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The photoaffinity labeling agent 5-azidoindole-3-acetic acid, an analog of the endogenous plant hormone indole-3-acetic acid (an auxin), was used to identify indole-3-acetic acid-binding proteins in maize. Two peptides with subunit molecular masses of 24 and 22 kilodaltons are specifically labeled in a saturable manner. Both peptides are slightly acidic and behave as dimers under nondenaturing conditions. The possibility that one of these peptides is the auxin receptor that mediates cell elongation in maize is discussed.

The mRNAs encoding two enzymes of phenylpropanoid metabolism, phenylalanine ammonia-lyase (PAL; EC 4.3.1.5) and 4-coumarate:CoA ligase (4CL; EC 6.2.1.12), were induced in cultured parsley cells ( Petroselinum hortense ) either by irradiation with UV light or by treatment with elicitor, a cell-wall fraction of the fungus Phytophthora megasperma f. sp. glycinea . Two-dimensional gel electrophoresis of the encoded PAL and 4CL proteins revealed that the mRNAs induced by either treatment were very similar if not identical. RNA blot hybridization with cDNAs complementary to these mRNAs was used to measure changes in the mRNA amounts at various times after either treatment. Total cellular PAL and 4CL mRNA amounts increased coordinately after UV irradiation to a maximum at 7 hr and then decreased to uninduced levels by 30 hr with the same kinetics as observed previously for the changes in the translational activities. Treatment with the fungal elicitor also caused coordinated, but more rapid, changes in PAL and 4CL mRNA translational activities, with a sharp peak occurring 3 hr after the addition of elicitor. Corresponding changes in mRNA amounts were observed only for 4CL, whereas the amount of PAL mRNA continued to increase at least up to 20 hr after elicitor addition. Our results suggest that parsley cells respond to UV irradiation or addition of fungal elicitor by increased rates of transcription of genes involved in the synthesis of compounds related to UV or disease resistance, respectively.

To investigate the mechanisms underlying activation of plant defenses against microbial attack we have studied elicitor regulation of a chimeric gene comprising the 5′ flanking region of a defense gene encoding the phytoalexin biosynthetic enzyme chalcone synthase fused to a bacterial chloramphenicol acetyltransferase gene. Glutathione or fungal elicitor caused a rapid, marked but transient expression of the chimeric gene electroporated into soybean protoplasts. The response closely resembled that of endogenous chalcone synthase genes in suspension cultured cells. Functional analysis of 5′ deletions suggests that promoter activity is determined by an elicitor-regulated activator located between the “TATA box” and nucleotide position -173 and an upstream silencer between -173 and -326. These cis-acting elements function in the transduction of the elicitation signal to initiate elaboration of an inducible defense response.

Phytophthora megasperma var. sojae (A. A. Hildebrand) is a fungal stem and root rot-causing pathogen of soybeans. Glycoproteins secreted into the medium of the aseptically cultured fungus have been partially purified by (NH 4 ) 2 SO 4 precipitation and by column chromatography on norleucine-substituted Sepharose 4B and on DEAE-cellulose. Glycoprotein preparations from P. megasperma var. sojae races 1, 2, and 3 have been tested on four cultivars of soybeans. The partially purified glycoproteins from incompatible races of the pathogen (races that cannot successfully infect the plant), but not those from compatible races (races that can kill the plant), protect soybean seedlings from attack by compatible races. The seedlings are protected by introducing the glycoproteins into hypocotyl wounds of seedlings either 90 min prior to or at the time of inoculation of the wounds with mycelia of one of the pathogens. The glycoprotein preparations are poor nonspecific elicitors of phytoalexin accumulation; the glycoproteins have less than 1.0% of the elicitor activity of the glucans present in the mycelial walls of the pathogen.

Sucrose is accumulated in response to NaCl-induced stress in the cyanobacterium Synechococcus 6311. Internal cell volume was measured by ESR spectra with 2,2,6,6-tetramethyl-4-oxopiperidinoxy free radical (TEMPONE) as a spin probe in order to calculate sucrose concentrations inside the cell. This method is rapid and reliable and provides an unambiguous measurement of absolute volumes in different osmotic environments. Because the osmolar concentration of sucrose does not counter-balance the osmolar concentrations of ions in the growth medium, we suggest that sucrose accumulation is one of the mechanisms involved in the process of adaptation to salt of Synechococcus 6311. The accumulation of sucrose in non-N 2 -fixing cyanobacteria such as Synechococcus 6311 and in N 2 -fixing cyanobacteria such as Nostoc muscorum suggests a common mechanism of osmoregulation of fresh water cyanobacteria in response to increasing NaCl concentrations in the growth medium.

Four endochitinases (poly[1,4-( N -acetyl-β-D-glucosaminide)] glycanohydrolase, EC 3.2.1.14) have been purified from leaves of Nicotiana tabacum cv. Samsun NN reacting hypersensitively to tobacco mosaic virus. Two of them are acidic proteins of molecular weights 27,500 and 28,500 and have been identified as 2 of the 10 pathogenesis-related proteins that are known to accumulate in tobacco in response to stress or pathogen attack. These two pathogenesis-related proteins, named “P” and “Q” when their biological function was unknown, account for one-third of tobacco mosaic virus-induced chitinase activity of tobacco leaves. They are serologically closely related to the two other chitinases, which can be considered as new basic pathogenesis-related proteins. These two basic chitinases exhibit higher molecular weights (32,000 and 34,000) and higher specific enzyme activity than the two acidic isoforms.

Turgor-mediated leaf movements of the legume Samanea saman are associated with the migration of K + and Cl - between opposing sides of the motor organs (pulvini). We have investigated the pathway of this ion migration by localizing K + and Cl - within the secondary pulvinus at various times during leaf movements. Ion distributions in freeze-dried cryosections of pulvini were determined by scanning electron microscopy/x-ray microanalysis. The results indicate that the apoplast is a major pathway for the migration of K + and Cl - within the pulvinus.

This paper presents a general model to describe coupled solute and water flow through plant roots when they are subjected to osmotic or hydrostatic pressure gradients, or both simultaneously. The model is based on well-established membrane transport equations derived from irreversible thermodynamic considerations. A variety of experimentally observed phenomena such as changes in root resistance with flow rate, apparent negative resistance effects, xylem sap dilution, and apparent non-osmotic water transport can be adequately explained with this model. The model also predicts that an exuding excised root system will be very insensitive to changes in the hydraulic conductivity coefficient. Previous work with a flat membrane of unit surface area and uniform properties is confirmed for a cylindrical coordinate system of nonuniform characteristics.

