUG triplets coding for Met1 and Met2 at the N-terminal end of SoPPO2, which are either deleted or mutagenized in SoPPO2^Δ26aa and SoPPO2^M27A, respectively. B) Homozygous Arabidopsis ppo1 mutants overexpressing the indicated SoPPO2 constructs under the control of the CaMV 35S promoter (left panel). For comparison, the wild-type (Col-0) and the ppo1 mutant complemented by overexpression of A. thaliana PPO2 (AtPPO2) under the control of pPPO1 (left panel, Hedtke et al. 2023) are shown. All plants were cultivated for 4 weeks under short-day conditions. The numbers depicted above the line designations were also used in the analyses shown in (C, D). The scale bar included in all images represents 1 cm. C) PCR analyses of plants depicted in B. Homozygosity of ppo1 was verified with the aid of primer combinations specific for the ppo1 T-DNA insertion (upper panel) and the PPO1 wild-type allele (middle). In addition, the presence of SoPPO2 overexpression constructs was confirmed (lower panel). Controls carried out in the absence of template (nc) and a DNA size marker (M, GeneRuler 1 kb DNA ladder, Thermo Scientific) are included. D) Immunoblot analyses of ppo1 plants complemented by SoPPO2 constructs. Specific antisera were used to determine levels of PPO1, amaranth PPO2, glutamyl tRNA reductase (GluTR), magnesium chelatase subunit I (CHLI), protochlorophyllide oxidoreductase B (PORB), and light-harvesting complex protein B1 (LHCB). Ponceau staining of the large subunit of ribulose bisphosphate carboxylase/oxygenase (RBCL) is depicted to illustrate equal loading. Total protein extracts obtained from 2 mg of leaf material were applied to each lane. E) Quantification of protoporphyrin IX (Proto) and magnesium protoporphyrin IX (MgP) by high-pressure liquid chromatography (HPLC). The values shown are each derived from three biological replicates, and standard deviations are indicated. Plant material used in (D, E) was grown for either two (lines 1, 2, and 4) or four weeks (lines 3 and 5) under short-day conditions.

Figure 2B depicts the ppo1 complementation phenotypes of T2 progenies for all three SoPPO2 gene constructs. For comparison, WT (Col-0) and partially complemented ppo1 mutants overexpressing AtPPO2 (Hedtke et al. 2023) are depicted. PCR analyses confirmed both the homozygosity of ppo1 and the presence of the expected SoPPO2 gene constructs (Fig. 2C). Immunodetection assays verified the absence of PPO1, together with the overexpression of SoPPO2 (Fig. 2D). Remarkably, in all overexpression lines, the SoPPO2 proteins detected with an amaranth-specific PPO2 antiserum showed identical mobilities in SDS-PAGE (Fig. 2D), even under extended running conditions (see also Supplementary Fig. 1 for ApPPO2). Since the calculated molecular masses of both SoPPO2 and SoPPO2^M27G (58.2 kDa) differ substantially from that of SoPPO^Δ26aa (55.5 kDa) this indicates that the N-terminal transit peptide in SoPPO2 as well as SoPPO2^M27G was successfully cleaved. Additional antibodies were used to determine the relative levels of key TPB enzymes in these lines. In partially complemented ppo1(p35S:SoPPO2^Δ26aa) plants, glutamyl tRNA reductase (GluTR) accumulates to lower levels in comparison to WT levels. Other TPB enzymes, as well as LHC proteins, are also affected, albeit to a lesser extent (Fig. 2D). In contrast, the fully complemented ppo1 mutants expressing SoPPO2 or SoPPO2^M27G did not exhibit differences in the accumulation of any of the tested proteins.

HPLC analyses of TPB intermediates and end-products revealed high levels of Proto in yellow–green ppo1(p35S:SoPPO2^Δ26aa) individuals (Fig. 2E), which were comparable to that seen in ppo1(pPPO1:AtPPO2) described earlier (Hedtke et al. 2023). The Proto values detected by HPLC include the contribution derived from Protogen, which is auto-oxidized during sample extraction. Levels of TPB intermediates downstream of the PPO enzymatic step are strongly reduced in ppo1(p35S:SoPPO2^Δ26aa) plants, as shown for magnesium protoporphyrin (MgP) in Fig. 2E. In agreement with their growth and pigmentation phenotypes, the transgenic lines ppo1(p35S:SoPPO2) and ppo1(p35S:SoPPO2^Δ27A) did not show any deviations from the steady-state levels of tetrapyrrole intermediates in WT Arabidopsis. Other intermediates of the chlorophyll branch, and chlorophyll itself, showed accumulation patterns comparable to that of MgP among the five analyzed lines (Supplementary Fig. 2).

PPO2 in Amaranthaceae is targeted to different membranes in plastids

To elucidate the bases for the different degrees of ppo1 complementation, chloroplasts were isolated from representative Arabidopsis plants overexpressing each of the three different SoPPO2 constructs. Purified chloroplasts were lysed and membranes separated into envelope- and thylakoid-enriched fractions (Fig. 3A). In agreement with the successful complementation of ppo1, all three overexpressed spinach PPO2 variants were detected in purified chloroplasts. However, immunoblot analyses revealed characteristic differences between the subplastid levels of SoPPO2^Δ26aa and those of the longer variants SoPPO2 and SoPPO2^Δ27A. While the latter two were detected in both envelope and thylakoid membrane fractions, the N-terminally truncated form is suggested to be exclusively located in the chloroplast envelope—as revealed by the respective distributions of the chloroplast envelope protein DnaJD12 and the thylakoid-localized LHCB1 (Fig. 3A).

Figure 3.

