amanda dowson - Academia.edu (original) (raw)

Papers by amanda dowson

Springer eBooks, 1997

The engineering of plants capable of fixing their own nitrogen is an extremely complex task, requ... more The engineering of plants capable of fixing their own nitrogen is an extremely complex task, requiring the coordinated and regulated expression of 16 nif genes in an appropriate cellular location. We suggest that plastids may provide a favourable environment for nif gene expression provided that the nitrogenase enzyme can be protected from oxygen damage. Using the non-heterocystous cyanobacteria as a model, we argue that photosynthesis could be temporally separated from nitrogen fixation in chloroplasts by restricting nitrogenase synthesis to the dark period. We report preliminary data on the introduction and expression of one of nitrogenase components, the Fe protein, in transgenic tobacco and Chlamydomonas reinhardtii. Finally we discuss potential avenues for further research in this area and the prospects for achieving the ultimate goal of expressing active nitrogenase in cereal crops such as rice.

Carolina Digital Repository (University of North Carolina at Chapel Hill), 2000

Light regulates various aspects of plant growth, and the photoreceptor phytochrome B (phyB) media... more Light regulates various aspects of plant growth, and the photoreceptor phytochrome B (phyB) mediates many responses to red light. In a screen for Arabidopsis mutants with phenotypes similar to those of phyB mutants, we isolated two new elf3 mutants. One has weaker morphological phenotypes than previously identified elf3 alleles, but still abolishes circadian rhythms under continuous light. Like phyB mutants, elf3 mutants have elongated hypocotyls and petioles, flower early, and have defects in the red light response. However, we found that elf3 mutations have an additive interaction with a phyB null mutation, with phyA or hy4 null mutations, or with a PHYB overexpression construct, and that an elf3 mutation does not prevent nuclear localization of phyB. These results suggest that either there is substantial redundancy in phyB and elf3 function, or the two genes regulate distinct signaling pathways.

Plant Science, 1987

During grain filling in wheat (Triticum aestivum L.) there is a progressive increase in the numbe... more During grain filling in wheat (Triticum aestivum L.) there is a progressive increase in the number of amyloplasts in the endosperm, as well as in cell number, DNA content and nuclear ploidy as the grain increases in size. The plastid DNA content also rises initially, and then there is a levelling off in the amount, with the percentage plastid DNA finally making up approximately 0.9% of the total endosperm DNA.

Plant Journal, 1999

Many endogenous and environmental signals control seedling growth, including several phototransdu... more Many endogenous and environmental signals control seedling growth, including several phototransduction pathways. We demonstrate that the circadian clock controls the elongation of the Arabidopsis hypocotyl immediately upon germination. The pattern of hypocotyl elongation in constant light includes a daily growth arrest spanning subjective dawn and an interval of rapid growth at subjective dusk. Maximal hypocotyl growth coincides with the phase during which the cotyledons are raised, in the previously described rhythm of cotyledon movement. The rhythm of hypocotyl elongation was entrained by light-dark cycles applied to the imbibed seed and its period was shortened in the toc1-1 mutant, indicating that it is controlled by a similar circadian system to other rhythmic markers. The daily growth arrest is abolished by the early flowering 3 (elf3) mutation, suggesting that this defect may cause its long-hypocotyl phenotype. Mutations that affect the circadian system can therefore cause gross morphological phenotypes, not because the wild-type gene functions pleiotropically in several signalling pathways, but rather because the circadian clock exerts widespread control over plant physiology.

Plant Molecular Biology Reporter, Dec 1, 1994

A number of plant expression cassettes have been constructed that are designed to ensure efficien... more A number of plant expression cassettes have been constructed that are designed to ensure efficient ribosome recruitment, thereby improving translational efficiency. For this purpose the cassettes employ the untranslated leader of the tobacco mosaic virus (TMV) RNA. They also comprise the cauhflower mosaic virus (CaMV) 35 S promoter, with duplicated upstream elements, and the nopaline synthase (nos) terminator. Cloning nests were included 3' to the TMV leader and 5' to the promoter to facilitate the construction of transcriptional or translational fusions and promoter modifications. In both a transient assay system and transgenic tobacco these cassettes were found to facilitate highexpression levels of a cytosolic and plastid-targeted reporter protein (~-glucuronidase). Also they enabled the detection of a hitherto very poorly expressed protein. by the method of Watts, Dawson and King (1980). 9 Remove the lower epidermis of a greenhouse-grown leaf using forceps. 1 9 Cut the leaf into 2-cm 2 pieces and transfer to 100 mL 0.7M mannitol in a 250-mL Erlenmeyer flask. 9 Replace 0.7 M mannitol with 30 mL Macerozyme solution and vacuum infiltrate the leaf pieces for 30 s at 10 mm Hg. 9 Shake on a reciprocating waterbath at 25~ stroke 2.5 cm. frequency 2 Hz, for 5 min. 9 Replace the solution with 35 mL Macerozyme solution and shake as before but for 20 rain. 9 Collect the cells by filtration through a 1-mm nylon mesh. 9 Return the leaf pieces to the flask, add 35 mL macerozyme solution and shake for a further 30 min. 9 Collect the cell suspension as before. 9 Wash the cells once with 0.7 M mannitol by centrifugation at 600 rpm for 2 rain. 9 Resuspend in 50 mL cellulase solution and shake at 35~ stroke 2.5 cm, frequency I Hz, for lh. 2 9 Filter the protoplasts through two layers of cheesecloth and wash with 0.7 M mannitol until the supernatant is only slightly coloured. 9 Resuspend the protoplasts to 2.5 x 10 s/mLinDNA solution at 6~ 3A 9 Electroporate protoplasts with a single square voltage pulse, at 500 V/cm, of 1-3 ms duration.

