Barley Etioplasts Research Articles

Aerobic Barley Mg-protoporphyrin IX Monomethyl Ester Cyclase is Powered by Electrons from Ferredoxin.

Chlorophyll is the light-harvesting molecule central to the process of photosynthesis. Chlorophyll is synthesized through 15 enzymatic steps. Most of the reactions have been characterized using recombinant proteins. One exception is the formation of the isocyclic E-ring characteristic of chlorophylls. This reaction is catalyzed by the Mg-protoporphyrin IX monomethyl ester cyclase encoded by Xantha-l in barley (Hordeum vulgare L.). The Xantha-l gene product (XanL) is a membrane-bound diiron monooxygenase, which requires additional soluble and membrane-bound components for its activity. XanL has so far been impossible to produce as an active recombinant protein for in vitro assays, which is required for deeper biochemical and structural analyses. In the present work, we performed cyclase assays with soluble and membrane-bound fractions of barley etioplasts. Addition of antibodies raised against ferredoxin or ferredoxin-NADPH oxidoreductase (FNR) inhibited assays, strongly suggesting that reducing electrons for the cyclase reaction involves ferredoxin and FNR. We further developed a completely recombinant cyclase assay. Expression of active XanL required co-expression with an additional protein, Ycf54. In vitro cyclase activity was obtained with recombinant XanL in combination with ferredoxin and FNR. Our experiment demonstrates that the cyclase is a ferredoxin-dependent enzyme. Ferredoxin is part of the photosynthetic electron-transport chain, which suggests that the cyclase reaction might be connected to photosynthesis under light conditions.

Assembly of NADPH:protochlorophyllide oxidoreductase complex is needed for effective greening of barley seedlings

NADPH:protochlorophyllide (Pchlide) oxidoreductase (POR) is the key enzyme in the light-induced greening of higher plants. A unique light-harvesting POR:Pchlide complexes (LHPP) has been found in barley etioplasts, but not in other plant species. Why PORs from barley, but not from other plants, can form LHPP? And its function is not well understood. We modeled the barley and Arabidopsis POR proteins and compared molecular surface. The results confirm the idea that barley PORA can form a five-unit oligomer that interacts with a single PORB. Chemical treatment experiments indicated that POR complex may be formed by dithiol oxidation of cysteines of two adjacent proteins. We further showed that LHPP assembly was needed for barley POR functions and seedling greening. On the contrary, Arabidopsis POR proteins only formed dimers, which were not related to the functions or the greening. Finally, POR complex assembly (including LHPP and POR dimers) did not affect the formation of prolamellar bodies (PLBs) that function for efficient capture of light energy for photo conversion in etioplasts.

Mass spectrometric characterization of membrane integral low molecular weight proteins from photosystem II in barley etioplasts

In Photosystem II (PSII), a high number of plastid encoded and membrane integral low molecular weight proteins smaller than 10 kDa, the proteins PsbE, F, H, I, J, K, L, M, N, Tc, Z and the nuclear encoded PsbW, X, Y1, Y2 proteins have been described. Here we show that all low molecular weight proteins of PSII already accumulate in the etioplast membrane fraction in darkness, whereas PsaI and PsaJ of photosystem I (PSI) represent the only low molecular weight proteins that do not accumulate in darkness. We found by BN-PAGE separation of membrane protein complexes and selective MS that the accumulation of one-helix proteins from PSII is light independent and occurs in etioplasts. In contrast, in chloroplasts isolated from light-grown plants, low molecular weight proteins were found to specifically accumulate in PSI and II complexes. Our results demonstrate how plants grown in darkness prepare for the induction of chlorophyll dependent photosystem assembly upon light perception. We anticipate that our investigation will provide the essential means for the analysis of protein assembly in any membrane utilizing low molecular weight protein subunits.

A new method for isolating physiologically active Mg-protoporphyrin monomethyl ester, the substrate of the cyclase enzyme of the chlorophyll biosynthetic pathway

Mg-protoporphyrin monomethyl ester (MPE) is a biosynthetic intermediate of chlorophyll and converted by MPE cyclase to protochlorophyllide. Limited availability of MPE has so far hampered cyclase research. In a new, simplified, method MPE was prepared from freeze dried bchE mutant Rhodobacter capsulatus DB575 cells by extraction with acetone/H 2O/25% NH 3. Isolated MPE was identified by absorption and fluorescence spectroscopy, and its purity was analyzed by HPLC. The extracted MPE was dried and redissolved in buffered DMSO and its substrate activity is shown by enzymatic cyclase assays. A linear time course was observed for MPE conversion to protochlorophyllide by enzymes from barley etioplasts. Our innovation of freeze drying the R. capsulatus cells before extraction provides a high yield method for MPE, which is significantly faster and more reproducible than previous extraction methods.