Distribution of Amaranthaceae PPO2 proteins in plastid membrane fractions. A) Immunoblot analyses of Arabidopsis lines overexpressing SoPPO2, SoPPO2^Δ26a, or SoPPO2^M27A. Chloroplasts (Cp) purified from leaf tissue were used to enrich for thylakoid (Thyl) and envelope (Env) membrane fractions. Following fractionation by SDS-PAGE, antisera directed against amaranth PPO2, DnaJD12, and LHCB1 were applied to compare protein abundances. A section of the Ponceau-stained membrane (lower panel) illustrates the levels of RBCL in purified chloroplasts (highlighted by asterisks). In the case of chloroplast and thylakoid fractions, 20-µg aliquots of protein were loaded; envelope fractions contained 2 µg of protein. B) Distribution of A. palmeri PPO2 in N. benthamiana plants transiently expressing ApPPO2, ApPPO2^Δ29aa or ApPPO2^M30A. The fractions and antisera applied are identical to those in (A), except for the light-harvesting complex protein B5 (LHCB5) used as the thylakoid reference protein in (B).

In order to rule out possible targeting artifacts of heterologously expressed SoPPO2 variants in Arabidopsis, comparable constructs based on A. palmeri PPO2 (ApPPO2) sequences were used to transiently transform leaves of the model species Nicotiana benthamiana (Solanaceae). After purification of chloroplasts and separation of plastid membranes, the N-terminally truncated form of amaranth PPO2 (ApPPO2^Δ30aa) was specifically enriched in envelope membrane fractions (Fig. 3B). In contrast, gene constructs enabling translation initiation at the first methionine residue (ApPPO2, ApPPO2^M31A) resulted in targeting of ApPPO2 to both envelope and thylakoid membranes.

Differential usage of PPO2 start codons in Amaranthaceae is regulated at the transcriptional level

Interestingly, the different Amaranthaceae PPO2 variants all displayed the same migration behavior on SDS gels, even though the SoPPO^Δ26aa construct is shorter than SoPPO2^M27A. We assume that the N-terminal part of Amaranthaceae PPO2 proteins is cleaved off in close proximity to the second methionine, most probably after import into the chloroplast. It is therefore impossible to decide, on the basis of SDS-PAGE, which of the two potential initiation codons is actually used. Since initiation at each of the two amino-terminal AUG codons results in a different distribution of PPO2 variants across the plastid membranes (Fig. 3A), with substantial functional consequences (Fig. 2B), the site of translation initiation employed turns out to be fundamental for an understanding of the control of TPB at this enzymatic step.

Selection of a specific initiation codon may result either from transcriptional regulation using different promoters or from translational control by skipping the initiation at the first AUG codon due to leaky ribosome scanning (Merchante et al. 2017). To examine the impact of transcriptional regulation, spinach PPO2 transcripts were investigated by reverse transcription-quantitative PCR (RT-qPCR). SoPPO2-specific primer pairs that amplify cDNA segments starting at either the first or second initiation codon were used to compare the amounts of PPO2 transcripts encompassing the full-length PPO2 reading frame (using qP2Met1) with those of N-terminally “truncated” PPO2 mRNAs (using qP2Met2) (Fig. 4A). As a reference, a third primer-pair binding downstream in the coding region SoPPO2 (qP2mid) was included. Amplification efficiencies of the three primer pairs were compared on a DNA template comprising the entire SoPPO2 cDNA sequence (Fig. 4B). While a slightly lower cycle threshold (Ct) value for qP2Met2 indicated increased amplification efficiency of the primer-pair specific for the cDNA segment starting at the second AUG codon, use of qP2Met1 and the internal reference pair qP2mid gave rise to nearly identical Ct values. Then, RNA isolated from transgenic Arabidopsis plants overexpressing SoPPO2 under control of p35S was analyzed by RT-qPCR and expression calculated relative to qP2mid after normalization to SAND (Czechowski et al. 2004) (Fig. 4C). As assumed for transcription under p35S, amounts of cDNA containing qP2Met1 were similar to those obtained using the internal reference qP2mid. An apparent over-accumulation of the qP2Met2 amplicon agreed well with the increased amplification efficiency shown for the respective primer pair in Fig. 4B. Analyses of RNA samples from spinach leaves, in contrast, revealed strongly decreased amounts of transcripts containing qP2Met1 (Fig. 4D). The relative expression of 0.28 detected using the primer-pair qP2Met1 implies that only about 30% of the total SoPPO2 transcripts in spinach leaves include the sequence that begins with the first initiation codon, i.e. these amplicons represent full-length SoPPO2 transcripts. Remarkably, a higher amount of full-length SoPPO2 transcripts was detected in RNA extracted from spinach roots (Fig. 4E). Here, a relative normalized expression of 0.51 suggests that about 50% of SoPPO2 transcripts contain the complete reading frame starting at the first AUG initiation codon.

Figure 4.

RT-qPCR analysis of the transcriptional regulation of PPO2 expression in Amaranthaceae. A) Scheme illustrating the locations of the amplicons qP2Met1, qP2Met2, and qP2mid in spinach PPO2 (SoPPO2). The arrows indicate primer-binding sites, and gray bars highlight the amplified sequence segments. B) Comparison of Ct values for spinach PPO2 amplicons. Diluted plasmid DNA encoding full-length SoPPO2 cDNA was employed to determine Ct values for the three SoPPO2 amplicons depicted in A. Analyses of spinach C to E) and Palmer amaranth F to H) PPO2 transcript accumulation by RT-qPCR. To validate the experimental approach, quantifications using the three PPO2-specific amplicons were performed with RNA from Arabidopsis plants expressing full-length cDNAs coding for SoPPO2 (C) and ApPPO2 (F), respectively. PPO2 transcripts in wild-type tissue were analyzed in RNA samples obtained from leaf D, G) or root E, H) RNA samples. All RT-qPCR results (C to H) are given as normalized expression levels relative to qP2mid (=1), using A. thaliana SAND mRNA (At2g28390, C, F) and actin transcripts of spinach (genebank accession XM_056837919, D, E) or Palmer amaranth (KT321447.1, G, H) as standards. Standard deviations are indicated (n ≥ 3).

