Acetohydroxyacid synthase was discovered as the site of action of imidazolinone and sulfonylurea herbicides over 6 yr ago. In recent years, advances have been made in the understanding of this enzyme as a herbicide target site. Derivatives of both imidazolinones and sulfonylureas have yielded new herbicide chemistry. AH of the herbicides display unusual “slow-binding” behavior with the enzyme, and this behavior may help explain efficacy of the herbicides. Resistance to these herbicides has been developed through a number of different procedures, and the mechanism of resistance is through changes in sensitivity of the enzyme to the herbicides. The changes are either selective to only one class of chemistry, or broad to a number of classes of chemistry. These data support the idea that binding sites for the herbicides on the enzyme are only partially overlapping. Progress in purification of AHAS from corn includes discovery of the existence of the enzyme in monomer and oligomer aggregation states. The interaction of the enzyme with the herbicides is affected by enzyme aggregation state.

Five classes of herbicides (carbamothioates, chloroacetamides, substituted pyridazinones, cyclohexanediones, and aryloxyphenoxypropionic acids) have been reported to inhibit lipid biosynthesis in higher plants. Carbamothioates impair the synthesis of surface lipids (waxes, cutin, suberin). These effects have been attributed to the ability of this herbicide class to inhibit one or more acyl-CoA elongases. Though as yet poorly characterized, these enzymes are associated with the endoplasmic reticulum and catalyze the condensation of malonyl-CoA with fatty acid acyl-CoA substrates to form very long-chain fatty acids used in the synthesis of surface lipids. There is contradictory evidence regarding the effects of chloroacetamide herbicides on de novo fatty acid biosynthesis. Selected substituted pyridazinones decrease the degree of unsaturation of plastidic galactolipids. This effect is attributed to the ability of selected members of this herbicide class to inhibit fatty acid desaturases which are thought to be located in the chloroplast envelope. Aryloxyphenoxypropionic acid and cyclohexanedione herbicides inhibit de novo fatty acid biosynthesis in grasses. The target site for these herbicide classes is the enzyme acetyl-CoA carboxylase which is found in the stroma of plastids. In most cases, selectivity between grasses and dicots is expressed at this site. Aryloxyphenoxypropionic acids and cyclohexanediones are reversible, linear, noncompetitive inhibitors of acetyl-CoA carboxylase from grasses. Both classes are also mutually exclusive inhibitors of grass acetyl-CoA carboxylase which suggests that they bind at a common domain on the enzyme.

Approximately one-quarter of all herbicides that have been marketed affect mitosis as a primary mechanism of action. All of these herbicides appear to interact directly or indirectly with the microtubule. Dinitroaniline and phosphoric amide herbicides inhibit microtubule polymerization from free tubulin subunits. Because of the loss of spindle and kinetochore microtubules, chromosomes cannot move to the poles during mitosis, resulting in cells exhibiting an arrested prometaphase configuration. Nuclear membranes re-form around the chromosomal masses to form lobed nuclei. Cortical microtubules, which influence cell shape, are also absent, and, as a result, the cell expands isodiametrically. In root tips and other structures that are normally elongated, these herbicides induce a characteristic club-shaped swelling. Pronamide and MON 7200 induce similar effects, except that tufts of microtubules remain at the kinetochore region of the chromosomes. The carbamate herbicides barban, propham, and chlorpropham alter the organization of the spindle microtubules so that multiple spindles are formed. Chromosomes move to many poles and multiple nuclei result. Abnormal branched cell walls partly separate the nuclei. Terbutol induces “star anaphase” chromosome configurations in which the chromosomes are drawn into an area at the poles in a star-like aggregation. DCPA's most dramatic effect is on phragmoplast microtubule arrays. Multiple, branched, and curved phragmoplasts are found after herbicide treatment. These disrupters should prove to be useful tools in investigations of the proteins and structures required for a successful cell division.

The two primary sites of herbicide action in photosynthetic electron transport are the inhibition of photosystem II (PS II) electron transport and diversion of electron flow through photosystem I (PS I). PS II electron transport inhibitors bind to the D1 protein of the PS II reaction center, thus blocking electron transfer to plastoquinone. Inhibition of PS II electron transport prevents the conversion of absorbed light energy into electrochemical energy and results in the production of triplet chlorophyll and singlet oxygen which induce the peroxidation of membrane lipids. PS I electron acceptors probably accept electrons from the iron-sulfur protein, Fa/Fb. The free radical form of the herbicide leads to the production of hydroxyl radicals which cause the peroxidation of lipids. Herbicide-induced lipid peroxidation destroys membrane integrity, leading to cellular disorganization and phototoxicity.