The complete nucleotide sequences of the gene and the mRNA coding for a specific phaseolin type French bean major storage protein have been determined. Comparison of these sequences reveals a phaseolin gene structure consisting of 80 base pairs (bp) of 5′ untranslated DNA, 1,263 bp of protein-encoding DNA which is interrupted by five intervening sequences (IV S1 , 72 bp; IV S2 , 88 bp; IV S3 , 124 bp; IV S4 , 128 bp; and IV S5 , 103 bp), and 135 bp of 3′ untranslated DNA. Sequences characteristic of eukaryotic promoters “CCAAT” and “TATA” are present in the 5′ flanking DNA, and the eukaryotic poly(A) addition signal A-A-T-A-A-A occurs 16 bp before the first nucleotide of poly(A). The derived amino acid sequence yields an amino acid composition and a molecular weight compatible with those found for the β-type phaseolin protein. Two regions that probably serve as carbohydrate-peptide linkage recognition sites have been identified. A region of highly hydrophobic amino acids at the NH 2 terminus of the protein suggests the presence of a signal peptide in the newly synthesized phaseolin protein.

The respiration of carrot ( Daucus carota L.) roots is stimulated by ethylene. Polyribosomes were shown to proliferate concomitantly with the increase in respiration, and the extent of polyribosome augmentation was closely correlated to the amount of respiratory stimulation. In addition to the increase in quantity, ethylene caused a 2-fold increase in the average polyribosome size, suggesting tighter packing of ribosomes on RNA. In vitro translation of carrot polyadenylylated RNA with the rabbit reticulocyte lysate system followed by electrophoresis of the resulting translation products showed that ethylene treatment results in the appearance of new mRNAs.

A loss of electron transport capacity in chloroplast membranes was induced by high-light intensities (photoinhibition). The primary site of inhibition was at the reducing side of photosystem II (PSII) with little damage to the oxidizing side or to the reaction center core of PSII. Addition of herbicides (atrazine or diuron) partially protected the membrane from photoinhibition; these compounds displace the bound plastoquinone (designated as Q B ), which functions as the secondary electron acceptor on the reducing side of PSII. Loss of function of the 32-kilodalton Q B apoprotein was demonstrated by a loss of binding sites for [ 14 C]atrazine. We suggest that quinone anions, which may interact with molecular oxygen to produce an oxygen radical, selectively damage the apoprotein of the secondary acceptor of PSII, thus rendering it inactive and thereby blocking photosynthetic electron flow under conditions of high photon flux densities.

An enzyme system is described which oxidizes 1-aminocyclopropane-1-carboxylic acid (ACC) to ethylene under physiological conditions. It comprises linoleic acid, pyridoxal phosphate, manganese, and lipoxygenase (linoleate:oxygen oxidoreductase, EC 1.13.11.12). It requires oxygen and is specific for manganese; it can operate but only with greatly reduced yield in the absence of pyridoxal phosphate. An enzyme with the same properties was prepared from microsomal membranes of the seedling shoots of peas. Both have similar reactions to a variety of inhibitors and other reagents. The properties also resemble those of at least two of the in vivo systems recorded in the literature. Intact green oat leaves also contain a similar system. Because there is a growing body of evidence that ethylene formation is associated with cell membranes and because the yields of ethylene from the complete system are much higher than those recorded for other enzymes, it may be identical with the in vivo system acting in senescent leaves.

The molecular and genetic bases of salinity tolerance in plants are not understood. Gene expression at the mRNA level was investigated in a salt-tolerant and a salt-sensitive genotype of barley. Seedlings were exposed to NaCl stress and translatable mRNAs were isolated from root and shoot tissues. A reticulocyte cell-free system was programed with barley mRNAs and the in vitro products were resolved on two-dimensional polyacrylamide gels following isoelectric focusing or nonequilibrium pH gradient gel electrophoresis in the first dimension. The functional mRNAs in unstressed seedlings were qualitatively almost indistinguishable in the two genotypes. However, salinity stress triggered differential transcription of specific mRNAs depending upon genotype and tissue. In roots, 12 new mRNAs were induced that encoded proteins of 21-34 kDa, with a pI range of 6.1-7.7. In shoots, the 9 new mRNAs coded for proteins of 18-50.5 kDa, with a pI range of 5.4-7.8. These new stress mRNAs represented one of two main classes. Class I consisted of mRNAs shared by both genotypes. Class II represented mRNAs specific to each genotype; unique mRNAs of roots accumulated preferentially in the salt-tolerant genotype, whereas those of shoots accumulated in the salt-sensitive genotype. The findings suggest that transcriptional as well as posttranscriptional mechanisms regulate gene expression in barley during salinity stress.

Previous research from this laboratory has demonstrated CO 2 -fixing and H 2 -uptake capacities of certain strains of Rhizobium japonicum . In this report we have shown that SR, a H 2 -uptake-positive (Hup + ) strain of R. japonicum , is capable of autotrophic growth with H 2 as the energy source. Growth occurred on mineral salts/vitamins/Noble agar, mineral salts/vitamins liquid medium (0.27 μg of C as vitamins per ml), and in mineral salts liquid medium with no added vitamins when cultures were provided with NH 4 Cl and incubated in an atmosphere containing H 2 , CO 2 , O 2 , and N 2 . Little or no growth occurred when either H 2 or CO 2 was omitted from the atmosphere or when the culture was inoculated with SR3, a Hup - mutant of SR. Growth was measured by protein synthesis, fixed organic carbon, and increase in cell number in liquid cultures. The organism that grew autotrophically was verified as R. japonicum by ( i ) apparent purity on streak plates; ( ii ) retention of the double antibiotic resistance markers; and ( iii ) its capability to nodulate soybeans. H 2 - and CO 2 -supported growth was demonstrated for three additional Hup + wild-type R. japonicum strains (USDA 136, 3I1b 6, and 3I1b 143), while three Hup - wild-type strains (USDA 120, 3I1b 144, and USDA 117) were incapable of growth on the Noble agar medium containing mineral salts/vitamins in the H 2 /CO 2 /O 2 /N 2 atmosphere. This demonstrated capability of Hup + R. japonicum strains to grow autotrophically requires revision of current concepts regarding conditions for survival and competition of these bacteria in the soil and their relationships to other microorganisms.