A very similar pattern was observed using A. palmeri-specific primers and templates. Here, a transgenic A. thaliana line that overexpressed full-length ApPPO2 from the p35S promoter was used as a control and resulted for qP2Met1 in numbers of amplicons similar to those obtained for total ApPPO2 cDNA quantified using qP2mid (Fig. 4F). Analyses of WT A. palmeri leaf and root transcripts revealed in both tissues a lower abundance of ApPPO2 transcripts that include the coding sequence for the N-terminal extension (qP2Met1) and thus enable translation initiation at the first AUG triplet relative to those that encode qP2Met and qP2mid (Fig. 4, G and H). As in spinach, the relative contribution of full-length PPO2 cDNAs to total PPO2 transcript amounts was higher in amaranth root tissue (30%, Fig. 4H) than in leaves (12%, Fig. 4G).

Thylakoid-targeted PPO2 contributes to herbicide resistance

Amaranthaceae have recently drawn attention, because PPO2 point mutations in several species of this group were reported to confer resistance to PPO-inhibiting herbicides (Dayan et al. 2018). To investigate the possible impact of differential subplastidic targeting of PPO2 on herbicide resistance, transgenic Arabidopsis lines that overexpress envelope- or thylakoid-targeted PPO2 isoforms were tested for their ability to germinate and develop on growth media containing different concentrations of the PPO-inhibiting compound saflufenacil (Fig. 5). While WT A. thaliana plants can tolerate inhibitor concentration up to 10 nM, control lines that overexpressed the thylakoid-targeted PPO isoform AtPPO1 were, in agreement with earlier findings (Lermontova et al. 1997), able to grow in the presence of concentrations of up to 100 nM (Fig. 5, A and B). Amounts of AtPPO1 and total PPO activity in crude leaf extracts in the overexpressing line were about 15-fold and 21-fold higher, respectively, than in WT plants (Fig. 5 and Supplementary Fig. 3). An increase in herbicide tolerance in comparison to WT was also observed when AtPPO2, an exclusively envelope-targeted PPO2 variant, was expressed under the control of p35S, while moderate overexpression driven by the PPO1 promoter (pPPO1) did not contribute substantially to resistance (Fig. 5, C and D). Remarkably, the level of tolerance achieved under an approximately 50-fold increase in the AtPPO2 concentration corresponds to a 500-fold increase in overall leaf PPO activity (Fig. 5D, Supplementary Fig. 3).

Figure 5.

Herbicide resistance depends on PPO activity in thylakoids. Wild-type Arabidopsis plants (Col-0, A), as well as A. thaliana PPO-overexpressing transgenic lines (B to F), were cultivated on agar plates containing increasing concentrations of the herbicide saflufenacil. The degree of overexpression of the respective PPO isoforms (in parenthesis), as well as total PPO activity, were determined in all depicted lines (Supplementary Fig. 3) and values relative to wild-type are listed on the right. B) Overexpression of AtPPO1 under the control of the CaMV 35S promoter (p35S). C, D) Expression of AtPPO2 driven by the PPO1 promoter (pPPO1, C) and p35S (D). E, F) Fusion of the chloroplast transit peptide (cTP) of AtPPO1 to the amino-terminus of AtPPO2 results in predominantly thylakoid localization the resulting cTP-PPO2 polypeptide (Hedtke et al. 2023). Lines expressing the fusion under the control of pPPO1 (E) or p35S (F) were analyzed. Owing to the necrotic effects of strong cTP-PPO2 overexpression (p35S:cTP-AtPPO2) observed under day–night cycles, all plates were incubated in continuous light for 12 d. n.a, not applicable.

In contrast, when AtPPO2 was fused to a transit peptide conferring thylakoid targeting upon this isoform (cTP-AtPPO2; see Hedtke et al. 2023), even low levels of overexpression under the control of pPPO1 resulted in an increased herbicide tolerance (Fig. 5E). Indeed, strong overexpressors that accumulated about 50-fold more cTP-AtPPO2 in comparison to WT PPO2, were able to grow on 300 nM saflufenacil (Fig. 5F), and thus much higher concentrations than the levels attainable with AtPPO1-overexpressing lines (Fig. 5B). In comparison to strictly envelope-localized AtPPO2, the thylakoid-targeted cTP-AtPPO2 fusion displayed enhanced herbicide tolerance. It is worth noting that the relative increase in PPO enzyme activities in all transgenic lines expressing AtPPO2 was far greater than the corresponding change in AtPPO2 protein levels (Fig. 5C to F, Supplementary Fig. 3). This contrasts with the observations made with AtPPO1 overexpressor lines (Fig. 5B) and is explained by the higher specific activity of the PPO2 isoform in comparison to PPO1.

Discussion

We recently investigated the function and subcellular localization of the PPO isoform PPO2 in A. thaliana (Hedtke et al. 2023). Remarkably, a ppo2 knock-out mutation showed no visible phenotype, while the loss of PPO1 function is seedling-lethal (Zhang et al. 2014). In Arabidopsis, PPO2 accumulated exclusively in plastid envelope membranes, casting doubts on earlier reports of a mitochondrial localization of PPO2 in N. tabacum, since both proteins have a similar amino-terminal structure (Fig. 1A) (Lermontova et al. 1997). It was particularly striking that AtPPO2 lacks the N-terminal transit peptide required for the canonical targeting of plastid-localized proteins. While the N-terminal structure of PPO2 in A. thaliana is shared by the vast majority of dicotyledonous plants, it clearly differs from this norm in members of the Amaranthaceae such as spinach and amaranth, as it does in monocotyledonous plants (Fig. 1A). We have shown here that PPO2 sequences of the Amaranthaceae family possess an N-terminal stretch of about 30 amino acids, as well as a conserved second methionine that corresponds to the translation start in Arabidopsis PPO2 (Fig. 1A). This N-terminal extension was described previously as a chloroplast transit peptide, while initiation of translation at the second Met residue was reported to result in targeting to mitochondria (Watanabe et al. 2001).

