Lee McIntosh

Molecular Basis of Herbicide Resistance in Amaranthus hybridus.

Resistance of different species of weeds to s-triazines, a commonly used class of herbicides, has been shown to involve a change in the binding affinity of the herbicide to a chloroplast polypeptide of 32,000 daltons. A single amino acid difference in this 32,000-dalton protein appears to be responsible for resistance to the herbicide in Amaranthus hybridus.

Higher Plant Mitochondria

Over the past 20 years, researchers investigating the mitochondria of plants have been astonished by the phenomenal variation these organelles display relative to their mammalian and fungal counterparts. Plant mitochondria have evolved distinct strategies for genome maintenance, genetic decoding,

Alternative Oxidase Activity in Tobacco Leaf Mitochondria (Dependence on Tricarboxylic Acid Cycle-Mediated Redox Regulation and Pyruvate Activation)

Transgenic Nicotiana tabacum (cv Petit Havana SR1) containing high levels of mitochondrial alternative oxidase (AOX) protein due to the introduction of a sense transgene(s) of Aox1, the nuclear gene encoding AOX, were used to investigate mechanisms regulating AOX activity. After purification of leaf mitochondria, a large proportion of the AOX protein was present as the oxidized (covalently associated and less active) dimer. High AOX activity in these mitochondria was dependent on both reduction of the protein by DTT (to the noncovalently associated and more active dimer) and its subsequent activation by certain [alpha]-keto acids, particularly pyruvate. Reduction of AOX to its more active form could also be mediated by intramitochondrial reducing power generated by the oxidation of certain tricarboxylic acid cycle substrates, most notably isocitrate and malate. Our evidence suggests that NADPH may be specifically required for AOX reduction. All of the above regulatory mechanisms applied to AOX in wild-type mitochondria as well. Transgenic leaves lacking AOX due to the introduction of an Aox1 antisense transgene or multiple sense transgenes were used to investigate the potential physiological significance of the AOX-regulatory mechanisms. Under conditions in which respiratory carbon metabolism is restricted by the capacity of mitochondrial electron transport, feed-forward activation of AOX by mitochondrial reducing power and pyruvate may act to prevent redirection of carbon metabolism, such as to fermentative pathways.

Salicylic Acid Regulation of Respiration in Higher Plants: Alternative Oxidase Expression.

Alternative respiratory pathway capacity increases during the development of the thermogenic appendix of a voodoo lily inflorescence. The levels of the alternative oxidase proteins increased dramatically between D-4 (4 days prior to the day of anthesis) and D-3 and continued to increase until the day of anthesis (D-day). The level of salicylic acid (SA) in the appendix is very low early on D-1, but increases to a high level in the evening of D-1. Thermogenesis occurs after a few hours of light on D-day. Therefore, the initial accumulation of the alternative oxidase proteins precedes the increase in SA by 3 days, indicating that other regulators may be involved. A 1.6-kb transcript encoding the alternative oxidase precursor protein accumulated to a high level in the appendix tissue by D-1. Application of SA to immature appendix tissue caused an increase in alternative pathway capacity and a dramatic accumulation of the alternative oxidase proteins and the 1.6-kb transcript. Time course experiments showed that the increase in capacity, protein levels, and transcript level corresponded precisely. The response to SA was blocked by cycloheximide or actinomycin D, indicating that de novo transcription and translation are required. However, nuclear, in vitro transcription assays indicated that the accumulation of the 1.6-kb transcript did not result from a simple increase in the rate of transcription of aox1.

Nucleotide sequence of the ribulosebisphosphate carboxylase gene from Rhodospirillum rubrum

SummaryThe nucleotide sequence of a cloned DNA fragment encoding ribulose bisphosphate carboxylase from the purple non-sulfur bacterium Rhodospirillum rubrum has been determined. The deduced amino acid sequence of a 1398 nucleotide open reading frame exhibits weak overall homology to the sequences reported for analogous enzymes from cyanobacteria, algae and angiosperms. Thus knowledge of the sequence is useful in attempts to identify structural features of the enzyme which are essential to catalysis. The gene is flanked by nucleotide sequences similar to those implicated in the initiation of translation and termination of transcription in other bacteria.

Molecular Localization of a Redox-Modulated Process Regulating Plant Mitochondrial Electron Transport

Using in organellar assays, we found that significant tobacco alternative oxidase (AOX) activity is dependent on both reduction of a putative regulatory disulfide bond and the presence of pyruvate, which may interact with a Cys sulfhydryl. This redox modulation and pyruvate activation thus may be important in determining the partitioning of electrons to AOX in vivo. To investigate these regulatory mechanisms, we generated tobacco plants expressing mutated AOX proteins. Mutation of the most N-terminal Cys residue (Cys-126) to an Ala residue produced an AOX that could not be converted to the disulfide-linked form, thus identifying this Cys residue as being responsible for redox modulation. Although this mutation might be expected to produce an AOX with constitutive high activity in the presence of pyruvate, we found it to have minimal in organellar activity in the presence of pyruvate. Nonetheless, the Cys-126 mutation did not appear to have compromised the catalytic function of AOX, given that cells expressing the protein displayed high rates of cyanide-resistant respiration in vivo. The striking difference between in vivo and in organellar results suggests that an additional mechanism(s), as yet unidentified by in organellar assays, may promote activity in vivo. Mutation of the Cys residue nearest the presumptive active site (Cys-176) to an Ala residue did not prevent disulfide bond formation or affect the ability of AOX to be stimulated by pyruvate, indicating that this Cys residue is involved in neither redox modulation nor pyruvate activation.