Preparations of allophycocyanin isolated from the alga Fremyella diplosiphon show light-induced optical absorbance changes that suggest the presence of a photoconvertible component [Formula: see text] similar to the algal pigments described by J. Scheibe [(1972) Science 176, 1037-1039]. At pH < 4 the allophycocyanin has an absorption maximum at 620 nm. Red illumination causes a loss of absorbance in the red, centered at 620 nm, and subsequent green illumination restores the lost absorbance. We have studied this photoconversion at temperatures between 200 K and 307 K, analyzing the results in terms of photostationary states established under red (640 nm) and green (550 nm) light. As the temperature was lowered to 260 K, the state P r became progressively favored; the reaction P r → P g induced by red light was attenuated but the reaction P g → P r induced by green light was not. Decreasing the temperature from 260 K to 200 K had no further effect. Two distinct and simple models can account for this curious temperature dependence. By analyzing the kinetic and steady-state data, with reasonable estimates of the molar extinction coefficients of P r and P g , we computed quantum efficiencies greater than 15% for the photoconversion at 300 K. We deduced that a conversion of “all P r ” to “all P g ” should produce a fractional absorbance change Δ A/A at 620 nm equal to 0.1. If the chromatic adaptation response of intact F. diplosiphon shows the unusual temperature dependence reported here, the system P r ⇌ P g will be implicated in mediating this response.

The species richness of fungi associated with British trees is described by a significant species/area curve ( r = 0.53, 0.01 > P > 0.001). Introduced tree species cannot be shown to have fewer fungal species than natives, per unit distributional range about Britain. Also, among natives and among introductions, older host taxa do not have more fungal species than do younger ones. This indicates the species richness of fungi to rapidly reach the limit set by host range, within ecological time. The slope of the species/area relationship for fungi is one-fourth that for insects; we propose that this is due to the inherently greater dispersability of fungi. Finally, the species/area regression residuals for fungi are correlated with those for insects ( r = 0.65, 0.001 > P ), suggesting plant defense mechanisms may be generally effective to plant parasites, be they insects or fungi.

Etiolated tissues of several plants are multiple bundles of fiber optics capable of coherent transfer of light over at least 20 mm. The acceptance angles (the angles at which light can be intercepted and then internally reflected longitudinally) for mung beans, oats, and corn are 47 degrees , 59 degrees , and 52 degrees -54 degrees , respectively. The shapes of the curves that describe the acceptance angles are the same for various tissues of the same plant but differ between species. The pattern of light transmitted longitudinally through a tissue is dependent on the angle at which the light intercepts the side of the tissue and is strongly influenced by the tissue geometry. When 0.5 mm of the tip is irradiated, the amount of light traveling down the "shaded" side of the coleoptile is equal to or 2- to 3-fold greater than the amount traveling down the "lighted" side.

Tobacco mesophyll protoplasts were previously shown to respond to naphthaleneacetic acid by modifying their transmembrane potential difference. In the present work, evacuolated protoplasts were used to show that this response resides only at the plasmalemma. This electrical response was investigated by using polyclonal antibodies directed against plasma membrane antigens presumably involved in the reception and transduction of the auxin signal. An IgG fraction from an antiserum directed against the membrane auxin-binding protein from maize coleoptile completely inhibited the naphthaleneacetic acid-induced response of tobacco protoplasts. The suppression of the auxin-induced variation in the transmembrane potential difference by an IgG preparation directed against the plasmalemma ATPase from yeast demonstrated the involvement of the ATPase in the electrical response. Variation induced by fusicoccin in the transmembrane potential difference of tobacco protoplasts was unaffected by the anti-auxin-binding protein IgG fraction but was completely suppressed by the anti-ATPase IgG preparation. These results demonstrate the presence of a membrane receptor for auxin at the plasmalemma, the binding of the hormone to this receptor leading to the activation of the proton-pumping ATPase. They also show that at least the primary steps of activation by naphthaleneacetic acid are distinct from those of the fusicoccin-induced response.

The endogenous gibberellins (GAs) were examined from young vegetative shoots of the dominant mutant, Dwarf-8 , a GA-nonresponder, and normal maize; GA 44 , GA 17 , GA 19 , GA 20 , GA 29 , GA 1 , and GA 8 , members of the early-13-hydroxylation pathway, were identified from both kinds of shoots by full-scan mass spectra and Kovats retention indices. In addition, we report the identification of 3-epi-GA 1 , GA 3 , GA 4 , GA 5 , GA 7 , GA 9 , GA 12 , GA 15 , GA 24 , GA 34 , and GA 53 by using the same criteria. [1,7,12,18- 14 C 4 ]GA 53 and -GA 44 , [17- 2 H 2 ]GA 19 , and [17- 13 C, 3 H 2 ]GA 20 , -GA 29 , -GA 1 , -GA 8 , and -GA 5 were used as internal standards to determine the endogenous levels of these GAs by measurement of isotope dilution, using capillary gas chromatography and selected ion monitoring. Shoots of Dwarf-8 accumulate relatively high levels of GA 20 , GA 1 , and GA 8 . The accumulation of GA 1 appears to be related to gene dosage. Since Dwarf-8 contains the same pattern of GAs as normals (including GA 1 and GA 3 ), the genetic control point probably lies after GA 1 (and GA 3 ). Thus Dwarf-8 may be a GA receptor mutant or a mutant that controls a product downstream from the binding of the bioactive GA to a receptor.

A gene bank of Rhizobium japonicum DNA was constructed by using the broad host range conjugative cosmid pLAFR1. Eighty-three percent of the clones in the bank contained cosmids with insert DNA averaging 22.6 kilobase pairs in length. A series of cosmids containing a hydrogen uptake ( hup ) gene was identified by transferring the gene bank into a H 2 uptake-negative (Hup - ) R. japonicum point mutant (PJ17nal) and screening tetracycline-resistant colonies for the ability to grow chemolithotrophically and to reduce methylene blue in a recently devised colony assay. Hup + transconjugants arose at a frequency of approximately 6 × 10 -3 . Plasmid DNAs from II of the Hup + transconjugants were isolated and used to transform Escherichia coli. Eco RI digests of all plasmids isolated from Hup + transconjugants had three DNA fragments in common. Eight of the E. coli transformants containing hup gene cosmids were conjugated with PJ17nal and another Hup - point mutant, PJ18nal. All PJ17nal transconjugants were Hup + . The frequency of Hup + transconjugants with PJ18nal was approximately 10 -3 . The results indicate that the hup gene cosmids may contain one gene and a portion of another.

Helminthosporoside is a host-specific toxin produced by Helminthosporium sacchari , both in culture and in infected sugarcane tissue. The susceptibility of any given clone of sugarcane to disease is invariably associated with the presence of a toxin-binding protein. This report presents evidence for the presence of the toxin-binding protein on the plasma membrane of the plant cell. This evidence includes, among others: ( 1 ) The protection of susceptible plant tissues by prior treatment with antiserum to the binding protein, ( 2 ) the successful pyridoxylation and reduction of the binding protein in vivo followed by its isolation, ( 3 ) the reaction of sugarcane cells and free protoplasts with [ 3 H]antiserum prepared against the binding protein, and ( 4 ) the agglutination of sugarcane protoplast preparations with antiserum to the binding protein.