Nitrogen Fixation, 1991

Nicotiana tabacum has been examined by construction of hybrid nifH and nifM genes which should ta... more Nicotiana tabacum has been examined by construction of hybrid nifH and nifM genes which should target their polypeptide products to the plant chloroplast. In vitro import of these proteins into isolated chloroplasts has been demonstrated both aerobically and anaerobically. Initial attempts to express the engineered genes in transformed plants were unsuccessful, possibly because of the low translational efficiency of the constructs used. Modified plant vectors have now been constructed using viral leader sequences. After electroporation into tobacco mesophyll protoplasts these vectors show significantly improved expression as measured by transient expression assays wi th 8-glucuronidase as the reporter. The improved vectors are now being assessed in transformed plants.

Plant Physiology

Accumulating evidence suggests that peptidoglycan, consistent with a bacterial cell wall, is synt... more Accumulating evidence suggests that peptidoglycan, consistent with a bacterial cell wall, is synthesized around the chloroplasts of many photosynthetic eukaryotes, from glaucophyte algae to early-diverging land plants including pteridophyte ferns, but the biosynthetic pathway has not been demonstrated. Here, we employed mass spectrometry and enzymology in a two-fold approach to characterize the synthesis of peptidoglycan in chloroplasts of the moss Physcomitrium (Physcomitrella) patens. To drive the accumulation of peptidoglycan pathway intermediates, P. patens was cultured with the antibiotics fosfomycin, D-cycloserine, and carbenicillin, which inhibit key peptidoglycan pathway proteins in bacteria. Mass spectrometry of the trichloroacetic acid-extracted moss metabolome revealed elevated levels of five of the predicted intermediates from uridine diphosphate N-acetylglucosamine (UDP-GlcNAc) through the uridine diphosphate N-acetylmuramic acid (UDP-MurNAc)-D,L-diaminopimelate (DAP)-p...

Current Plant Science and Biotechnology in Agriculture

Over the past two decades, strategies for the engineering of nitrogen fixation (nif) genes into h... more Over the past two decades, strategies for the engineering of nitrogen fixation (nif) genes into higher plants plastids have been proposed, but the O2 sensitivity of the nitrogenase component proteins has been considered to be a major problem because nitrogen fixation is not compatible with photosynthetic O2 evolution (1). We have used homologous recombination events to replace precisely the entire chlL coding region in the C. reinhardtii plastome with the eubacterial genes nifH (Figure 1). Surprisingly, nifH can partially restore the capacity for chlorophyll biosynthesis in the dark. Recent in vitro reconstitution of DPOR with purified BchL, BchN-BchB subunits (2) also indicate the structural and mechanistic similarity between DPOR and nitrogenase. Since K. peumoniae nifH can complementarily replace the function of C. reinhardtii chlL, by analogy ChlL might also function in ATP-coupled electron transfer to the other components of the light-independent protochlorophyllide reductase (DPOR) encoded by ChlN and ChlB, which have sequence similarities with NifD and NifK (3). However, the details of the subunit structures, biochemical properties and requirements for biosynthesis of DPOR are as yet largely unknown. It has been proposed that the “chlorophyll iron proteins” evolved from the nitrogenase Fe protein as a consequence of a gene duplication event (4). Our results may have provided in vivo evidence that these two proteins are similar in function as well as structure.

Post-Transcriptional Control of Gene Expression, 1990

The primary function of a viral genome is to replicate to produce progeny virions. To achieve thi... more The primary function of a viral genome is to replicate to produce progeny virions. To achieve this, the exquisitely compact genetic information must perform several functions very efficiently and it is not uncommon for one sequence to fulfil several unrelated tasks. One such pleiotropic sequence, the 5′-untranslated leader of tobacco mosaic virus (TMV) RNA, is the main subject of this article.

The Plant Journal, 1999

Many endogenous and environmental signals control seedling growth, including several phototransdu... more Many endogenous and environmental signals control seedling growth, including several phototransduction pathways. We demonstrate that the circadian clock controls the elongation of the Arabidopsis hypocotyl immediately upon germination. The pattern of hypocotyl elongation in constant light includes a daily growth arrest spanning subjective dawn and an interval of rapid growth at subjective dusk. Maximal hypocotyl growth coincides with the phase during which the cotyledons are raised, in the previously described rhythm of cotyledon movement. The rhythm of hypocotyl elongation was entrained by light-dark cycles applied to the imbibed seed and its period was shortened in the toc1-1 mutant, indicating that it is controlled by a similar circadian system to other rhythmic markers. The daily growth arrest is abolished by the early flowering 3 (elf3) mutation, suggesting that this defect may cause its long-hypocotyl phenotype. Mutations that affect the circadian system can therefore cause gross morphological phenotypes, not because the wild-type gene functions pleiotropically in several signalling pathways, but rather because the circadian clock exerts widespread control over plant physiology.