We first used antibodies directed against amaranth PPO2 to clarify the subplastidal distribution of the protein in WT spinach and A. palmeri plants. The specificity of an immunoreactive band observed in chloroplast envelope fractions from both species was supported by measurements of PPO enzyme activity in plastid envelope fractions isolated from spinach (Fig. 1B to D). Since the mature PPO2 proteins resulting from translation initiation at the first and second AUG triplet of Amaranthaceae migrate identically in SDS-PAGE analyses (see below, Fig. 2D and Supplementary Fig. 1), the smaller, thylakoid-specific bands observed in WT extracts (Fig. 1, B and C) were deduced to result from cross-reactions of the amaranth PPO2 antiserum. This agrees with previous descriptions of chloroplast-localized PPO2 in spinach to be mainly associated with plastid inner envelope membranes (Watanabe et al. 2001).

Next, we re-examined the possibility of a mitochondrial localization for PPO2 in Amaranthaceae, using fractions enriched in mitochondria from both amaranth and spinach. No accumulation of PPO2-specific bands was observed in either of these mitochondrial fractions, and weak signals detected in the extracts from amaranth were demonstrated to associate with plastidic contaminations (Fig. 1, E and F). Moreover, proteinase K digestion of purified amaranth mitochondria confirmed that PPO2 was not located within the organelles (Fig. 1E).

Then we used the Arabidopsis ppo1 mutant (Hedtke et al. 2023) to elucidate the role of the two AUG codons located in the upstream segment of the sequence coding for spinach PPO2. With the aid of three different transgenic constructs (Fig. 2A), we demonstrated that translation initiation beginning with the first methionine residue results in a full-length protein (SoPPO2) that fully complements the ppo1 mutant. In contrast, the N-terminally truncated SoPPO2 protein, i.e. an overexpression construct that utilizes the second AUG codon for initiation, complemented ppo1 only partially (Fig. 2B). Moreover, the yellow–green slow-growing phenotype of the corresponding line was very similar to that seen when the Atppo1 mutant was complemented by Arabidopsis PPO2 (Hedtke et al. 2023). Identical ppo1 complementations were obtained using analogous ApPPO2 constructs (Supplementary Fig. 4).

Immunodetection of SoPPO2 revealed that the overexpressed protein accumulated to similar levels in all investigated lines and confirmed the knockout of Arabidopsis PPO1 (Fig. 2D). The yellow–green ppo1(p35S:SoPPO2^Δ26aa) plants, on the other hand, show strongly increased levels of Proto, which are most probably attributable to nonspecific oxidation of excess Protogen. In parallel, downstream tetrapyrrole intermediates are strongly reduced in yellow–green mutants, which also points to the perturbations in the enzymatic conversion of Protogen (Fig. 2E and Supplementary Fig. 2). The macroscopic phenotype, the disturbed pattern of TPB intermediates and the downregulation of GluTR1 and other proteins in the pathway (Fig. 2D) all agree with the previously described complementation of ppo1 by overexpressed endogenous AtPPO2 (Hedtke et al. 2023).

The distributions of overexpressed SoPPO2 variants in subfractions of purified chloroplasts were also examined (Fig. 3A). Both of the transgenic constructs that included the N-terminal extension (SoPPO2 and SoPPO2^M27A, referred to as SoPPO2^long below) resulted in the accumulation of mature PPO2 in thylakoid and envelope membranes. In contrast, the SoPPO2^Δ26aa variant (SoPPO2^short) is specifically enriched in the chloroplast envelope fraction. These findings are compatible with the structural similarity to, and localization of Arabidopsis PPO2 (Fig. 1A) as well as with the results of ppo1 complementation reported for the latter (Hedtke et al. 2023).

Remarkably, the immunodetectable, mature SoPPO2^long is, according to SDS-PAGE analyses, identical in size to SoPPO2^short (Figs. 2D and 3A), while unprocessed in vitro translation products were previously shown to migrate differently (Watanabe et al. 2001). Thus, Amaranthaceae PPO2 proteins initiated at the first AUG codon can be deduced to form precursor proteins carrying an N-terminal extension that specifically mediates targeting to thylakoids and is cleaved off following plastid import.

Clearly, the thylakoid localization of spinach PPO2 depends on the presence of the N-terminal extension. However, a substantial portion of SoPPO2^long was also localized in envelope fractions. Since translation initiation at the second AUG codon can be excluded for SoPPO^M27A, this partial envelope localization could result either from integration from the stromal side following cTP-mediated import, or from noncanonical targeting to envelopes. In Arabidopsis, the mature PPO2 protein starts with the second, acetylated amino acid, proving the absence of a cleavable cTP (Bienvenut et al. 2012). Noncanonical import into the chloroplast inner envelope membrane in the absence of a cTP has been described earlier for proteins such as ceQORH and was demonstrated to depend on internal sequence motifs. If such an inner amino acid stretch determines targeting of SoPPO2^short to the plastid envelope membrane, the same mechanism can be assumed to mediate the localization of a subfraction of the mature form of SoPPO2^long polypeptides to the envelope.