Plasma membrane vesicles were isolated from zucchini ( Cucurbita pepo ) hypocotyl tissue by aqueous phase partitioning and assessed for homogeneity by the use of membrane-specific enzyme assays. The highly pure ( ca. 95%) plasma membrane vesicles maintained a pH differential across the membrane and accumulated a tritiated azido analogue of 3-indoleacetic acid (IAA), 5-azido-[7- 3 H]IAA ([ 3 H]N 3 IAA), in a manner similar to the accumulation of [ 3 H]IAA. The association of the [ 3 H]N 3 IAA with membrane vesicles was saturable and subject to competition by IAA and auxin analogues. Auxin-binding proteins were photoaffinity labeled by addition of [ 3 H]N 3 IAA to plasma membrane vesicles prior to exposure to UV light (15 sec; 300 nm) and detected by subsequent NaDodSO 4 /PAGE and fluorography. When the reaction temperature was lowered to - 196°C, high-specific-activity labeling of a 40-kDa and a 42-kDa polypeptide was observed. Triton X-100 (0.1%) increased the specific activity of labeling and reduced the background, which suggests that the labeled polypeptides are intrinsic membrane proteins. The labeled polypeptides are of low abundance, as expected for auxin receptors. Further, the addition of IAA and auxin analogues to the photoaffinity reaction mixture resulted in reduced labeling that was qualitatively similar to their effects on the accumulation of radiolabeled IAA in membrane vesicles. Collectively, these results suggest that the radiolabeled polypeptides are auxin receptors. The covalent nature of the label should facilitate purification and further characterization of the receptors.

The freezing tolerance of protoplasts isolated from nonacclimated rye leaves ( Secale cereale L. cv Puma) was significantly altered by using a pH-induced protoplastliposome fusion technique to modify the lipid composition of the plasma membrane. The increase in freezing tolerance was elicited by fusion with liposomes composed of either the total phospholipid fraction isolated from the plasma membrane of cold-acclimated leaves or single mono- or diunsaturated species of phosphatidylcholine (PtdCho). Of the PtdCho species tested, dilinoleoylphosphatidylcholine ([Lin 2 ]PtdCho) and dilinolenoylphosphatidylcholine ([Lnn 2 ]PtdCho) liposomes were the most effective; 1-palmitoyl-2-oleoylphosphatidylcholine, 1-palmitoyl-2-linoleoylphosphatidylcholine, or dioleoylphosphatidylcholine liposomes were somewhat less effective; dimyristoylphosphatidylcholine or dipalmitoylphosphatidylcholine liposomes had no effect. The increased freezing tolerance was the result of a transformation in the cryobehavior of the plasma membrane during freeze-induced osmotic contraction. In control nonacclimated protoplasts, osmotic contraction resulted in endocytotic vesiculation of the plasma membrane which was irreversible and resulted in lysis during osmotic expansion after melting of the suspending medium. In nonacclimated protoplasts fused with mono- or diunsaturated species of PtdCho, osmotic contraction resulted in the reversible formation of exocytotic extrusions of the plasma membrane—as normally occurs in protoplasts isolated from cold-acclimated leaves (acclimated protoplasts). In scanning electron micrographs, the morphology of the extrusions of nonacclimated protoplasts fused with [Lin 2 ]PtdCho was virtually indistinguishable from that of the extrusions formed in acclimated protoplasts. These studies provide direct evidence that changes in the lipid composition of the plasma membrane are causally related to one facet of the cold-acclimation process.

The diurnal change in activity of ribulose 1,5-bisphosphate (Rbu-1,5- P 2 ) carboxylase [3-phospho-D-glycerate carboxy-lyase (dimerizing); EC 4.1.1.39] of leaves of Phaseolus vulgaris is regulated (in part) by mechanisms that control the level of an endogenous inhibitor that binds tightly to the activated (carbamoylated) form of Rbu-1,5- P 2 carboxylase. This inhibitor was extracted from leaves and copurified with the Rbu-1,5- P 2 carboxylase of the leaves. Further purification by ion-exchange chromatography, adsorption to purified Rbu-1,5- P 2 carboxylase, barium precipitation, and HPLC separation yielded a phosphorylated compound that was a strong inhibitor of Rbu-1,5- P 2 carboxylase. The compound was analyzed by GC/MS, 13 C NMR, and 1 H NMR and shown to be 2-carboxyarabinitol 1-phosphate [(2- C -phosphohydroxymethyl)-D-ribonic acid]. Verification of structure was obtained by comparison of the inhibitory activity of the isolated compound with that of 2-carboxy-D-arabinitol 1-phosphate synthesized in vitro . This compound (but not 2-carboxy-D-arabinitol 5-phosphate) inhibited Rbu-1,5- P 2 carboxylase in a way that was kinetically identical to that of the isolated, naturally occurring compound. The structure of the isolated compound differs from the Rbu-1,5- P 2 carboxylase transition-state analogue 2-carboxyarabinitol 1,5-bisphosphate only by the lack of the C-5 phosphate group. This difference results in a higher binding constant for the monophosphate ( K d = 32 nM) compared with the bisphosphate ( K d < 10 pM). The less tightly bound compound acts in a light-dependent, reversible regulation of Rbu-1,5- P 2 carboxylase activity in vivo .

Purified oat phytochrome was labeled with 125 I without altering the photoreversibility or absorbance properties of the pigment. The radiolabeled phytochrome was used in experiments in vitro to quantitate the binding of the pigment to both crude and purified membrane preparations from oat tissue. After the membranes were allowed to react with 125 I-labeled phytochrome, washed free of unbound material, and pelleted, they were found to have significant levels of radioactivity bound to them. Qualitative identification of phytochrome as the bound radioactive species was confirmed by autoradiography of sodium dodecyl sulfate gels after electrophoresis of the proteins contained in the washed membranes. Data supporting the specificity of the binding are that the binding shows saturation kinetics and that unlabeled phytochrome, but not bovine serum albumin, will competitively inhibit the binding of labeled phytochrome. This technique permits the detection of less than a nanogram of phytochrome and provides a new method for quantifying bound phytochrome that is independent of the spectral detectability of the pigment. It should be useful in elucidating the nature of phytochrome attachment to cellular membranes.