Plant Physiology, 2004

Ethylene controls multiple physiological processes in plants, including cell elongation. Conseque... more Ethylene controls multiple physiological processes in plants, including cell elongation. Consequently, ethylene synthesis is regulated by internal and external signals. We show that a light-entrained circadian clock regulates ethylene release from unstressed, wild-type Arabidopsis (Arabidopsis thaliana) seedlings, with a peak in the mid-subjective day. The circadian clock drives the expression of multiple ACC SYNTHASE genes, resulting in peak RNA levels at the phase of maximal ethylene synthesis. Ethylene production levels are tightly correlated with ACC SYNTHASE 8 steady-state transcript levels. The expression of this gene is controlled by light, by the circadian clock, and by negative feedback regulation through ethylene signaling. In addition, ethylene production is controlled by the TIMING OF CAB EXPRESSION 1 and CIRCADIAN CLOCK ASSOCIATED 1 genes, which are critical for all circadian rhythms yet tested in Arabidopsis. Mutation of ethylene signaling pathways did not alter the ph...

Plant Physiology, 2000

Light regulates various aspects of plant growth, and the photoreceptor phytochrome B (phyB) media... more Light regulates various aspects of plant growth, and the photoreceptor phytochrome B (phyB) mediates many responses to red light. In a screen for Arabidopsis mutants with phenotypes similar to those ofphyB mutants, we isolated two new elf3mutants. One has weaker morphological phenotypes than previously identified elf3 alleles, but still abolishes circadian rhythms under continuous light. Like phyB mutants,elf3 mutants have elongated hypocotyls and petioles, flower early, and have defects in the red light response. However, we found that elf3 mutations have an additive interaction with a phyB null mutation, with phyA orhy4 null mutations, or with a PHYB overexpression construct, and that an elf3 mutation does not prevent nuclear localization of phyB. These results suggest that either there is substantial redundancy in phyB and elf3 function, or the two genes regulate distinct signaling pathways.

Ethylene controls multiple physiological processes in plants, including cell elongation. Conseque... more Ethylene controls multiple physiological processes in plants, including cell elongation. Consequently, ethylene synthesis is regulated by internal and external signals. We show that a light-entrained circadian clock regulates ethylene release from unstressed, wild-type Arabidopsis (Arabidopsis thaliana) seedlings, with a peak in the mid-subjective day. The circadian clock drives the expression of multiple ACC SYNTHASE genes, resulting in peak RNA levels at the phase of maximal ethylene synthesis. Ethylene production levels are tightly correlated withACC SYNTHASE 8 steady-state transcript levels. The expression of this gene is controlled by light, by the circadian clock, and by negative feedback regulation through ethylene signaling. In addition, ethylene production is controlled by the TIMING OF CAB EXPRESSION 1 and CIRCADIAN CLOCK ASSOCIATED 1 genes, which are critical for all circadian rhythms yet tested in Arabidopsis. Mutation of ethylene signaling pathways did not alter the pha...

Current Plant Science and Biotechnology in Agriculture

Biochemical and Biophysical Research Communications, 2005

The entire coding region of chlL, an essential chloroplast gene required for chlorophyll biosynth... more The entire coding region of chlL, an essential chloroplast gene required for chlorophyll biosynthesis in the dark in Chlamydomonas reinhardtii, was precisely replaced by either the Klebsiella pneumoniae nifH (encoding the structural component of nitrogenase Fe protein) or the Escherichia coli uidA reporter gene encoding b-glucuronidase. Homoplasmic nifH or uidA transformants were identified by Southern blots after selection on minimal medium plates for several generations. All the uidA transformants had the ''yellow-in-the-dark'' phenotype characteristic of chlL mutants, whereas homoplasmic nifH transformants exhibited a partial ''green-in-the-dark'' phenotype. NifH protein was detected in the nifH transformants but not in the wild-type strain by Western blotting. Fluorescence emission measurements also showed the existence of chlorophyll in the dark-grown nifH transformants, but not in the dark-grown uidA transformants. The nifH transplastomic form of C. reinhardtii that lacks the chlL gene can still produce chlorophyll in the dark, suggesting that the nifH product can at least partially substitute for the function of the putative ''chlorophyll iron protein'' encoded by chlL. Thus, introducing nitrogen fixation gene directly into a chloroplast genome is likely to be feasible and providing a possible way of engineering chloroplasts with functional nitrogenase. Notably, to introduce foreign genes without also introducing selective marker genes, a novel two-step chloroplast transformation strategy has been developed.

Opportunities for Biological Nitrogen Fixation in Rice and Other Non-Legumes, 1997

The engineering of plants capable of fixing their own nitrogen is an extremely complex task, requ... more The engineering of plants capable of fixing their own nitrogen is an extremely complex task, requiring the coordinated and regulated expression of 16 nif genes in an appropriate cellular location. We suggest that plastids may provide a favourable environment for nif gene expression provided that the nitrogenase enzyme can be protected from oxygen damage. Using the non-heterocystous cyanobacteria as a model, we argue that photosynthesis could be temporally separated from nitrogen fixation in chloroplasts by restricting nitrogenase synthesis to the dark period. We report preliminary data on the introduction and expression of one of nitrogenase components, the Fe protein, in transgenic tobacco and Chlamydomonas reinhardtii. Finally we discuss potential avenues for further research in this area and the prospects for achieving the ultimate goal of expressing active nitrogenase in cereal crops such as rice

An accumulation of evidence suggests that peptidoglycan, consistent with a bacterial cell wall, i... more An accumulation of evidence suggests that peptidoglycan, consistent with a bacterial cell wall, is synthesised around the chloroplasts of many photosynthetic eukaryotes, from glaucophyte algae to land plants at least as evolved as pteridophyte ferns, but the biosynthetic pathway has not been demonstrated. We employed mass spectrometry and enzymology in a twofold approach to characterize the synthesis of peptidoglycan in chloroplasts of the moss Physcomitrium (Physcomitrella) patens. To drive the accumulation of peptidoglycan pathway intermediates, P.patens was cultured with the antibiotics phosphomycin, D-cycloserine and carbenicillin, which inhibit key peptidoglycan pathway proteins in bacteria. Mass spectrometry of the TCA-extracted moss metabolome revealed elevated levels of five of the predicted intermediates from UDP-GlcNAc through to the UDP-MurNAc-D,L-diaminopimelate (DAP)-pentapeptide.Most Gram negative bacteria, including cyanobacteria, incorporate meso-diaminopimelate (D,L...