Hence, differential use of the first and second AUG codons present in PPO2 coding sequences of Amaranthaceae results in PPO2 proteins that are either located in both thylakoids and chloroplast envelopes (PPO2^long) or exclusively targeted to the envelope membrane (PPO2^short). This broader localization spectrum (in comparison to other dicotyledonous families) is accompanied by an increased dependence of TPB on the PPO2 gene product. While Arabidopsis ppo2 mutants do not display a macroscopic phenotype, PPO2 function is vital in Palmer amaranth (Porri et al. 2022; Hedtke et al. 2023). The parallel localization of PPO2 in thylakoid membranes of Amaranthaceae may facilitate a partial take-over of genuine PPO1 functions, for instance during de-etiolation. However, detailed analyses of tissue- and development-dependent expression of both PPO isoforms, and other TPB enzymes, in Amaranthaceae will be needed to shed light on the specific roles of PPO2 in this plant family.

Our initial studies of the regulation of PPO2 expression in Amaranthaceae focused on transcript analyses using an RT-qPCR-based approach (Fig. 4). Primer pairs specific for the N-terminal extension of PPO2^long transcripts (qP2Met1) were used in parallel with oligonucleotide pairs quantifying either all mRNAs that included the second methionine codon (qP2Met2) or an internal PPO2 amplicon located further downstream (qP2mid, Fig. 4A). Validations using DNA sequences (Fig. 4B) and cDNAs from Arabidopsis plants overexpressing the complete amaranth and spinach sequences coding for PPO2 under the control of p35S (Fig. 4, C and F) ensured similar amplification efficiencies for all primers.

Strikingly, when amplicons specific for PPO2^long transcripts (qP2Met1 in Fig. 4) were quantified and compared with all PPO2 mRNAs including the PPO2^short variants (qP2Met2 and qP2mid), the long transcripts represented only 28% and 12% of total PPO2 transcript amounts in spinach and amaranth leaf RNA, respectively (Fig. 4, D and G). A clearly different ratio of long to total PPO2 transcripts was observed in root tissue, where PPO2^long mRNAs accounted for 51% and 30% of total PPO2 transcripts in spinach and amaranth, respectively (Fig. 4, E and H). These findings reveal tissue-specific regulation of PPO2 expression at the transcriptional level. Only a subfraction of accumulating PPO2 mRNA codes for the PPO2^long variant, which is targeted to thylakoids as well as to plastid envelopes, while the majority of PPO2 transcripts, especially in leaf tissue, gives rise to the exclusively envelope-targeted PPO2^short. The envelope-localized PPO2 is probably destined for use in the synthesis of Proto for extraplastidal heme (Hedtke et al. 2023). We speculate that an increased ratio of PPO2^long transcripts in root plastids of Amaranthaceae enables PPO2 to largely replace the thylakoid-specific function of PPO1 in this tissue. Further studies are necessary to unveil the functional consequences of the simultaneous presence of both PPO2^long and PPO1 in thylakoid membranes of Amaranthaceae and explain the lethal phenotype reported recently for a PPO2 loss-of-function mutant in amaranth (Porri et al. 2022).

In recent large-scale studies in Arabidopsis, around 10% of genes were found to use more than one transcription start site (TSS) cluster to initiate mRNA synthesis (Thieffry et al. 2020). In addition, alternative transcription initiation has previously been shown to generate differently targeted protein variants (Daras et al. 2014). We therefore assume that the differential accumulation of PPO2^long and PPO2^short transcripts in Amaranthaceae results from the use of alternative TSS within the PPO2 gene.

The preferential accumulation of PPO2^short transcripts which encode exclusively plastid envelope-associated PPO variants in Amaranthaceae leaf samples is supported by immunoblot analyses (Fig. 1, B and C). Here, a PPO2-specific signal was enriched in both amaranth and spinach chloroplast envelopes (see above).

Herbicide sensitivity is dependent on thylakoid-localized PPO activity

The Amaranthaceae family has aroused particular interest in recent years owing to the fact that four out of 15 weed species that have developed tolerance against PPO-inhibiting herbicides belong to the genus Amaranthus (Dayan et al. 2018; Heap 2023). In these herbicide-tolerant Amaranthus mutants, target-site mutations in three different amino acid positions in PPO2 were associated with herbicide resistance (Porri et al. 2022). The reassessment of PPO2 localization in the amaranth family carried out in this study sheds light on the specific role of the different isoforms in this phylogenetic group. Importantly, two of the three known PPO2 target-site mutations that confer herbicide tolerance affect either substrate binding or enzymatic activity (Porri et al. 2022). Mutations with such strong side-effects are likely to be detrimental to PPO1, since this isoform accounts for the majority of PPO activity in green tissue (Hedtke et al. 2023). Downregulation of PPO1 impaired TPB and causes severe photodynamic lesions due to the accumulation of Proto. In Arabidopsis, PPO2 accumulates exclusively in chloroplast envelope membranes. Hence, only fusion to known cTP sequences can localize PPO2 to thylakoids and fully complement ppo1 mutants (Hedtke et al. 2023). To investigate the effect of PPO localization on herbicide resistance, we measured the sensitivity of Arabidopsis plants overexpressing PPO1 and differently targeted PPO2 variants to saflufenacil (Fig. 5). In each transgenic line, the degree of overexpression relative to the endogenous level of the respective Arabidopsis PPO isoform, and the increase in total PPO enzyme activity were determined (Fig. 5, Supplementary Fig. 3). Transgenic lines with increased PPO1 amounts exhibited a gain in total leaf PPO activity that resembles or equals the extent of overexpression (Fig. 5B) and, as reported earlier, they displayed increased herbicide tolerance (Lermontova and Grimm 2000).

Interestingly, the consequences of Arabidopsis PPO2 overexpression differ from the effects observed for PPO1. First, an increase in PPO2 protein has a stronger impact on total PPO activity. Even a 5-fold increase in PPO2 amounts results in an approximately 20- to 60-fold rise in activity (Fig. 5, C and E). Since PPO2 is less abundant than PPO1 in A. thaliana WT leaves, the 5-fold overexpression shown here represents PPO2 protein levels that resemble WT amounts of PPO1 (Hedtke et al. 2023). The marked increase in PPO activity observed in total plant extracts from all transgenic lines overexpressing PPO2 reveals a roughly 10-fold higher specific activity for Arabidopsis PPO2 relative to PPO1. The elevated specific activity of PPO2 in comparison to PPO1 observed in planta is supported by an even 30-fold increase of PPO activity detected using recombinant AtPPO proteins heterologously expressed in E. coli (Supplementary Fig. 5).