Several commercial and experimental herbicides such as p-nitrodiphenyl ethers, oxadiazoles, and cyclic imides inhibit protoporphyrinogen IX oxidase (Protox), the enzyme that converts protoporphyrinogen IX to protoporphyrin IX (Proto). This leads to uncontrolled autooxidation of the substrate and results in accumulation of Proto. Blockage of the porphyrin pathway at this site inhibits synthesis of both chlorophylls and heme. Heme is a feedback regulator of the porphyrin pathway. Thus, inhibition of Protox also deregulates the pathway, causing increased carbon flow to the accumulating pool of Proto. Proto is a potent photosensitizer that generates high levels of singlet oxygen in the presence of molecular oxygen and light. In plants treated with these herbicides, damage is light dependent and closely correlated with the level of Proto that accumulates. Proto accumulation is apparently largely extraplastidic, resulting in rapid photodynamic damage to the plasmalemma and tonoplast. After high levels of Proto accumulate in response to these herbicides, protochlorophyllide (PChlide) levels can increase also; however, Proto appears to be the primary photodynamic pigment responsible for the herbicidal activity.

Many bleaching herbicides with different core structures inhibit phytoene desaturase (PD), a membrane-bound enzyme in the carotenogenic pathway catalyzing the hydrogen abstraction step at the first C40 precursor of β-carotene. Prospects are good that new PD-active herbicides will be discovered by screening for bleaching activity. Accordingly, interest in PD enzymology and molecular genetics has increased. Although active carotenogenic cell-free systems are available, no isolation of PD has been achieved since the enzyme cannot be detected in its isolated form due to complete loss of activity. A portion of the Rhodobacter PD gene was incorporated into an appropriate plasmid which could be expressed in E. coli. This system was used to produce an antibody specific against PD from higher plants as well as Rhodobacter. All PDs assayed had an apparent molecular weight of 52 to 55 kDa. A Rhodobacter gene probe hybridized with a 3.1 kbBamH I fragment from Aphanocapsa which allowed us to sequence the PD gene from this cyanobacterium. Its DNA sequence matched with the apparent molecular weight of the PD band in the western blot, and a fusion-gene product was found to be immunoreactive with the Rhodobacter PD antibody, Anacystis mutants were produced exhibiting cross-resistance against norflurazon and fluorochloridone. Apparently, this resistance is due to an altered PD with concurrent decrease of inhibitor binding affinity. Cloning of the resistant gene into the wild type is in progress.

Rapid strides are being made in understanding the fundamental regulation of plant growth, development, and responses to the environment due to recent advances in molecular biology. Current questions in weed science such as herbicide mechanisms of action, biodegradation, and mechanisms of weed resistance are equally approachable using such methodology. Efforts to introduce herbicide resistance into agronomically important crops are possible because of successful isolation and transfer of genes. Investigations of weed survival and competitive strategies based on developmental processes, such as seed dormancy, are currently underway using techniques designed to monitor and characterize differential gene expression. Molecular methodology also plays a key role in taxonomic studies of weed populations using restriction fragment length polymorphism (RFLP) mapping. The future potential for these and other techniques such as nucleic acid hybridization, polymerase chain reaction (PCR), gene transfer, and the use of transgenic plants is described.

Genetics can be a powerful adjunct to just about any kind of physiological study, including weed physiology or weed/herbicide interactions. Making, mapping, and reverting mutations is simple and straightforward. Making mutants can be as simple as isolating variant individuals from the “wild”, as uncomplicated as doing seed mutagenesis in your laboratory, or as sneaky as recovering mutants as sectors in whole plants. The overall principles for successful development of a protocol for seed mutagenesis of weeds are described and potential problem areas noted. These generalities are illustrated with a specific case history, that of chlorsulfuron. Although chlorsulfuron is accurately described as an inhibitor of the synthesis of branched chain amino acids, careful physiological examination suggests that it kills plant cells, not by starvation for amino acids, but by active toxicity of a metabolite, α-amino butyric acid, produced from a precursor available for diversion in cells with inhibited acetolactate synthase (EC 4.1.3.18, ALS). The story of dominant resistance due to an altered ALS enzyme is well known; analysis using additional mutants fleshes out the story of how chlorsulfuron works. Such analysis has the potential to help unravel other problems in weed physiology.