ACS Chemical Biology

Bacteria exhibit a myriad of different morphologies, through the synthesis and modification of th... more Bacteria exhibit a myriad of different morphologies, through the synthesis and modification of their essential peptidoglycan (PG) cell wall. Our discovery of a fluorescent D-amino acid (FDAA)-based PG labeling approach provided a powerful method for observing how these morphological changes occur. Given that PG is unique to bacterial cells and a common target for antibiotics, understanding the precise mechanism(s) for incorporation of (F)DAA-based probes is a crucial determinant in understanding the role of PG synthesis in bacterial cell biology and could provide a valuable tool in the development of new antimicrobials to treat drug-resistant antibacterial infections. Here, we systematically investigate the mechanisms of FDAA probe incorporation into PG using two model organisms Escherichia coli (Gramnegative) and Bacillus subtilis (Gram-positive). Our in vitro and in vivo data unequivocally demonstrate that these bacteria incorporate FDAAs using two extracytoplasmic pathways: through activity of their D,D-transpeptidases, and, if present, by their L,D-transpeptidases and not via cytoplasmic incorporation into a D-Ala-D-Ala dipeptide precursor. Our data also revealed the unprecedented finding that the DAA-drug, D-cycloserine, can be incorporated into peptide stems by each of these transpeptidases, in addition to its known inhibitory activity against D-alanine racemase and D-Ala-D-Ala ligase. These mechanistic findings enabled development of a new, FDAA-based, in vitro labeling approach that reports on subcellular distribution of muropeptides, an especially important attribute to enable the study of bacteria with poorly defined growth modes. An improved understanding of the incorporation mechanisms utilized by DAAbased probes is essential when interpreting results from high resolution experiments and highlights the antimicrobial potential of synthetic DAAs.

ACS Chemical Biology, Nov 19, 2019

Bacteria exhibit a myriad of different morphologies, through the synthesis and modification of th... more Bacteria exhibit a myriad of different morphologies, through the synthesis and modification of their essential peptidoglycan (PG) cell wall. Our discovery of a fluorescent D-amino acid (FDAA)-based PG labeling approach provided a powerful method for observing how these morphological changes occur. Given that PG is unique to bacterial cells and a common target for antibiotics, understanding the precise mechanism(s) for incorporation of (F)DAA-based probes is a crucial determinant in understanding the role of PG synthesis in bacterial cell biology and could provide a valuable tool in the development of new antimicrobials to treat drug-resistant antibacterial infections. Here, we systematically investigate the mechanisms of FDAA probe incorporation into PG using two model organisms Escherichia coli (Gram-negative) and Bacillus subtilis (Gram-positive). Our in vitro and in vivo data unequivocally demonstrate that these bacteria incorporate FDAAs using two extracytoplasmic pathways: through activity of their D,D-transpeptidases, and, if present, by their L,D-transpeptidases and not via cytoplasmic incorporation into a D-Ala-D-Ala dipeptide precursor. Our data also revealed the unprecedented finding that the DAA-drug, D-cycloserine, can be incorporated into peptide stems by each of these transpeptidases, in addition to its known inhibitory activity against D-alanine racemase and D-Ala-D-Ala ligase. These mechanistic findings enabled development of a new, FDAA-based, in vitro labeling approach that reports on subcellular distribution of muropeptides, an especially important attribute to enable the study of bacteria with poorly defined growth modes. An improved understanding of the incorporation mechanisms utilized by DAA-based probes is essential when interpreting results from high resolution experiments and highlights the antimicrobial potential of synthetic DAAs.

Plant Molecular Biology, 1993

In order to optimise expression of a foreign protein in transgenic plants we investigated the pot... more In order to optimise expression of a foreign protein in transgenic plants we investigated the potential benefits of including a viral untranslated leader sequence within a plant transformation vector. A variety of 5 leaders, including the tobacco mosaic virus (TMV) leader sequence and 31 nucleotides of the cauliflower mosaic virus (CaMV) 35S RNA leader, were compared. Viral leader constructs employing the 35S promoter and the reporter β-glucuronidase (GUS) were tested by electroporation into tobacco mesophyll protoplasts and against a cointroduced chloramphenicol acetyl transferase (CAT) gene in transgenic tobacco leaves. In the transient assay system, GUS activities from the viral leaders were compared with those from either a short, random leader or a translational fusion of the CaMV 19S RNA ORF VI to GUS. A two-to-three-fold enhanced level of expression resulted when these leaders were substituted with either the 35S RNA or the TMV leader sequences. This enhancement was further increased, to four-to five-fold, by inclusion of four or seven of the bases from the 35S transcription initiation site adjacent to the TMV leader. In transgenic tobacco the improved GUS levels were maintained from constructs including either the TMV leader (eight-fold) or this sequence with the addition of the 35S transcription initiation site bases (ten-fold). A comparison of GUS enzyme amounts with GUS mRNA amounts, using the CAT gene as an internal standard, revealed that TMV leader-bearing mRNA was translated from four-to six-fold more efficiently than the random leader control.