Second, despite the pronounced increase in total PPO activity, herbicide tolerance is only moderately enhanced in transgenic lines that overexpress Arabidopsis PPO2. Thus, even a 50-fold increase in PPO2 amounts, which is accompanied by an about 500-fold rise in total PPO activity, has only a limited impact on herbicide sensitivity. However, when AtPPO2 is artificially targeted to thylakoid membranes using a cTP fusion, herbicide tolerance is increased. Here, even a low level of overexpression results in substantially improved germination on plates containing 100 nM saflufenacil. Strong expression driven by p35S enables growth on up to 300 nM saflufenacil. This effect not only exceeds the herbicide tolerance induced by PPO1 overexpression under the same p35S promoter, it also differs substantially from that achieved by envelope-targeted PPO2. Hence, thylakoid-targeted PPO proteins have a greater impact on tolerance to PPO-inhibiting herbicides than comparable amounts of envelope-localized enzymes. This is consistent with the idea that PPO1 is the main target of PPO-inhibiting herbicide action in green tissue which is supported by the similar phenotypical consequences of PPO1 deficiency and herbicide treatment (Lermontova and Grimm 2006). Since PPO1 is thylakoid-localized, an effective increase in herbicide tolerance can be hypothesized to depend on targeting to the same subplastidal compartment. TPB in photoautotrophic cells was reported earlier to be mainly dedicated to chlorophyll synthesis and hence associated with internal plastid membranes (Yaronskaya et al. 2003).

However, in contrast to Arabidopsis and the majority of dicotyledonous plant species, Amaranthaceae harbor PPO2 genes that encode PPO2^long as well as PPO2^short proteins, with distinct subplastidal localizations. The unique thylakoid targeting of the PPO2^long form in Amaranthaceae enables PPO2 mutations that confer herbicide tolerance to counterbalance herbicide inhibition of PPO1 on the thylakoid membranes. In addition, the higher specific activity of PPO2 (Fig. 5) results in sufficient Protogen conversion even in PPO2 mutants which strongly affect Protogen binding or turnover. Similarly, a reduction of up to 97% in PPO2 enzyme activity did not affect plant growth and development in A. palmeri G399A mutants (Noguera et al. 2021). Hence, while low PPO2 activity can be tolerated, the presence of inhibitor-tolerant PPO2^long in thylakoid membranes may confer a decisive selective advantage under herbicide pressure.

PPO2 distribution in chloroplasts differs among subgroups of eudicotylodonous plants

The subplastidal distribution shown here for spinach and amaranth PPO2 may be a feature of all members of the Amaranthaceae family. Available sequence data confirm similar N-terminal extensions for sugar beet and quinoa, to name only two further examples. How widespread this specific PPO2 structure is within the order Caryophyllales is presently unknown, but N-terminal PPO2 extensions that confer thylakoid targeting can be excluded for specific members of the orders Brassicales (A. thaliana) and Solanales (N. tabacum) (Hedtke et al. 2023). Since PPO2 is characteristic for land plants (Kobayashi et al. 2014) and sequences described for mosses (Physcomitrium patens) and lycophytes (Selaginella moellendorfii) do not possess N-terminal extensions, the described cTPs of Amaranthaceae PPO2 are hypothesized to represent a “late” acquisition based on genomic rearrangements that led to an exchange of exon 1 (Fig. 6A). Interestingly, a similar extension coding for a hypothetical cTP is found in monocotyledonous PPO2 sequences. However, the absence of intron 1 in the latter points to a sequence reorganization that occurred independently from cTP acquisition in Amaranthaceae.

Figure 6.

Unique distribution of PPO enzymes in eudicotyledonous plants of the Amaranthaceae family. A) Alignment of the amino termini of PPO2 protein sequences from various groups of embryophytes. Physcomitrium patens (Physco), representing bryophytes, is compared with angiosperms of the eudicotyledonous (Arabidopsis, A.t, and spinach, S.o.) and monocotyledonous (O. sativa, O.s.) clades. Filled triangles indicate the positions of introns present within the depicted PPO2 coding region; methionine residues are boxed. B, C) Models of PPO targeting in plastids of Amaranthaceae (B) and Arabidopsis (C). In all plants, isoform PPO1 is encoded as a preprotein that includes a chloroplast transit peptide (cTP). Plastid import (dashed arrow) is assumed to engage the translocons on the outer and inner chloroplast membranes (TOC and TIC), followed by cTP cleavage in the stroma and association with thylakoid membranes. Amaranthaceae (B), uniquely among eudicotyledonous species, encode two variants of plastid-localized PPO2. Like PPO1, the form that includes the entire PPO2 sequence (PPO2^long) has an amino-terminal cTP. This N-terminal extension enables targeting of PPO2^long to thylakoids. PPO2^short proteins in Amaranthaceae (B) result from translation of shorter transcripts that lack the first AUG codon, which represented the major fraction of PPO2 mRNA in leaf tissue. PPO2^short is structurally equivalent to Arabidopsis PPO2 (C), which was deduced to integrate into the inner chloroplast envelope via a noncanonical import mechanism (Hedtke et al. 2023). Arabidopsis (C), representing the vast majority of dicotyledonous species, lacks an N-terminal PPO2 extension encoding a cTP sequence. Consequently, PPO2 is exclusively associated with plastid envelope membranes (Hedtke et al. 2023). Tetrapyrrole synthesis is depicted by key intermediates and its main products, heme and chlorophyll. Envelope-localized PPO2 is thought to synthesize heme intended for export.