Tissue and cell culture offer weed scientists many opportunities to research herbicide effects on plants. This review will discuss examples in which plant cells grown in vitro have been used to study herbicide action. Plant cell and tissue culture have many advantages over the use of whole plants; however, several disadvantages that exist are discussed. Cell cultures can be established for most plant species and provide a relatively homogeneous system for studying herbicide action. Responses of plant cells to herbicides are usually correlated with responses at the whole plant level, and cells have the advantage of posing fewer physical barriers to herbicide uptake and translocation. Cell culture techniques discussed include: screening candidate herbicide compounds; investigating herbicide efficacy, mechanism of action, metabolism, and uptake; and ascertaining mechanisms of herbicide resistance, selecting for resistance, and regenerating crops.

The utility, advantages, and disadvantages of several spectrofluorometric and in vivo spectrophotometric methods in plant science are reviewed, with emphasis on their use in weed science. Examples of the use of in vivo, dual-wavelength spectrophotometry to probe the effects of herbicides on cytochrome f oxidation/reduction, the P515 chromatic shift, phytochrome synthesis, and other processes are discussed. Use of in vivo spectrophotometry to examine effects of herbicides on phytylation of chlorophyllide, protochlorophyllide photoconversion, porphyrin accumulation, and other processes that result in spectral changes is discussed and examples are provided. Spectrofluorometric methods for pigment identification and quantification and for enzyme assays are illustrated with examples. Spectrofluorometric methods used in conjunction with high-pressure liquid chromatography (HPLC) and thin-layer chromatography (TLC) are discussed.

Many problems in weed science may be solved by utilizing immunochemical techniques, although these techniques are currently underutilized by weed scientists. In this review, we describe some of the methods of greatest use to weed scientists. Antibodies may be raised against proteins, such as the targets of herbicide action or against the herbicide itself which can be linked to a larger molecule so that antibodies are elicited. Once specific antibodies are obtained, several techniques may be performed utilizing the binding phenomena of antibody-antigen interactions. Immunofluorescence and immunogold electron microscopy are utilized to obtain tissue and subcellular distributions of the protein or herbicide of interest. Quantitation of either protein or herbicide in a given sample may be performed by ELISA or “slot” and “dot” blotting. These protocols are less costly, more sensitive, and much less labor-intensive than most analytical methods. Molecular mass or charge alterations may be determined by electrophoresis and subsequent immunoblotting. With the increased exposure to immunological techniques by weed scientists and their potential utility, we predict that many more weed science problems will be addressed using these protocols.

Weed scientists are trained broadly in agronomy, botany, chemistry, soils, and other agricultural disciplines. The study of weeds, rather than the techniques used or the questions asked, unifies weed scientists around a common focus. It is often difficult for weed scientists to identify closely with any one scientific discipline, since the techniques and questions of many disciplines are needed to address problems posed by weeds. One discipline with relevance and potential for addressing weed science problems is physiological ecology. The study of the functioning or adaptation of plants in relation to environmental influences has its roots in both classical ecology and experimental physiology. Application of this discipline to weed science may take an environmental approach (e.g., studying limiting factors in the environment), a physiological approach (e.g., studying the responses of critical plant processes to environmental stress), or a more autecological approach (e.g., studying the physiological basis for the adaptation of a particular weed to a particular habitat). Many methodologies and technologies are available for both field and laboratory investigations. For example, photosynthesis, a major determinant of plant growth, can be investigated in the field at the leaf, plant, or canopy level using plant growth analysis or a portable infrared gas analyzer (IRGA) and appropriate assimilation chambers. Investigations of photosynthesis in the laboratory can focus on the plant, leaf, chloroplast, or thylakoid level using an IRGA or the techniques of polarography (measurement of evolved oxygen) or fluorometry. Application of such approaches to weed science should improve our understanding of the basis for particular weed problems and thus broaden our options for management.