Springer eBooks, 1997

The engineering of plants capable of fixing their own nitrogen is an extremely complex task, requ... more The engineering of plants capable of fixing their own nitrogen is an extremely complex task, requiring the coordinated and regulated expression of 16 nif genes in an appropriate cellular location. We suggest that plastids may provide a favourable environment for nif gene expression provided that the nitrogenase enzyme can be protected from oxygen damage. Using the non-heterocystous cyanobacteria as a model, we argue that photosynthesis could be temporally separated from nitrogen fixation in chloroplasts by restricting nitrogenase synthesis to the dark period. We report preliminary data on the introduction and expression of one of nitrogenase components, the Fe protein, in transgenic tobacco and Chlamydomonas reinhardtii. Finally we discuss potential avenues for further research in this area and the prospects for achieving the ultimate goal of expressing active nitrogenase in cereal crops such as rice.

Carolina Digital Repository (University of North Carolina at Chapel Hill), 2000

Light regulates various aspects of plant growth, and the photoreceptor phytochrome B (phyB) media... more Light regulates various aspects of plant growth, and the photoreceptor phytochrome B (phyB) mediates many responses to red light. In a screen for Arabidopsis mutants with phenotypes similar to those of phyB mutants, we isolated two new elf3 mutants. One has weaker morphological phenotypes than previously identified elf3 alleles, but still abolishes circadian rhythms under continuous light. Like phyB mutants, elf3 mutants have elongated hypocotyls and petioles, flower early, and have defects in the red light response. However, we found that elf3 mutations have an additive interaction with a phyB null mutation, with phyA or hy4 null mutations, or with a PHYB overexpression construct, and that an elf3 mutation does not prevent nuclear localization of phyB. These results suggest that either there is substantial redundancy in phyB and elf3 function, or the two genes regulate distinct signaling pathways.

Plant Science, 1987

During grain filling in wheat (Triticum aestivum L.) there is a progressive increase in the numbe... more During grain filling in wheat (Triticum aestivum L.) there is a progressive increase in the number of amyloplasts in the endosperm, as well as in cell number, DNA content and nuclear ploidy as the grain increases in size. The plastid DNA content also rises initially, and then there is a levelling off in the amount, with the percentage plastid DNA finally making up approximately 0.9% of the total endosperm DNA.

Plant Journal, 1999

Many endogenous and environmental signals control seedling growth, including several phototransdu... more Many endogenous and environmental signals control seedling growth, including several phototransduction pathways. We demonstrate that the circadian clock controls the elongation of the Arabidopsis hypocotyl immediately upon germination. The pattern of hypocotyl elongation in constant light includes a daily growth arrest spanning subjective dawn and an interval of rapid growth at subjective dusk. Maximal hypocotyl growth coincides with the phase during which the cotyledons are raised, in the previously described rhythm of cotyledon movement. The rhythm of hypocotyl elongation was entrained by light-dark cycles applied to the imbibed seed and its period was shortened in the toc1-1 mutant, indicating that it is controlled by a similar circadian system to other rhythmic markers. The daily growth arrest is abolished by the early flowering 3 (elf3) mutation, suggesting that this defect may cause its long-hypocotyl phenotype. Mutations that affect the circadian system can therefore cause gross morphological phenotypes, not because the wild-type gene functions pleiotropically in several signalling pathways, but rather because the circadian clock exerts widespread control over plant physiology.

Plant Molecular Biology Reporter, Dec 1, 1994

A number of plant expression cassettes have been constructed that are designed to ensure efficien... more A number of plant expression cassettes have been constructed that are designed to ensure efficient ribosome recruitment, thereby improving translational efficiency. For this purpose the cassettes employ the untranslated leader of the tobacco mosaic virus (TMV) RNA. They also comprise the cauhflower mosaic virus (CaMV) 35 S promoter, with duplicated upstream elements, and the nopaline synthase (nos) terminator. Cloning nests were included 3' to the TMV leader and 5' to the promoter to facilitate the construction of transcriptional or translational fusions and promoter modifications. In both a transient assay system and transgenic tobacco these cassettes were found to facilitate highexpression levels of a cytosolic and plastid-targeted reporter protein (~-glucuronidase). Also they enabled the detection of a hitherto very poorly expressed protein. by the method of Watts, Dawson and King (1980). 9 Remove the lower epidermis of a greenhouse-grown leaf using forceps. 1 9 Cut the leaf into 2-cm 2 pieces and transfer to 100 mL 0.7M mannitol in a 250-mL Erlenmeyer flask. 9 Replace 0.7 M mannitol with 30 mL Macerozyme solution and vacuum infiltrate the leaf pieces for 30 s at 10 mm Hg. 9 Shake on a reciprocating waterbath at 25~ stroke 2.5 cm. frequency 2 Hz, for 5 min. 9 Replace the solution with 35 mL Macerozyme solution and shake as before but for 20 rain. 9 Collect the cells by filtration through a 1-mm nylon mesh. 9 Return the leaf pieces to the flask, add 35 mL macerozyme solution and shake for a further 30 min. 9 Collect the cell suspension as before. 9 Wash the cells once with 0.7 M mannitol by centrifugation at 600 rpm for 2 rain. 9 Resuspend in 50 mL cellulase solution and shake at 35~ stroke 2.5 cm, frequency I Hz, for lh. 2 9 Filter the protoplasts through two layers of cheesecloth and wash with 0.7 M mannitol until the supernatant is only slightly coloured. 9 Resuspend the protoplasts to 2.5 x 10 s/mLinDNA solution at 6~ 3A 9 Electroporate protoplasts with a single square voltage pulse, at 500 V/cm, of 1-3 ms duration.