PPO2 targeting in Amaranthaceae is summarized in Fig. 6B. PPO2 is synthesized in two forms that result from translation initiation at different start sites. PPO2 mRNAs including the first initiation codon give rise to PPO2^long precursor proteins that include a cTP and are assumed to be imported via the TOC and TIC complexes, like PPO1 and the majority of nuclear-encoded plastid proteins. Following translocation, the cTP is removed by the stromal processing peptidase (SPP) and the mature protein integrates into thylakoid membranes. Although not depicted in Fig. 6B, a minor portion of PPO2^long is targeted to the envelope (Fig. 3, A and B). Since PPO2^long precursors also include all the residues found in PPO2^short, it is assumed that the mature form of PPO2^long is translocated to the envelope via an unknown pathway or through the noncanonical import suggested for PPO2^short. In agreement with recent studies in Arabidopsis, PPO2 transcripts lacking the coding sequence for the N-terminal extension give rise to PPO2^short polypeptides, which are integrated exclusively into the chloroplast envelope via noncanonical import (Fig. 6B; Hedtke et al. 2023). Based on RT-qPCR analyses, this short PPO2 form represents the predominant form of PPO2 in spinach and amaranth leaves (Fig. 4, D and G).

For comparison, the mode of PPO import and localization in Arabidopsis chloroplasts emphasizes strict spatial separation of the two PPO isoforms seen in the majority of eudicotyledonous plants (Fig. 6C). While the stringent partitioning described for A. thaliana establishes spatial branching of TPB within plastids and generates an exclusively envelope-localized fraction of TPB intermediates (Hedtke et al. 2023), other phylogenetic groups such as the Amaranthaceae have extended the targeting spectrum of their PPO2 isoform. Here, PPO2 is able to catalyze Protogen conversion at both envelope and thylakoid membranes (Fig. 6B). Thus, PPO2^long is able to functionally substitute for PPO1, and developmental stages or tissues with limited demand for Proto may even dispense with PPO1 activity entirely. The different division of labor between the two PPO isoforms in Amaranthaceae is assumed to provide an explanation for the embryo lethality triggered by the functional loss of PPO2 in Palmer amaranth (Porri et al. 2022). Future transcript and protein analyses will provide a more detailed understanding of the contributions of both PPO isoforms to TPB in the amaranth family.

Materials and methods

Plant material and growth conditions

The Arabidopsis (A. thaliana) genotypes used in this study included the wild-type (Col-0), ppo1 (GK_539C07), and ppo1(pPPO1:PPO2) (Hedtke et al. 2023). Plants were grown in soil at 23°C under short-day conditions [10 h light (100 µmol photons m⁻² s⁻¹)/14 h dark]. Spinach (S. oleraceae), Palmer amaranth (A. palmeri), and N. benthamiana plants were cultivated on soil in a greenhouse under a long-day light regime (16 h light, 8 h dark). For isolation of root tissue, surface-sterilized seeds were grown on agar plates (0.8%) containing Murashige and Skoog medium (4.4 g/L) supplemented with 0.05% [w/v] 2-(N-morpholino)ethanesulfonic acid (pH 5.7) for 10 d in the dark. Herbicide tolerance was assessed on the same medium supplemented with the indicated concentrations of saflufenacil (BASF, Germany) under continuous light (100 µmol photons m⁻² s⁻¹). Transgenic lines overexpressing AtPPO isoforms used in herbicide tests are described in Hedtke et al. (2023).

Genotyping PCRs

Analyses of the Arabidopsis ppo1 mutant were performed with the primers PPO1 down fw/Gabi LB (T-DNA) and AtPPO1_genot.WtFw/AtPPO1_genot.WtRv (WT allele). Primer sequences are listed in Supplementary Table S1.

Cloning

The primer combinations P2long_FW and P2_RV and P2short_Fw and P2_RV were used to amplify the long (SoPPO2, ApPPO2) and short forms (SoP2^Δ26aa, ApPPO2^Δ30aa) of PPO2 from spinach (SoP2) and amaranth (ApP2) cDNAs, respectively. PCRs were performed using S7 Fusion High-Fidelity DNA Polymerase (Biozym, Germany) and products were cloned into pJET (Thermo Scientific). Nucleotide exchanges that convert the second in-frame methionine into alanine were introduced into SoPPO2 and ApPPO2 in pJET by site-specific mutagenesis (Laible and Boonrod 2009) using specific primer pairs P2mut_FW and P2mut_RV to generate SoPPO2^Δ27A and ApPPO2^Δ31A. After confirmation of their sequences (LGC, Germany), the resulting PPO2 variants were excised by restriction with XbaI/XmaI (SoPPO2) or NheI/XmaI (ApPPO2) and transferred into the binary vector pGL1 (Apitz et al. 2014) to enable expression under the control of CAMV p35S in planta.

Plant transformation

Arabidopsis plants were transformed with the Agrobacterium tumefaciens strain pGV2260 (McBride and Summerfelt 1990) by the floral-dip method (Clough and Bent 1998). Nicotiana benthamiana plants used for transient in planta expression were grown for 8 weeks under greenhouse conditions. Leaves were infiltrated with A. tumefaciens pGV2260 cultures that had been resuspended in 10 mM MgCl₂, 10 mM MES, and 100 μM acetosyringone (pH 5.7).