Protochlorophyllide photo-transformation and chlorophyll(ide) formation in barley etioplast fractions

Localization of protochlorophyll(ide) (Pchlide) forms and chlorophyllide (Chlide) transformation process were studied by using comparative analyses of de-convoluted 77 K fluorescence spectra of barley etioplast stroma and different membrane fractions obtained by sucrose gradient centrifugation. Non-photoactive 633 nm Pchlide form was mainly located in the envelope-prothylakoid membrane mixture while the photoactive 657 nm Pchlide was dominant pigment in the prolamellar body membrane and in the soluble etioplast fraction (stroma). When these fractions were exposed to a saturating flash, conversion of photoactive Pchlide into 697 nm Chlide was preferential in the prolamellar body and in the stroma, while the 676 nm Chlide was dominant pigment form in the envelope-prothylakoid fraction. These spectral characteristics are considered to reflect molecular composition and organization of the pigment-protein complexes specific for each etioplast compartment.

Irradiation-induced in vivo re-localization of NADPH-protochlorophyllide oxidoreductase from prolamellar body to stroma of barley etioplast

Distribution of NADPH-protochlorophyllide oxidoreductase (POR) in etioplast of etiolated barley leaf was studied by using Western blot analyses of etioplast fractions isolated on a sucrose gradient. When the leaf was exposed to light, POR content decreased in the etioplast inner membrane and prolamellar body sub-membrane fraction while it was simultaneously increased in the stroma. By using 77 K fluorescence spectroscopy analyzes, we found for irradiated etiolated leaf that the POR protein in the stroma was co-localized with chlorophyllide (Chlide) emitting at 678 nm. Relocalization of the POR-Chlide complex induced by irradiation suggests that POR participates in the pigment transport processes during early stages of the thylakoid membrane development.

The outer plastid envelope protein Oep16: role as precursor translocase in import of protochlorophyllide oxidoreductase A.

A 16-kDa plastid envelope protein was identified by chemical crosslinking that interacts with the precursor of NADPH:protochlorophyllide oxdidoreductase A (pPORA) during its posttranslational import into isolated barley chloroplasts. Protein purification and subsequent protein sequencing showed that the 16-kDa protein is an ortholog of a previously identified outer plastid envelope protein, Oep16. A protein of identical size was present in barley etioplasts and interacted with pPORA. Similar 16-kDa protein-dependent crosslink products of pPORA were detected in wheat, pea, and Arabidopsis chloroplasts. Database analyses revealed that the 16-kDa protein belongs to a family of preprotein and amino acid transporters found in free-living bacteria and endosymbiotic mitochondria and chloroplasts. Antibodies raised against the 16-kDa protein inhibited import of pPORA, highlighting its role in protein import.

Chloroplast Biogenesis 88: PROTOCHLOROPHYLLIDE b OCCURS IN GREEN BUT NOT IN ETIOLATED PLANTS

It has recently been reported that protochlorophyllide (Pchlide) b is an abundant pigment in barley etioplasts but is rather unstable, as it is rapidly converted to Pchlide a by 7-formyl reductase during pigment extraction with conventional 80% acetone (Reinbothe, S., Pollmann, S., and Reinbothe, C. (2003) J. Biol. Chem. 278, 800-806). It has also been claimed that extraction of barley etioplasts with 100% acetone containing 0.1% diethyl pyrocarbonate prevents the conversion of Pchlide b to Pchlide a and leads to the detection of large amounts of Pchlide b in the isolated etioplasts. In this work the extraction protocol of Reinbothe et al. is compared with the more conventional 80% aqueous acetone extraction method. No Pchlide b was detected either in etiolated barley leaves or isolated barley etioplasts irrespective of the extraction protocol. On the other hands, small amounts of Pchlide b were detected in green barley leaves and isolated chloroplasts, extracted either with 80% acetone or 100% acetone containing 0.1% diethyl pyrocarbonate. It is concluded that the proposed occurrence of a light-harvesting POR-Pchlide-a,b complex in etiolated plant tissues is untenable, and its ensuing consequences and implications, for the greening process, are irrelevant.