Nitrogen Fixation, 1991

Nicotiana tabacum has been examined by construction of hybrid nifH and nifM genes which should ta... more Nicotiana tabacum has been examined by construction of hybrid nifH and nifM genes which should target their polypeptide products to the plant chloroplast. In vitro import of these proteins into isolated chloroplasts has been demonstrated both aerobically and anaerobically. Initial attempts to express the engineered genes in transformed plants were unsuccessful, possibly because of the low translational efficiency of the constructs used. Modified plant vectors have now been constructed using viral leader sequences. After electroporation into tobacco mesophyll protoplasts these vectors show significantly improved expression as measured by transient expression assays wi th 8-glucuronidase as the reporter. The improved vectors are now being assessed in transformed plants.

Plant Physiology

Accumulating evidence suggests that peptidoglycan, consistent with a bacterial cell wall, is synt... more Accumulating evidence suggests that peptidoglycan, consistent with a bacterial cell wall, is synthesized around the chloroplasts of many photosynthetic eukaryotes, from glaucophyte algae to early-diverging land plants including pteridophyte ferns, but the biosynthetic pathway has not been demonstrated. Here, we employed mass spectrometry and enzymology in a two-fold approach to characterize the synthesis of peptidoglycan in chloroplasts of the moss Physcomitrium (Physcomitrella) patens. To drive the accumulation of peptidoglycan pathway intermediates, P. patens was cultured with the antibiotics fosfomycin, D-cycloserine, and carbenicillin, which inhibit key peptidoglycan pathway proteins in bacteria. Mass spectrometry of the trichloroacetic acid-extracted moss metabolome revealed elevated levels of five of the predicted intermediates from uridine diphosphate N-acetylglucosamine (UDP-GlcNAc) through the uridine diphosphate N-acetylmuramic acid (UDP-MurNAc)-D,L-diaminopimelate (DAP)-p...

Current Plant Science and Biotechnology in Agriculture

Over the past two decades, strategies for the engineering of nitrogen fixation (nif) genes into h... more Over the past two decades, strategies for the engineering of nitrogen fixation (nif) genes into higher plants plastids have been proposed, but the O2 sensitivity of the nitrogenase component proteins has been considered to be a major problem because nitrogen fixation is not compatible with photosynthetic O2 evolution (1). We have used homologous recombination events to replace precisely the entire chlL coding region in the C. reinhardtii plastome with the eubacterial genes nifH (Figure 1). Surprisingly, nifH can partially restore the capacity for chlorophyll biosynthesis in the dark. Recent in vitro reconstitution of DPOR with purified BchL, BchN-BchB subunits (2) also indicate the structural and mechanistic similarity between DPOR and nitrogenase. Since K. peumoniae nifH can complementarily replace the function of C. reinhardtii chlL, by analogy ChlL might also function in ATP-coupled electron transfer to the other components of the light-independent protochlorophyllide reductase (DPOR) encoded by ChlN and ChlB, which have sequence similarities with NifD and NifK (3). However, the details of the subunit structures, biochemical properties and requirements for biosynthesis of DPOR are as yet largely unknown. It has been proposed that the “chlorophyll iron proteins” evolved from the nitrogenase Fe protein as a consequence of a gene duplication event (4). Our results may have provided in vivo evidence that these two proteins are similar in function as well as structure.

Post-Transcriptional Control of Gene Expression, 1990

The primary function of a viral genome is to replicate to produce progeny virions. To achieve thi... more The primary function of a viral genome is to replicate to produce progeny virions. To achieve this, the exquisitely compact genetic information must perform several functions very efficiently and it is not uncommon for one sequence to fulfil several unrelated tasks. One such pleiotropic sequence, the 5′-untranslated leader of tobacco mosaic virus (TMV) RNA, is the main subject of this article.

The Plant Journal, 1999

Many endogenous and environmental signals control seedling growth, including several phototransdu... more Many endogenous and environmental signals control seedling growth, including several phototransduction pathways. We demonstrate that the circadian clock controls the elongation of the Arabidopsis hypocotyl immediately upon germination. The pattern of hypocotyl elongation in constant light includes a daily growth arrest spanning subjective dawn and an interval of rapid growth at subjective dusk. Maximal hypocotyl growth coincides with the phase during which the cotyledons are raised, in the previously described rhythm of cotyledon movement. The rhythm of hypocotyl elongation was entrained by light-dark cycles applied to the imbibed seed and its period was shortened in the toc1-1 mutant, indicating that it is controlled by a similar circadian system to other rhythmic markers. The daily growth arrest is abolished by the early flowering 3 (elf3) mutation, suggesting that this defect may cause its long-hypocotyl phenotype. Mutations that affect the circadian system can therefore cause gross morphological phenotypes, not because the wild-type gene functions pleiotropically in several signalling pathways, but rather because the circadian clock exerts widespread control over plant physiology.