RT-qPCR

RNA was extracted from frozen plant material by homogenization in a mixer mill MM 400 (Retsch, Germany) using the citric-acid protocol (Oñate-Sánchez and Vicente-Carbajosa 2008). Following DNase I treatment, total RNA was transcribed using Moloney Murine Leukemia Virus reverse transcriptase and an oligo dT(18) primer according to the manufacturer's protocol (Thermo Scientific). qPCR analysis was carried out in a CFX96-C1000 96-well plate thermocycler (Bio-Rad, CA) using ChamQ Universal SYBR qPCR master mix (Vazyme). Gene expression levels were detected using the Bio-Rad CFX Maestro 2.0 Software. Expression was normalized using SAND [AT2G28390 (Czechowski et al. 2004)] or actin-specific primers, resulting in ΔCt. Following conversion to 2^−ΔCt, expression was calculated relative to qP2mid (=1). Average and standard deviation are calculated for n ≥ 3 biological samples.

Protein extraction and western-blot procedures

Total leaf protein was extracted from homogenized leaf material using 10 µL of 2 × Laemmli buffer (Sambrook 2001) per mg fresh weight. Samples were incubated for 5 min at 95°C, and centrifuged for 5 min (16,000 × g, RT). Samples representing either identical fresh weights (1 mg) or adjusted to Chl/protein content were fractionated by electrophoresis on SDS-polyacrylamide gels (10% or 12% [w/v]) and blotted onto nitrocellulose membranes (Amersham Protran, GE Healthcare, UK). Protein sizes were estimated using Pierce unstained protein molecular weight marker or prestained molecular weight marker (Thermo Scientific). Membranes were stained with Ponceau S and probed with protein-specific antibodies using standard procedures (Sambrook 2001). Polyclonal antisera directed against the His-tagged Arabidopsis proteins DnaJD12, CHLI, GluTR1, PORB, and PPO2, and the N. tabacum proteins GSAT and PPO1, were raised in the authors’ laboratory and affinity-purified using the appropriate antigen if necessary. Sera specific for MnSOD, LHCB1.6, LHCB5, TIC110, and VDAC were purchased from Agrisera (Sweden). Antisera directed against Amaranthus tuberculatus PPO2 were kindly provided by Dr. J. Lerchl (BASF SE Germany).

HPLC analyses

Tetrapyrroles were extracted from homogenized leaves using 300 µL of acetone:0.2 M NH₄OH (9:1), incubated for 30 min at −20°C and centrifuged for 30 min (16,000 × g, 4°C). The supernatant was analyzed by HPLC for (Mg) porphyrins and Chls. Noncovalently bound (ncb) heme was extracted from the remaining pellet by resuspension in 200 µL of AHD (acetone:HCl:DMSO, 10:0.5:2) and incubated for 15 min at RT. After centrifugation for 15 min (16,000 × g, RT), the amount of ncb heme in the supernatant was quantified by HPLC. HPLC analyses were performed on Agilent LC systems following previously described methods (Richter et al. 2019) and using authentic standards for peak quantification.

Purification of organelles

Chloroplasts and mitochondria were purified from leaves of 12-week-old spinach and Palmer amaranth plants cultivated under greenhouse conditions. Transiently transformed N. benthamiana plants were incubated for 4 d in the dark before leaf material was harvested. Arabidopsis chloroplasts were isolated from plants grown for 6 weeks under short-day conditions. Purification of organelles was performed as described previously (Hedtke et al. 2023). Proteinase K digestion assays contained 2 µg of mitochondrial protein in WB (0.3 M sucrose, 10 mM MOPS, 1 mM EDTA, pH 7.2), supplemented with the indicated concentrations of the enzyme. Incubations were carried out for 5 min at 4°C and terminated by adding Laemmli protein loading buffer (Sambrook 2001) and heating to 95°C.

PPO activity assays

Plant material was either homogenized under liquid N₂ in a mixer mill MM 400 (Retsch, Germany) or obtained from organelle purifications and dissolved in assay buffer (AB) containing 0.5 M Bis–Tris pH 7.5, 4 mM DTT, 2.5 mM EDTA, and 0.004% (v/v) Tween on ice. Aliquots (50 µL) of the extracts were mixed with 150 µL of AB containing 4 µM Protogen at room temperature, and Proto formation was recorded over a period of 20 min on a Hitachi F-700 fluorescence spectrophotometer (excitation 405 nm, emission 635 nm). Protogen was produced by reduction of Proto using sodium amalgam (Lermontova et al. 1997). Conversion of relative fluorescence values was based on an authentic Proto standard dissolved in AB.

Accession numbers

PPO2 protein sequences used in this article can be found in data libraries under accession numbers ATE88443.1 (A. palmeri), NP_001105004.2 (S. oleraceae), NP_001105004.2 (Z. mays), XP_025880545.1 (O. sativa), XP_024396127.1 (P. patens), NP_196926.2 (A. thaliana), and NP_001312887.1 (N. tabacum). Nucleotide sequences for PPO2 coding sequences are available under MF583744.1 (A. palmeri) and AB046993.1 (S. oleraceae).

Author contributions

B.H. and B.G. designed the research; D.T.W., F.E.P., S.M.S., and P.O.R. performed the experiments. B.H. and B.G. wrote the article.

Supplementary data

The following materials are available in the online version of this article.

Supplementary Figure S1 . Electrophoretic mobility of Palmer amaranth PPO2 variants.

Supplementary Figure S2 . Quantification of additional tetrapyrroles in complemented ppo1 mutants.

Supplementary Figure S3 . Determination of PPO abundance and activity in Arabidopsis PPO. overexpression lines.

Supplementary Figure S4 . Complementation of Arabidopsis ppo1 by Amaranthus palmeri PPO2 (ApPPO2) sequences.

Supplementary Figure S5 . Enzyme activity of recombinant Arabidopsis PPO proteins.

Supplementary Table S1 . Oligonucleotides used in the present study.

Funding

The work of D.T.W. was supported by BASF (Germany) and a Deutsche Forschungsgemeinschaft grant (GR936 17-1/2 in the SPP 1710 “Thiol Switches” to B.G.). P.O.R. was supported by the German Academic Exchange Service (DAAD).

Data availability

The data underlying this article are available in the article and in its online supplementary material.