Plants from several families (Chenopodiaceae, Gramineae, Compositae) accumulate betaine (glycine betaine) in response to salt or water stress via the pathway: choline → betainal (betaine aldehyde) → betaine. Betaine accumulation is probably a metabolic adaptation to stress. Intact protoplasts from leaves of spinach ( Spinacia oleracea ) oxidized [ 14 C]choline to betainal and betaine, as did protoplast lysates. Upon differential centrifugation, the [ 14 C]choline-oxidizing activity of lysates sedimented with chloroplasts. Chloroplasts purified from protoplast lysates by a Percoll cushion procedure retained strong [ 14 C]choline-oxidizing activity (1-3 nmol/mg of chlorophyll per hr), although the proportion of the intermediate, [ 14 C]betainal, in the reaction products was usually higher than for protoplasts. Isolated chloroplasts also readily oxidized [ 14 C]betainal to betaine (20-100 nmol/mg of chlorophyll per hr). Light increased the oxidation of both [ 14 C]choline and [ 14 C]betainal by isolated chloroplasts ≈3-fold; this light-stimulation was abolished by 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU). Similar results were obtained with another chenopod ( Beta vulgaris ) but not with pea ( Pisum sativum ), a species that accumulates no betaine. The chloroplast site for betaine synthesis in chenopods contrasts with the mitochondrial site in mammals.

The influence of 6-benzylaminopurine (BAP) on the respiration by mitochondria from bush bean ( Phaseolus vulgaris L.), mung bean ( P. aureus Roxburgh), soybean [ Glycine max (L.) Merrill], maize ( Zea mays L.), pea ( Pisum sativum L.), and wheat ( Triticum aestivum L.) was examined. BAP, a synthetic cytokinin, consistently inhibited oxygen uptake by mitochondria from all species when malate was used as the substrate. The decrease in respiration was especially evident in the presence of ADP or an uncoupler of oxidative phosphorylation. 6-Isopentenylaminopurine and 6-furfurylaminopurine also inhibited malate oxidation, but zeatin and adenine did not. In certain instances, BAP reduced succinate and NADH oxidation. With succinate as the substrate and with antimycin A present, inhibition by BAP paralleled that caused by salicylhydroxamic acid, an inhibitor of alternative respiration. A suggested scheme features a cytokinin-inhibited point located between NADH dehydrogenase and cytochrome b of the electron transport system. Electrons from the NADH generated by malate oxidation are assumed to flow through this point, with electrons from externally supplied or cytosolic NADH and succinate doing so only under certain conditions such as when alternative respiration is occurring. Cytokinin effects on respiration and perhaps on other phenomena may be mediated by this mechanism.

Genes specifically induced during somatic embryogenesis may play key roles in plant embryo development. An antiserum against an extract of carrot somatic embryos revealed a few rare antigens induced at the onset of embryogenesis. Through differential immunoadsorption techniques, we purified antibodies against the embryo-specific antigens and probed a phage λ gt11 library of cDNA from carrot somatic embryos. This paper describes three distinct cDNA clones that hybridize to embryo-specific RNAs. Monospecific antibodies, purified by affinity to the recombinant phage fusion proteins, confirm that the cloned cDNAs encode unique embryo-specific peptide antigens. One 50-kDa protein correlates with embryogenic ability in cultures of other plant species, including cereals.

The ability of N,N ′-diphenylurea (Ph 2 urea) to substitute for cytokinin-active adenine derivatives in promoting callus growth of Phaseolus lunatus has been examined. In general, Ph 2 urea stimulated callus growth at high concentrations, although the growth of most callus tissues was irregular. Variability in the sensitivity and uniformity of the growth response to Ph 2 urea was found among different genotypes of P. lunatus . Most importantly, tissues cultured on Ph 2 urea-containing medium for one passage had acquired the ability to proliferate in subsequent passages in the absence of either Ph 2 urea or cytokinin-active adenine derivatives. Corresponding tissues maintained on kinetin-containing medium remained cytokinin-dependent. It appears that the effect of Ph 2 urea in promoting the growth of P. lunatus callus tissue resides in its ability to induce cytokinin autonomy. This result suggests that the cytokinin activity of Ph 2 urea may be due to promotion of endogenous cytokinin biosynthesis in the bioassay systems in which it is active.

Zinniol [1,2-bis(hydroxymethyl)-3-methoxy-4-methyl-5-(3-methyl-2-butenyloxy)benzene], a toxin produced by fungi of the Alternaria group, causes symptoms in plants that resemble those induced by the fungi. The phytotoxin binds to carrot protoplasts and isolated membranes in a saturable and reversible manner. Receptor occupancy stimulates entry of calcium into protoplasts. Zinniol can partially reverse the effects and binding of the calcium-channel blockers desmethoxyverapamil and bepridil. Selected cell lines that are insensitive to zinniol lose part of their binding capacity and sensitivity to the action of the agonist-like compound but are still able to bind calcium-channel blockers. We conclude that zinniol acts on calcium entry but that the targets of the toxin and of calcium-channel blockers are dissimilar, suggesting the occurrence of sites affected both by zinniol and by channel blockers and of sites affected only by zinniol.

Xanthomonas campestris pv. vesicatoria is the causal agent of leaf spot disease on pepper and tomato. On non-host plants, such as bean, soybean, cowpea, alfalfa, and cotton, X. campestris pv. vesicatoria is unable to cause disease, inducing instead a hypersensitive resistance response (HR). Since avirulence genes from X. campestris pv. vesicatoria specifically induce HR in several pepper cultivars, we investigated whether there were avirulence genes governing induction of resistance in non-host species. We report on the molecular cloning and characterization of a non-host avirulence gene from X. campestris pv. vesicatoria . A cosmid clone isolated from a library of DNA from X. campestris pv. vesicatoria tomato race 1 converted X. campestris pv. phaseoli to avirulence by inducing HR on the bean cultivar Sprite, but not on Bush Blue Lake. The HR-inducing activity was localized to a 2.1-kilobase Pst I fragment of DNA, designated avrRxv . In addition, we demonstrate that avrRxv inhibited disease production by several X. campestris pathovars on their normally susceptible hosts: glycines on soybean, vignicola on cowpea, alfalfae on alfalfa, holcicola on corn, and malvacearum on cotton. The HR resistance in bean induced by avrRxv segregated as a single incompletely dominant gene, designated Rxv . These results indicate that the avirulence gene avrRxv and the resistance gene Rxv partially control the outcome of the interaction between X. campestris pv. vesicatoria and the non-host bean.

Antisense nopaline synthase (nos) (D-nopaline synthase; EC 1.5.1.19) RNA is stably expressed from the cauliflower mosaic virus 35S promoter in transformed tobacco plants. The expression of a previously introduced wild-type nos gene is inhibited by the antisense RNA, with less nos enzyme activity detected (by a factor of 8-50) depending on the tissue analyzed. The steady-state levels of nos mRNA are reduced in the presence of the antisense RNA, implying that mRNA degradation is probably the main mode of action for the decrease in expression in this system. The antisense RNA-expressing gene and its inhibition of nos expression are shown to be heritable, demonstrating that it is a potentially useful method for the modification of phenotype.