Plant Physiology, 2004

Ethylene controls multiple physiological processes in plants, including cell elongation. Conseque... more Ethylene controls multiple physiological processes in plants, including cell elongation. Consequently, ethylene synthesis is regulated by internal and external signals. We show that a light-entrained circadian clock regulates ethylene release from unstressed, wild-type Arabidopsis (Arabidopsis thaliana) seedlings, with a peak in the mid-subjective day. The circadian clock drives the expression of multiple ACC SYNTHASE genes, resulting in peak RNA levels at the phase of maximal ethylene synthesis. Ethylene production levels are tightly correlated with ACC SYNTHASE 8 steady-state transcript levels. The expression of this gene is controlled by light, by the circadian clock, and by negative feedback regulation through ethylene signaling. In addition, ethylene production is controlled by the TIMING OF CAB EXPRESSION 1 and CIRCADIAN CLOCK ASSOCIATED 1 genes, which are critical for all circadian rhythms yet tested in Arabidopsis. Mutation of ethylene signaling pathways did not alter the ph...

Plant Physiology, 2000

Light regulates various aspects of plant growth, and the photoreceptor phytochrome B (phyB) media... more Light regulates various aspects of plant growth, and the photoreceptor phytochrome B (phyB) mediates many responses to red light. In a screen for Arabidopsis mutants with phenotypes similar to those ofphyB mutants, we isolated two new elf3mutants. One has weaker morphological phenotypes than previously identified elf3 alleles, but still abolishes circadian rhythms under continuous light. Like phyB mutants,elf3 mutants have elongated hypocotyls and petioles, flower early, and have defects in the red light response. However, we found that elf3 mutations have an additive interaction with a phyB null mutation, with phyA orhy4 null mutations, or with a PHYB overexpression construct, and that an elf3 mutation does not prevent nuclear localization of phyB. These results suggest that either there is substantial redundancy in phyB and elf3 function, or the two genes regulate distinct signaling pathways.

Ethylene controls multiple physiological processes in plants, including cell elongation. Conseque... more Ethylene controls multiple physiological processes in plants, including cell elongation. Consequently, ethylene synthesis is regulated by internal and external signals. We show that a light-entrained circadian clock regulates ethylene release from unstressed, wild-type Arabidopsis (Arabidopsis thaliana) seedlings, with a peak in the mid-subjective day. The circadian clock drives the expression of multiple ACC SYNTHASE genes, resulting in peak RNA levels at the phase of maximal ethylene synthesis. Ethylene production levels are tightly correlated withACC SYNTHASE 8 steady-state transcript levels. The expression of this gene is controlled by light, by the circadian clock, and by negative feedback regulation through ethylene signaling. In addition, ethylene production is controlled by the TIMING OF CAB EXPRESSION 1 and CIRCADIAN CLOCK ASSOCIATED 1 genes, which are critical for all circadian rhythms yet tested in Arabidopsis. Mutation of ethylene signaling pathways did not alter the pha...

Current Plant Science and Biotechnology in Agriculture

Biochemical and Biophysical Research Communications, 2005

The entire coding region of chlL, an essential chloroplast gene required for chlorophyll biosynth... more The entire coding region of chlL, an essential chloroplast gene required for chlorophyll biosynthesis in the dark in Chlamydomonas reinhardtii, was precisely replaced by either the Klebsiella pneumoniae nifH (encoding the structural component of nitrogenase Fe protein) or the Escherichia coli uidA reporter gene encoding b-glucuronidase. Homoplasmic nifH or uidA transformants were identified by Southern blots after selection on minimal medium plates for several generations. All the uidA transformants had the ''yellow-in-the-dark'' phenotype characteristic of chlL mutants, whereas homoplasmic nifH transformants exhibited a partial ''green-in-the-dark'' phenotype. NifH protein was detected in the nifH transformants but not in the wild-type strain by Western blotting. Fluorescence emission measurements also showed the existence of chlorophyll in the dark-grown nifH transformants, but not in the dark-grown uidA transformants. The nifH transplastomic form of C. reinhardtii that lacks the chlL gene can still produce chlorophyll in the dark, suggesting that the nifH product can at least partially substitute for the function of the putative ''chlorophyll iron protein'' encoded by chlL. Thus, introducing nitrogen fixation gene directly into a chloroplast genome is likely to be feasible and providing a possible way of engineering chloroplasts with functional nitrogenase. Notably, to introduce foreign genes without also introducing selective marker genes, a novel two-step chloroplast transformation strategy has been developed.

Opportunities for Biological Nitrogen Fixation in Rice and Other Non-Legumes, 1997

The engineering of plants capable of fixing their own nitrogen is an extremely complex task, requ... more The engineering of plants capable of fixing their own nitrogen is an extremely complex task, requiring the coordinated and regulated expression of 16 nif genes in an appropriate cellular location. We suggest that plastids may provide a favourable environment for nif gene expression provided that the nitrogenase enzyme can be protected from oxygen damage. Using the non-heterocystous cyanobacteria as a model, we argue that photosynthesis could be temporally separated from nitrogen fixation in chloroplasts by restricting nitrogenase synthesis to the dark period. We report preliminary data on the introduction and expression of one of nitrogenase components, the Fe protein, in transgenic tobacco and Chlamydomonas reinhardtii. Finally we discuss potential avenues for further research in this area and the prospects for achieving the ultimate goal of expressing active nitrogenase in cereal crops such as rice

An accumulation of evidence suggests that peptidoglycan, consistent with a bacterial cell wall, i... more An accumulation of evidence suggests that peptidoglycan, consistent with a bacterial cell wall, is synthesised around the chloroplasts of many photosynthetic eukaryotes, from glaucophyte algae to land plants at least as evolved as pteridophyte ferns, but the biosynthetic pathway has not been demonstrated. We employed mass spectrometry and enzymology in a twofold approach to characterize the synthesis of peptidoglycan in chloroplasts of the moss Physcomitrium (Physcomitrella) patens. To drive the accumulation of peptidoglycan pathway intermediates, P.patens was cultured with the antibiotics phosphomycin, D-cycloserine and carbenicillin, which inhibit key peptidoglycan pathway proteins in bacteria. Mass spectrometry of the TCA-extracted moss metabolome revealed elevated levels of five of the predicted intermediates from UDP-GlcNAc through to the UDP-MurNAc-D,L-diaminopimelate (DAP)-pentapeptide.Most Gram negative bacteria, including cyanobacteria, incorporate meso-diaminopimelate (D,L...