In Situ Conversion of Protochlorophyllideb to Protochlorophyllide a in Barley: EVIDENCE FOR A NOVEL ROLE OF 7-FORMYL REDUCTASE IN THE PROLAMELLAR BODY OF ETIOPLASTS

We recently put forth a model of a protochlorophyllide (Pchlide) light-harvesting complex operative during angiosperm seedling de-etiolation (Reinbothe, C., Lebedev, N., and Reinbothe, S. (1999) Nature 397, 80-84). This model, which was based on in vitro reconstitution experiments with zinc analogs of Pchlide a and Pchlide b and the two NADPH:protochlorophyllide oxidoreductases (PORs), PORA and PORB, of barley, predicted a 5-fold excess of Pchlide b, relative to Pchlide a, in the prolamellar body of etioplasts. Recent work (Scheumann, V., Klement, H., Helfrich, M., Oster, U., Schoch, S., and Rüdiger, W. (1999) FEBS Lett. 445, 445-448), however, contradicted this model and reported that Pchlide b would not be present in etiolated plants. Here we demonstrate that Pchlide b is an abundant pigment in barley etioplasts but is rather metabolically unstable. It is rapidly converted to Pchlide a by virtue of 7-formyl reductase activity, an enzyme that had previously been implicated in the chlorophyll (Chl) b to Chl a reaction cycle. Our findings suggest that etiolated plants make use of 7-formyl reductase to fine tune the levels of Pchlide b and Pchlide a and thereby may regulate the steady-state level of light-harvesting POR-Pchlide complex.

Comparative Absorption, Translocation, and Metabolism of Foliar-Applied Oxyfluorfen in Wheat and Barley

Wheat is known to be relatively tolerant to diphenyl ether herbicides. The absorption, translocation, and metabolism of foliar-applied oxyfluorfen in wheat were examined in comparison with those of oxyfluorfen-susceptible barley. Epicuticular wax contents in the first fully expanded leaves were similar in wheat and barley, but the wheat leaves had a 1.74-fold higher cuticle content than the barely leaves. Absorption of [ 14 C]oxyfluorfen, as estimated by the amount of [ 14 C]oxyfluorfen penetrating the cuticle, appeared to be similar in the leaves of both species, although most of the radioactivity remained in the epicuticular wax. Little translocation of the herbicide out of the treated leaf was observed in either species, but slightly more translocation of [ 14 C]oxyfluorfen to shoots other than the treated leaf or to roots was found in barley than in wheat leaves. Autoradiographs of the [ 14 C]oxyfluorfen-treated leaves of both species also showed that the radioactivity was distributed mainly in the treated site. Thin-layer chromatographic analysis of leaf extracts revealed that oxyfluorfen metabolism did not occur in intact leaves of either species. The binding constants for the herbicide were estimated as 44.2 and 191 nM, respectively, for wheat and barley etioplasts, showing that the herbicide had higher affinity to barley than to wheat etioplasts. These results indicate that the differential susceptibilities of wheat and barley to oxyfluorfen are not due to the differential absorption, translocation, and metabolism of the herbicide, but to the differential affinity of the herbicide.

{Stable Insertion of the Early Light-induced Proteins into Etioplast Membranes Requires Chlorophyll a

Etiolated plant seedlings exposed to light respond by transient accumulation of the nucleus-encoded, plastid-located early light-inducible proteins (Elips). These proteins are distant relatives of the light-harvesting chlorophyll a/b-binding gene family and bind pigments with unusual characteristics. To investigate whether accumulation of Elips in plastid membranes is post-translationally regulated by pigments, reconstitution studies were performed, where in vitro transcribed and translated low molecular mass Elip precursors of barley were combined with lysed barley etioplasts complemented with various compositions of isolated pigments. We showed that the membrane insertion of Elips, as proven by protease protection assays and washes with a chaotropic salt or alkali, depended strictly on chlorophyll a but not on chlorophyll b or xanthophyll zeaxanthin. The amount of inserted Elips increased almost linearly with the chlorophyll a concentration, and the insertion efficiency was not significantly influenced by a light intensity between 1 and 1,000 micromol x m(-2) x s(-1). In contrast, in vitro import of Elip precursors into greening plastids was enhanced by high intensity light. Thus, we conclude that although chlorophylls bound to Elips seem to not be involved in light harvesting, they are crucial for a stable insertion of these proteins into the plastid membrane.