Targeted genetic inactivation of the photosystem I reaction center in the cyanobacterium Synechocystis sp. PCC 6803.

We describe the first complete segregation of a targeted inactivation of psaA encoding one of the P700‐chlorophyll a apoproteins of photosystem (PS) I. A kanamycin resistance gene was used to interrupt the psaA gene in the unicellular cyanobacterium Synechocystis sp. PCC 6803. Selection of a fully segregated mutant, ADK9, was performed under light‐activated heterotrophic growth (LAHG) conditions; complete darkness except for 5 min of light every 24 h and 5 mM glucose. Under these conditions, wild‐type cells showed a 4‐fold decrease in chlorophyll (chl) per cell, primarily due to a decrease of PS I reaction centers. Evidence for the absence of PS I in ADK9 includes: the lack of EPR (electron paramagnetic resonance) signal I, from P700+; undetectable P700‐apoprotein; greatly reduced whole‐chain photosynthesis rates; and greatly reduced chl per cell, resulting in a turquoise blue phenotype. The PS I peripheral proteins PSA‐C and PSA‐D were not detected in this mutant. ADK9 does assemble near wild‐type levels of functional PS II per cell, evidenced by: EPR signal II from YD+; high rates of oxygen evolution with 2,6‐dichloro‐p‐benzoquinone (DCBQ), an electron acceptor from PS II; and accumulation of D1, a PS II core polypeptide. The success of this transformation indicates that this cyanobacterium may be utilized for site‐directed mutagenesis of the PS I core.

Molecular Genetic Alteration of Plant Respiration (Silencing and Overexpression of Alternative Oxidase in Transgenic Tobacco).

The alternative oxidase (AOX) of plant mitochondria is encoded by the nuclear gene Aox1. Sense and antisense DNA constructs of Nicotiana tabacum Aox1 were introduced into tobacco, and transgenic plants with both increased and decreased levels of mitochondrial AOX protein were identified. Suspension cells derived from wild-type and transgenic plants were grown in heterotrophic batch culture. Transgenic cells with increased AOX protein had an increased capacity for cyanide-resistant, salicylhydroxamic acid-sensitive respiration compared to wild-type cells, whereas transgenic cells with decreased AOX protein had a decreased capacity for such respiration. Thus, genetic alteration of the level of AOX protein was sufficient to alter the capacity for electron transport through the alternative pathway. Under our standard growth conditions, “antisense” cells with dramatically reduced levels of AOX protein had growth and respiration rates similar to the wild type. However, whereas wild-type cells were able to grow under conditions that severely suppressed cytochrome pathway activity, antisense cells could not survive this treatment. This suggests that a critical function of AOX may be to support respiration when the cytochrome pathway is impaired. The much higher level of AOX protein in “sense” cells compared to the wild type did not appreciably alter the steady-state partitioning of electrons between the cytochrome path and the alternative pathway in vivo, suggesting that this partitioning may be subject to additional regulatory factors.

The Molecular Basis of Triazine-Herbicide Resistance in Higher-Plant Chloroplasts

Triazine herbicides inhibit photosynthesis by blocking electron transport in photosystem II. The target site of the herbicide was identified as a chloroplast thylakoid polypeptide (the Qв protein) of 32,000 daltons. Studies of triazine-resistant weed biotypes suggested that a subtle change in the Qв protein caused the resistance. We have cloned the chloroplast gene (psbA) that codes this protein from herbicide-resistant and herbicide-susceptible biotypes of Solanum nigrum. By DNA sequencing we detected a single base substitution in the psbA gene of the resistant plants, resulting in an amino acid change (serine to glycine for the susceptible to resistance conversion). This mutation is exactly the same one which we have described in a herbicide-resistant biotype of Amaranthus hybridus.

Mitochondrial events during development of thermogenesis in Sauromatum guttatum (Schott)

Changes in the mitochondrial electrontransport chain were followed in the thermogenic inflorescence ofSauromatum guttatum Schott from 5d before thermogenesis to 3d thereafter. The capacities of the alternative and cytochrome pathways of mitochondrial electron transport were found to be developmentally coordinated to contribute to the thermogenic events in the appendix and the sterile floral regions. Electron flow through the alternative pathway, is believed primarily responsible for heat production, and this pathway was expressed to the highest degree in both tissues during thermogenesis. In the appendix, the cytochrome chain was shut down considerably during thermogenesis, forcing electron flow through the alternative pathway and thus yielding maximum heat production. The shut-down of the cytochrome chain does not occur in the sterile floral region which may explain why this region is not as thermogenic as the appendix. Cytochrome-oxidase difference spectra indicated that the cytochrome oxidase of appendix mitochondria was not capable of accepting electrons on the day of thermogenesis, and that this capacity was partially restored by the following day even though the tissue was senescing at this time point. Relative levels of messenger RNAs for cytochrome-oxidase subunits I and II were found to decrease the day before thermogenesis, which could result in lower levels of these proteins in appendix mitochondria on the day of thermogenesis.The capacity for overall mitochondrial protein synthesis was also investigated and was found to drop continuously from 5d before thermogenesis to 3d thereafter, even though the capacities of the electron-transport chain were changing dramatically. The levels of mitochondrial ribosomal RNA levels decreased during development, which could explain the overall drop in mitochondrial translational efficiency. Experiments concerning the synthesis of the alternative-oxidase proteins indicated that they were most likely nuclearly encoded, and that their expression could be induced by salicylic acid.