ACS Chemical Biology

Bacteria exhibit a myriad of different morphologies, through the synthesis and modification of th... more Bacteria exhibit a myriad of different morphologies, through the synthesis and modification of their essential peptidoglycan (PG) cell wall. Our discovery of a fluorescent D-amino acid (FDAA)-based PG labeling approach provided a powerful method for observing how these morphological changes occur. Given that PG is unique to bacterial cells and a common target for antibiotics, understanding the precise mechanism(s) for incorporation of (F)DAA-based probes is a crucial determinant in understanding the role of PG synthesis in bacterial cell biology and could provide a valuable tool in the development of new antimicrobials to treat drug-resistant antibacterial infections. Here, we systematically investigate the mechanisms of FDAA probe incorporation into PG using two model organisms Escherichia coli (Gramnegative) and Bacillus subtilis (Gram-positive). Our in vitro and in vivo data unequivocally demonstrate that these bacteria incorporate FDAAs using two extracytoplasmic pathways: through activity of their D,D-transpeptidases, and, if present, by their L,D-transpeptidases and not via cytoplasmic incorporation into a D-Ala-D-Ala dipeptide precursor. Our data also revealed the unprecedented finding that the DAA-drug, D-cycloserine, can be incorporated into peptide stems by each of these transpeptidases, in addition to its known inhibitory activity against D-alanine racemase and D-Ala-D-Ala ligase. These mechanistic findings enabled development of a new, FDAA-based, in vitro labeling approach that reports on subcellular distribution of muropeptides, an especially important attribute to enable the study of bacteria with poorly defined growth modes. An improved understanding of the incorporation mechanisms utilized by DAAbased probes is essential when interpreting results from high resolution experiments and highlights the antimicrobial potential of synthetic DAAs.

ACS Chemical Biology, Nov 19, 2019

Bacteria exhibit a myriad of different morphologies, through the synthesis and modification of th... more Bacteria exhibit a myriad of different morphologies, through the synthesis and modification of their essential peptidoglycan (PG) cell wall. Our discovery of a fluorescent D-amino acid (FDAA)-based PG labeling approach provided a powerful method for observing how these morphological changes occur. Given that PG is unique to bacterial cells and a common target for antibiotics, understanding the precise mechanism(s) for incorporation of (F)DAA-based probes is a crucial determinant in understanding the role of PG synthesis in bacterial cell biology and could provide a valuable tool in the development of new antimicrobials to treat drug-resistant antibacterial infections. Here, we systematically investigate the mechanisms of FDAA probe incorporation into PG using two model organisms Escherichia coli (Gram-negative) and Bacillus subtilis (Gram-positive). Our in vitro and in vivo data unequivocally demonstrate that these bacteria incorporate FDAAs using two extracytoplasmic pathways: through activity of their D,D-transpeptidases, and, if present, by their L,D-transpeptidases and not via cytoplasmic incorporation into a D-Ala-D-Ala dipeptide precursor. Our data also revealed the unprecedented finding that the DAA-drug, D-cycloserine, can be incorporated into peptide stems by each of these transpeptidases, in addition to its known inhibitory activity against D-alanine racemase and D-Ala-D-Ala ligase. These mechanistic findings enabled development of a new, FDAA-based, in vitro labeling approach that reports on subcellular distribution of muropeptides, an especially important attribute to enable the study of bacteria with poorly defined growth modes. An improved understanding of the incorporation mechanisms utilized by DAA-based probes is essential when interpreting results from high resolution experiments and highlights the antimicrobial potential of synthetic DAAs.

Plant Molecular Biology, 1993

In order to optimise expression of a foreign protein in transgenic plants we investigated the pot... more In order to optimise expression of a foreign protein in transgenic plants we investigated the potential benefits of including a viral untranslated leader sequence within a plant transformation vector. A variety of 5 leaders, including the tobacco mosaic virus (TMV) leader sequence and 31 nucleotides of the cauliflower mosaic virus (CaMV) 35S RNA leader, were compared. Viral leader constructs employing the 35S promoter and the reporter β-glucuronidase (GUS) were tested by electroporation into tobacco mesophyll protoplasts and against a cointroduced chloramphenicol acetyl transferase (CAT) gene in transgenic tobacco leaves. In the transient assay system, GUS activities from the viral leaders were compared with those from either a short, random leader or a translational fusion of the CaMV 19S RNA ORF VI to GUS. A two-to-three-fold enhanced level of expression resulted when these leaders were substituted with either the 35S RNA or the TMV leader sequences. This enhancement was further increased, to four-to five-fold, by inclusion of four or seven of the bases from the 35S transcription initiation site adjacent to the TMV leader. In transgenic tobacco the improved GUS levels were maintained from constructs including either the TMV leader (eight-fold) or this sequence with the addition of the 35S transcription initiation site bases (ten-fold). A comparison of GUS enzyme amounts with GUS mRNA amounts, using the CAT gene as an internal standard, revealed that TMV leader-bearing mRNA was translated from four-to six-fold more efficiently than the random leader control.