Identification of the Ndh (NAD(P)H-plastoquinone-oxidoreductase) complex in etioplast membranes of barley: changes during photomorphogenesis of chloroplasts.

In the last few years the presence in thylakoid membranes of chloroplasts of a NAD(P)H-plastoquinone oxidoreductase complex (Ndh complex) homologous to mitochondrial complex I has been well established. Herein, we report the identification of the Ndh complex in barley etioplast membranes. Two plastid DNA-encoded polypeptides of the Ndh complex (NDH-A and NDH-F) were relatively more abundant in etioplast membranes than in thylakoids from greening chloroplasts. Conversion of etioplast into chloroplast, after light exposure of barley seedlings grown in the dark, was accompanied by a decrease in the NADH dehydrogenase activity associated to plastid membranes. Using native-PAGE and immunolabelling techniques we have determined that a NADH specific dehydrogenase activity associated with plastid membranes, which was more active in etioplasts than in greening chloroplasts, contained the NDH-A and NDH-F polypeptides. These results complemented by those obtained through blue-native-PAGE indicated that NDH-A and NDH-F polypeptides are part of a 580 kDa NADH dependent dehydrogenase complex present in etioplast membranes. This finding proves that accumulation of the Ndh complex is independent of light. The decrease in the relative levels and specific activity of this complex during the transition from etioplast to chloroplasts was accompanied by a parallel decrease in the specific activity of peroxidase associated to plastid membranes. Based on the mentioned observations it is proposed that an electron transport chain from NADH to H2O2 could be active in barley etioplasts.

Differential Susceptibilities of Wheat and Barley to Diphenyl Ether Herbicide Oxyfluorfen

Wheat is known to be relatively tolerant to diphenyl ether herbicides. Growth and physiological responses of wheat to diphenyl ether herbicide oxyfluorfen were examined in comparison with those of oxyfluorfensusceptible barley. Compared to barley, wheat was significantly less susceptible to the herbicide with preemergence and postemergence treatments. The differential susceptibilities of wheat and barley to the herbicide were more apparent with postemergence than with preemergence treatment. The effects of the herbicide on causing cellular leakage, chlorophyll loss, and lipid peroxidation were much lower in wheat than in barley leaves. Protoporphyrin IX accumulated in both wheat and barley leaves treated with oxyfluorfen, in a concentration-dependent manner. However, the magnitude of the protoporphyrin IX accumulation was much lower in wheat than in barley leaves treated with oxyfluorfen at a concentration of higher than 0.33 μM. Protoporphyrinogen oxidase from wheat leaves was found to be less susceptible to oxyfluorfen than that from barley leaves. The I50 concentrations of oxyfluorfen on protoporphyrinogen oxidase activity were approximately 0.2 and 0.06 μM for wheat and barley etioplasts, respectively. Whereas the inhibition of protoporphyrinogen oxidase in barley etioplasts increased with increasing concentration of oxyfluorfen, protoporphyrinogen oxidase in wheat etioplasts was not further inhibited beyond 0.33 μM oxyfluorfen concentrations. In addition, both plant species similarly responded to superoxide anion-generating paraquat and to singlet oxygen-generating rose bengal, implying that the relative tolerance of wheat to oxyfluorfen is not due to protective mechanism against active oxygen species. Our results suggest that the differential susceptibilities of wheat and barley to oxyfluorfen are at least partly due to the differential inhibition of protoporphyrinogen oxidase by the herbicide, and thereby wheat is relatively tolerant to the herbicide.

Protochlorophyllide b does not occur in barley etioplasts

Barley ( Hordeum vulgare L.) etioplasts were isolated, and the pigments were extracted with acetone. The extract was analyzed by HPLC. Only protochlorophyllide a and no protochlorophyllide b was detected (limit of detection <1% of protochlorophyllide a). Protochlorophyllide b was synthesized starting from chlorophyll b and incubated with etioplast membranes and NADPH. In the light, photoconversion to chlorophyllide b was observed, apparently catalyzed by NADPH:protochlorophyllide oxidoreductase. In darkness, reduction of the analogue zinc protopheophorbide b to zinc 7 1-hydroxy-protopheophorbide a was observed, apparently catalyzed by chlorophyll b reductase. We conclude that protochlorophyllide b does not occur in detectable amounts in etioplasts, and even traces of it as the free pigment are metabolically unstable. Thus the direct experimental evidence contradicts the idea by Reinbothe et al. (Nature 397 (1999) 80–84) of a protochlorophyllide b-containing light-harvesting complex in barley etioplasts.