Two RNA fractions have been isolated and purified from both oncogenic and nononcogenic strains of Agrobacterium tumefaciens . Both RNAs are capable of inducing the formation of transplantable tumors when introduced at wound sites in stems of Datura stramonium plants. One of these RNA fractions was found to be bound to an RNA-directed DNA polymerase, while the other was associated with the bacterial DNA. Physical evidence suggests that both are single stranded and small in size; linear sucrose gradients show that their size corresponds to a value of 5-6 S. A concentration of 4-5 μg of the RNAs dissolved in 0.01 ml of water is effective in initiating the formation of transplantable tumors in Datura plants.

The activity of ribulose 1,5-bisphosphate carboxylase [RuBPCase; 3-phospho-D-glycerate carboxylyase (dimerizing), EC 4.1.1.39] in leaf extracts of a number of species kept in the dark overnight was found to be very low. This was not the result of a change in the activation state or in the amount of enzyme that could be extracted from “dark” leaves. Rather, in Phaseolus vulgaris it was due to an inhibitor of catalysis that occupied the catalytic site of the enzyme. This inhibitor was compartmentalized in the chloroplast and its maximum concentration in both dark leaves and in intact chloroplasts made from such leaves was slightly in excess of the RuBPCase catalytic site concentration. The inhibitor (a phosphate ester) was bound preferentially to the activated form of the enzyme, apparently functioning as a positive effector of activation. Treatment of the enzyme-inhibitor complex in vitro with alkaline phosphatase could restore RuBPCase activity. In vivo , both the initial rate of disappearance and the final concentration of inhibitor in intact leaves was found to vary with light intensity, and these changes could account for observed light-dependent changes in RuBPCase activity, indicating that light modulation of inhibitor concentration controlled RuBPCase activity. Recovery of activity in vivo could be inhibited by 3-(3′,4′,4-dichlorophenyl)-1,1-dimethylurea.

In experiments carried out to investigate the acid secretion theory of auxin action, we utilized sodium orthovanadate, an agent found to be a selective inhibitor of a plasma membrane-associated H + -pumping ATPase in Neurospora [Bowman, B. J. & Slayman, C. W. (1979) J. Biol. Chem. 245, 2928-2934]. At 1 mM, vanadate inhibited auxin-enhanced medium acidification by pea epicotyl segments within 5 min, whether added 0.5 or 2.5 hr after auxin. Inhibition of acidification was total after 10-15 min but could be reversed within 10 min after vanadate removal. When given as a 40-min pretreatment, vanadate completely prevented any auxin-enhanced acidification. Vanadate inhibition of medium acidification by oat coleoptile segments was also total and reversible, but both inhibition and reversal occurred after longer lag times than in pea. Inhibitory effects of vanadate on elongation in pea and oat tissue closely paralleled its effects on acidification, and the inhibitory effect of vanadate on elongation could be reversed by an acidic buffer. Vanadate did not inhibit respiration or protein synthesis in pea epicotyl segments, although it strongly inhibited L-[ 14 C]leucine uptake. These results indicate the importance of cell wall acidification for short- and long-term auxin-enhanced growth and suggest the participation in wall acidification of a plasma membrane-associated ATPase acting as an H + pump.

2-Azido-4-ethylamino-6-isopropylamino- s -triazine (azido-atrazine) inhibits photosynthetic electron transport at a site identical to that affected by atrazine (2-chloro-4-ethylamino-6-isopropylamino- s -triazine). The latter is a well-characterized inhibitor of photosystem II reactions. Azido-atrazine was used as a photoaffinity label to identify the herbicide receptor protein; UV irradiation of chloroplast thylakoids in the presence of azido[ 14 C]atrazine resulted in the covalent attachment of radioactive inhibitor to thylakoid membranes isolated from pea seedlings and from a triazine-susceptible biotype of the weed Amaranthus hybridus . No covalent binding of azido-atrazine was observed for thylakoid membranes isolated from a naturally occurring triazine-resistant biotype of A. hybridus . Analysis of thylakoid polypeptides from both the susceptible and resistant A. hybridus biotypes by sodium dodecyl sulfate/polyacrylamide gel electrophoresis, followed by fluorography to locate 14 C label, demonstrated specific association of the azido[ 14 C]atrazine with polypeptides of the 34- to 32-kilodalton size class in susceptible but not in resistant membranes.

Rhizobium leguminosarum bv. trifolii T24 produces a potent anti-rhizobial compound, trifolitoxin, and exclusively nodulates clover roots when in mixed inoculum with trifolitoxin-sensitive strains of R. leguminosarum bv. trifolii [Schwinghamer, E. A. & Belkengren R. P. (1968) Arch. Mikrobiol. 64, 130-145]. In the present study, the isolation of trifolitoxin production and resistance genes is described. A cosmid genomic library of T24 was prepared in pLAFR3. No trifolitoxin expression was observed in the resulting Escherichia coli cosmid clones. One cosmid clone was identified that restored trifolitoxin production and nodulation competitiveness in three nonproducing mutants of T24. The recombinant plasmid from this cosmid clone, pTFX1, also conferred trifolitoxin production and resistance when transferred to symbiotically effective strains of R. leguminosarum bvs. trifolii, phaseoli , and viceae . Cosmid pTFX1 also conferred expression of trifolitoxin production when present in strains of Rhizobium meliloti and Agrobacterium tumefaciens . No trifolitoxin expression was observed in strains of Bradyrhizobium japonicum or Rhizobium sp. (cowpea) with pTFX1. Southern blot analysis with a biotinylated pTFX1 probe did not suggest that these genes were plasmid-borne. Transfer of pTFX1 to T24 or its derivatives resulted in 6- to 10-fold higher level of trifolitoxin production than wild-type T24.