Light-harvesting Chlorophyll a/b-binding Protein Stably Inserts into Etioplast Membranes Supplemented with Zn-pheophytina/b

Light-harvesting chlorophyll a/b-binding protein, LHCP, or its precursor, pLHCP, cannot be stably inserted into barley etioplast membranes in vitro. However, when these etioplast membranes are supplemented with the chlorophyll analogs Zn-pheophytin a/b, synthesized in situ from Zn-pheophorbide a/b and digeranyl pyrophosphate, pLHCP is inserted into a protease-resistant state. This proves that chlorophyll is the only component lacking in etioplast membranes that is necessary for stable LHCP insertion. Synthesis of Zn-pheophytin b alone promotes insertion of LHCP in vitro into a protease-resistant state, whereas synthesis of Zn-pheophytin a alone does not. Insertion of pLHCP into etioplast membranes can also be stimulated by adding chlorophyll a and chlorophyll b to the membranes, albeit at a significantly lower efficiency as compared with Zn-pheophytin a/b synthesized in situ. When pLHCP is inserted into chlorophyll- or Zn-pheophytin-supplemented etioplast membranes and then assayed with protease, only the protease digestion product indicative of the monomeric major light-harvesting chlorophyll a/b complex (LHCII) is found but not the one indicating trimeric complexes. In this respect, chlorophyll- or Zn-pheophytin-supplemented etioplast membranes resemble thylakoid membranes at an early greening stage: pLHCP inserted into plastid membranes from greening barley is assembled into trimeric LHCII only after more than 1 h of greening.

Substrate Specificity of Chlorophyll(ide) b Reductase in Etioplasts of Barley (Hordeurn Vulgare L.)

Enzyme activity of chlorophyll(ide) b reductase is present in etioplasts. Recently the conversion of chlorophyllide b to chlorophyll a via 7(1)-hydroxychlorophyll a was demonstrated in barley etioplasts. We used zinc pheophorbide b for a detailed investigation of the reduction of the 7-formyl group to the 7(1)-hydroxy compound in intact barley etioplasts. The reaction proceeded likewise before esterification and after esterification with phytyl diphosphate. The metal-free pheophorbide b, that is not accepted by chlorophyll synthase for esterification, is reduced to 7(1)-hydroxypheophorbide a to a small extent. The zinc (13(2)S)-pheophorbide b is at least equally well accepted for reduction as the epimer with the 13(2)R configuration of natural chlorophyll b. The reaction requires NADPH or NADH, although the latter is less effective. ATP is not required for the first step to the 7(1)-hydroxy compound. The significance of chlorophyll b reduction for acclimation from shade to sun leaves and for chlorophyll degradation is discussed.

Conversion of Chlorophyll b to Chlorophyll a via 7-Hydroxymethyl Chlorophyll

Chlorophyll b is synthesized from chlorophyll a by the oxidation of the methyl group on the ring B of the tetrapyrrole ring to the formyl group. Previously, we reported that chlorophyllide b could be converted to chlorophyll a in isolated cucumber etioplasts indicating the conversion of chlorophyll b to chlorophyll a. To identify the intermediate molecule, we used barley etioplasts instead of cucumber. Chlorophyll a and an additional pigment were found after incubation of chlorophyllide b with isolated barley etioplasts. The pigment has the same retention time and absorption spectrum as 7-hydroxymethyl chlorophyll, which has the hydroxymethyl group on ring B instead of the formyl group of chlorophyll b. Authentic 7-hydroxymethyl chlorophyll was prepared by reduction of chlorophyll b by NaBH4. Chlorophyll a accumulated during the incubation of 7-hydroxymethyl chlorophyllide with etioplasts. These findings indicate that chlorophyll b is converted to chlorophyll a via 7-hydroxymethyl chlorophyll. Chlorophyll b and 7-hydroxymethyl chlorophyll accumulated within a short period of incubation of chlorophyllide b with etioplasts. However, chlorophyll a accumulated with a concomitant decrease of chlorophyll b and 7-hydroxymethyl chlorophyll. These observations also suggest that chlorophyll b is converted to 7-hydroxymethyl chlorophyll and then to chlorophyll a. Both steps required ATP.