This report describes a process for delivering foreign genes into maize cells that does not require the removal of cell walls and is capable of delivering DNA into embryogenic and nonembryogenic tissues. Plasmid harboring a chimeric chloramphenicol acetyltransferase (CAT) gene was adsorbed to the surface of microscopic tungsten particles (microprojectiles). These microprojectiles were then accelerated to velocities sufficient for penetrating the cell walls and membranes of maize cells in suspension culture. High levels of CAT activity were consistently observed after bombardment of cell cultures of the cultivar Black Mexican Sweet, which were comparable to CAT levels observed after electroporation of protoplasts. Measurable increases in CAT levels were also observed in two embryogenic cell lines after bombardment. Gene expression was observed only when an intron from the alcohol dehydrogenase 1 gene of maize was ligated between the 35S promoter and the CAT coding region. CAT activity was detected in cell cultures bombarded with microprojectiles with an average diameter of 1.2 μm, but not after bombardment with microprojectiles 0.6 or 2.4 μm in diameter. Bombarding the same sample several times was found to markedly enhance CAT activity. These results demonstrate that the particle bombardment process can be used to deliver foreign DNA into intact cells of maize. Because this process circumvents the difficulties associated with regenerating whole plants from protoplasts, the particle bombardment process may provide significant advantages over existing DNA delivery methods for the production of transgenic maize plants. In addition, the process should be of value for studying transient and stable gene expression within intact cells and tissues.

Addition of 1.5 mM NADH tripled the O 2 consumption in corn root protoplasts. The stimulation was temperature and pH dependent, specific to NADH, and accompanied by a 2- to 3-fold increase in K + and P i uptake into protoplasts. The increase in ion uptake was not due to the accumulation of NADH into protoplasts. The effect of exogenous NADH on O 2 consumption and ion uptake was also evident in corn root segments but to a lesser extent. A 20-mV hyperpolarization of protoplast membrane potential occurred on addition of NADH and was abolished by the uncoupler carbonyl cyanide p -trifluoromethoxyphenylhydrazone. Increases in cell volume of 30% and 40% were detected in response to NADH/H + and NADH/H + /K + , respectively. The data are discussed in terms of a transmembrane redox reaction and the possibility that some part of the energy-linked ion transport may be driven by a NADH → O 2 electron-transport system in the plasmalemma.

The regulatory pigment phytochrome induces rapid and opposite growth changes in different regions of etiolated maize seedling: it stimulates the elongation rate of coleoptiles and inhibits that of mesocotyls. As measured by a quantitative immunoassay, phytochrome also promotes rapid and opposite changes in the extractable content of a M r 98,000 anionic isoperoxidase in the cell walls of these same organs: it induces a decrease of this peroxidase in coleoptiles and an increase in mesocotyls. The peroxidase changes precede the growth changes. As measured by video stereomicroscopy or a position transducer, red light (R), which photoactivates phytochrome, stimulates coleoptile elongation with a lag of about 15-20 min and suppresses mesocotyl growth with a lag of 45-50 min. R also induces a 50% reduction in the extractable level of the anionic peroxidase in coleoptile walls in less than 10 min and a 40% increase in the level of this peroxidase in mesocotyl walls within 30 min. Ascorbic acid, an inhibitor of peroxidase activity, blocks the effects of R on mesocotyl section growth. These results are relevant to hypotheses that postulate that certain wall peroxidases can participate in light-induced changes in growth rate by their effects on wall extensibility.

Two cell-division-promoting factors, which have partition coefficients of 1.9 and 2.75 determined by 500-tube countercurrent distribution in a butanolwater system, have been repeatedly isolated in this laboratory from crown gall tumor tissues of Vinca rosea L. These substances have been given the trivial names cytokinesin I and cytokinesin II, respectively. Chemical and mass spectrometric analyses suggest that both cytokinesins are substituted hypoxanthines and are thus very different compounds from the 6-substituted adenyl cytokinins. Carlos Miller, using a very different and far more drastic isolation procedure, obtained one main cell-division-promoting factor from these same tumor tissues, which he identified as ribosyl-trans-zeatin. On the basis of this finding, and without an attempt to repeat our studies, questions have been raised by Miller concerning the existence of the cytokinesins as biologically active substances. We have, therefore, compared some pertinent physical, chemical, and biological properties of the cytokinesins with those of zeatin riboside, have demonstrated that these three substances can be cleanly separated from one another by a number of different methods and that each behaves as a pure substance in the several systems, and, finally, we have shown that the cytokinesins are not contaminated with ribosyl-trans-zeatin and thus do not owe their biological activity to such a contaminant.

Tobacco plants expressing constitutive chloramphenicol acetyltransferase (CAT; EC 2.3.1.28) activity were obtained by transformation with a chimeric CAT gene driven by the cauliflower mosaic virus 19S promoter. Plants expressing different levels of CAT activity were retransformed with vectors containing CAT sequences transcriptionally fused in the antisense orientation between the coding region of the hygromycin-resistance gene and the 3′ end of the nopaline synthase gene. Several plants regenerated on high concentrations of hygromycin exhibited a loss of CAT activity, whereas plants retransformed with a vector conferring hygromycin resistance but lacking the antisense CAT sequence showed no reduction in CAT activity. RNA blot analysis revealed a strong correlation between the degree of CAT gene inactivation and the levels of stable antisense transcripts accumulated. The possibility that CAT gene inactivation was due to transferred DNA instability was discounted since a kanamycin-resistance gene contiguous with the CAT gene was expressed normally, and DNA blot analysis indicated no loss or rearrangements of the transferred DNA fragments. Thus, the imposed selection pressure enabled the selection of plants expressing high levels of stable bifunctional antisense transcripts that inhibited the activity of the targeted gene.

tRNA·DNA hybridization studies indicate that Euglena chloroplast tRNAs are transcriptional products of the chloroplast genome, which contains approximately 26 tRNA cistrons. Hybridization with purified chloroplast tRNA Phe and tRNA Asp shows that the chloroplast genome contains one cistron for each of these two species. No hybridization of chloroplast tRNA with nuclear DNA was observed. tRNAs from Euglena cytoplasm, Escherichia coli , and Agmenellum quadraduplicatum do not compete with chloroplast tRNA for hybridization with chloroplast DNA. Evidence is presented that photoinduction of chloroplast tRNAs is at the level of transcription rather than maturation of tRNA precursor molecules.

We have recently purified calorigen, the natural trigger for heat production in the inflorescences of Sauromatum guttatum Schott (voodoo lily), a thermogenic plant, and identified it as salicylic acid. Since then an analytical assay was developed that allows the quantitation of salicylic acid in plant tissues. This assay was used to demonstrate that on the day preceding the day of blooming the levels of salicylic acid in the thermogenic organs (appendix and lower spadix) of the voodoo lily increased almost 100-fold, reaching a level of 1 μg/g of fresh weight. The level of salicylic acid in the appendix started to rise in the afternoon and reached its maximum in the late evening, whereas the maximum accumulation of salicylic acid in the lower spadix occurred late at night. The increase in salicylic acid level in the appendix was followed the next morning by a spectacular metabolic burst that lasted for about 7 hr and at its peak increased the appendix temperature by over 12°C. The second, 14-hr-long, thermogenic episode in the lower spadix started late at night and ended on the following morning, after maximum temperature increases of more than 10°C. The concentration of salicylic acid in both thermogenic tissues promptly returned to basal, preblooming levels at the end of the thermogenic periods. The thermogenic response was under strong photoperiodic and developmental control, with salicylic acid eliciting much stronger thermogenic responses in light than in darkness. Similar surges in salicylic acid occurred in nonthermogenic male and female flowers, while the concentration of salicylic acid in the spathe remained consistently below 20 ng/g of fresh weight. Of 33 analogs of salicylic acid tested, only 2,6-dihydroxybenzoic acid and acetylsalicylic acid (aspirin) were thermogenic. The activity of 2,6-dihydroxybenzoic acid exceeded that of salicylic acid.