Amitrole effects on barley etioplasts

Summary The effects of amitrole-inhibited carotenogenesis on the differentiation of etioplasts have been investigated in barley plants grown either at 20°C or 30°C in darkness. Amitrole drastically impaired carotenoid synthesis in plants grown at 20°C and this effect was paralleled by extensive uitrastructural alterations in the etioplasts. Protochlorophyllide oxidoreductase was found in the badly arranged membranes together with abnormally high quantities of protochlorophyllide, essentially present as non-phototransformable pigment. Growing plants at 30°C caused a remarkable improvement in the carotenoid synthesis, which was accompanied by a normalization of all the other parameters analysed. The parallelism observed between correct carotenoid synthesis and normal etioplast development suggests that, as in chloroplasts, also in the dark-grown organelles, the carotenoids carry out a structural function, playing a role in the establishment of the regular organization of the inner membrane system.

Herbicidal Activity of UCC-C4243 and Acifluorfen is Due to Inhibition of Protoporphyrinogen Oxidase

Laboratory and greenhouse studies were conducted to determine the mode of action of soil- and foliar-applied UCC-C4243. Experiments demonstrated that UCC-C4243 required light for phytotoxicity, phytotoxic symptoms were similar to inhibitors of porphyrin synthesis such as acifluorfen, and UCC-C4243 potently inhibited protoporphyrinogen oxidase. Germination and emergence of field pennycress and lentil in the dark were not affected by soil-incorporated UCC-C4243 at rates more than 10 times greater than like treatments that killed all plants in the light. Soil-incorporated UCC-C4243 required light for activity and killed seedlings within 1 d after emergence; sublethal doses caused desiccation, veinal necrosis, and leaf deformation. Field pennycress and lentil were susceptible to soil-incorporated UCC-C4243 and acifluorfen in the light, but were 5 to 93 times less sensitive to the herbicides in the dark. Wheat was not affected by either herbicide in the light or dark. Injury symptoms from UCC-C4243 applied POST to redroot pigweed were similar to symptoms from diphenyl ether and bipyridinium herbicides: rapid, light-dependent chlorophyll bleaching, desiccation, and necrosis. UCC-C4243, acifluorfen-methyl, and acifluorfen acid caused light- and concentration-dependent chlorophyll bleaching and electrolyte leakage from cucumber leaf disks (I50= 1.0, 1.8, and 4.3 μM, respectively). An inhibitor of the porphyrin synthesis pathway, 4,6-dioxoheptanoic acid, almost completely inhibited herbicide-induced electrolyte leakage. δ-Aminolevulinic acid, a tetrapyrrole precursor and stimulator of the porphyrin synthesis pathway, caused synergistic effects with each herbicide. Protoporphyrinogen oxidase from barley etioplast preparations was inhibited 50% by 40 nM UCC-C4243. Barley leaf sections treated with 100 μM UCC-C4243 accumulated protoporphyrin IXin vivoto levels &gt; 75 times non-treated controls. These data indicate the light-requiring herbicide activity of UCC-C4243, like acifluorfen, is due to inhibition of protoporphyrinogen oxidase.

Enantioselectivity of protoporphyrinogen oxidase‐inhibiting herbicides

AbstractProtoporphyrinogen oxidase (Protox) was inhibited stereoselectively by three pairs of enantiomers belonging to diphenyl ether (DPE) and pyrazole phenyl ether (PPE) herbicide classes. The (R) enantiomers were 10‐ to 44‐fold more active than the (S) enantiomers as inhibitors of Protox from barley etioplasts. Similarly, the (R) enantiomers caused green barley tissue to accumulate greater amounts of porphyrins and caused greater tissue damage than the (S) enantiomers. The (R) enantiomers competed more successfully with [ 14 C]acifluorfen than the (S) enantiomers for the binding sites on Protox. In the PPE class, the in‐vitro and in‐vivo activity differences were wider in the isopropyl pairs than in the n‐propyl pairs. The DPE enantiomers were tested on ten dicotyledonous and six monocotyledonous weed species and ten crops for weed control and crop safety. In general, neither enantiomer had pre‐emergence activity on monocotyledons, but the (R) enantiomer provided some monocotyledonous weed control when applied post‐emergence. On dicotyledons, the (R) enantiomer provided excellent pre‐emergence control, whereas the (S) enantiomer was inactive. The (R) enantiomer caused no injury to corn, cotton, peanuts, rice, sorghum, or soybeans applied pre‐emergence, but it severely injured crops when applied post‐emergence. There was a positive correlation between the activities of the compounds at the molecular, cellular and whole plant levels. The only molecular property differences found to account for differences in activity between members of chiral pairs were steric parameters.