Cultured tobacco cells ( Nicotiana tabacum L. cv. Wisconsin 38) synthesize a predominant 26-kDa protein upon exposure to abscisic acid (ABA). ABA also accelerates the rate of adaptation of unadapted cells to NaCl stress. The ABA-induced 26-kDa protein is immunologically cross-reactive to, and produces a similar pattern of peptides after partial proteolysis as, the major 26-kDa protein associated with NaCl adaptation. Both have pI values of >8.2. The synthesis of the ABA-induced 26-kDa protein is transient unless the cells are simultaneously exposed to NaCl stress. There is an association between increased intracellular accumulation of ABA during cell growth and commencement of synthesis of the 26-kDa protein. ABA induces the synthesis of an immunologically cross-reactive 26-kDa protein in cultured cells of several plant species. In tobacco plants, synthesis of the 26-kDa protein could be detected in several tissues but the highest level of expression was seen in outer stem tissue. In root tissues, exogenous ABA greatly stimulated the synthesis of 26-kDa protein as compared to outer stem tissue and leaf. We suggest that ABA is involved in the normal induction of the synthesis of 26-kDa protein and that the presence of NaCl is necessary for the protein to accumulate.

Eleocharis vivipara Link, a freshwater amphibious leafless plant belonging to the Cyperaceae can grow in both terrestrial and submersed aquatic conditions. Two forms of E. vivipara obtained from these contrasting environments were examined for the characteristics associated with C 4 and C 3 photosynthesis. In the terrestrial form (δ 13 C values = -13.5 to -15.4‰, where ‰ is parts per thousand), the culms, which are photosynthetic organs, possess a Kranz-type anatomy typical of C 4 plants, and well-developed bundle-sheath cells contain numerous large chloroplasts. In the submersed form (δ 13 C value = -25.9‰), the culms possess anatomical features characteristic of submersed aquatic plants, and the reduced bundle-sheath cells contain only a few small chloroplasts. 14 C pulse- 12 C chase experiments showed that the terrestrial form and the submersed form fix carbon by way of the C 4 pathway, with aspartate (40%) and malate (35%) as the main primary products, and by way of the C 3 pathway, with 3-phosphoglyceric acid (53%) and sugar phosphates (14%) as the main primary products, respectively. The terrestrial form showed photosynthetic enzyme activities typical of the NAD-malic enzyme-C 4 subtype, whereas the submersed form showed decreased activities of key C 4 enzymes and an increased ribulose 1,5-bisphosphate carboxylase (EC 4.1.1.39) activity. These data suggest that this species can differentiate into the C 4 mode under terrestrial conditions and into the C 3 mode under submersed conditions.

In the phototropic response of Arabidopsis thaliana seedlings, the shape of the fluence-response relation depends on fluence rate and wavelength. At low fluence rates, the response to 450-nm light is characterized by a single maximum at about 0.3 μmol·m -2 . At higher fluence rates, the response shows two distinct maxima, I and II, at 0.3 and 3.5 μmol·m -2 , respectively. The response to 500-nm light shows a single maximum at 2 μmol·m -2 , and the response to 510-nm light shows a single maximum at 4.5 μmol·m -2 , independent of fluence rate. The response to 490-nm light shows a maximal at 4.5 μmol·m -2 and a shoulder at about 0.6 μmol·m -2 . Preirradiation with high-fluence 510-nm light from above, immediately followed by unilateral 450-nm light, eliminates maximum II but not maximum I. Preirradiation with high-fluence 450-nm light from above eliminates the response to subsequent unilateral irradiation with either 450-nm or 510-nm light. The recovery of the response following high-fluence 450-nm light is considerably slower than the recovery following high-fluence 510-nm light. Unilateral irradiation with low-fluence 510-nm light followed by 450-nm light results in curvature that is approximately the sum of those produced by either irradiation alone. Based on these results, it is proposed that phototropism in A. thaliana seedlings is mediated by at least two blue-light photoreceptor pigments.

DNAs complementary to poly(A) + RNA present in elicitor-treated cells of Phaseolus vulgaris L. were inserted into pBR325 and used to transform Escherichia coli strain JA221. A clone was identified that contained sequences complementary to mRNA encoding chalcone synthase, a regulatory enzyme of phenylpropanoid biosynthesis, which catalyzes the first reaction of a branch pathway specific to flavonoid and isoflavonoid biosynthesis. Rapid, marked but transient increases in chalcone synthase mRNA in response to elicitor treatment were observed by RNA blot hybridization with 32 P-labeled chalcone synthase cDNA sequences. Induction of chalcone synthase mRNA governs the rate of enzyme synthesis throughout the phase of rapid increase in enzyme activity at the onset of accumulation of isoflavonoid-derived phytoalexins. The data are consistent with the hypothesis that elicitor causes a rapid transient stimulation of transcription of chalcone synthase gene(s) as an early event in the expression of the phytoalexin defense response.

The stomatal response to blue light was analyzed with gas-exchange techniques in Commelina communis L. leaves by using high-fluence-rate short pulses. Pulses of blue light were given under a background of high-fluence-rate red light, which maintained photosynthesis at near saturation and stomatal conductance at a steady state. A single blue light pulse of 1-100 sec induced an increase in stomatal conductance, which peaked after 15 min and then returned to the initial steady-state level within 50-60 min after the pulse. The response could be repeatedly induced in the same leaf. Red light pulses on a red background did not induce any comparable response. The stomatal response quantified by integrating the conductance increases after pulse application approached saturation with increasing pulse duration ( t ½ ≈9 sec with 250 μmol·m -2 ·sec -1 of blue light). After a saturating pulse, sensitivity to a second pulse was restored slowly. This recovery response, quantified from the conductance increases caused by the two pulses, approached saturation with a t ½ of ≈9 min. These results were used to test a model in which a molecular component in the sensory transduction process is considered to exist in two interconvertible forms, A and B . If B is the physiologically active form inducing stomatal opening, then A is the inactive form. The A to B conversion is a light-induced reaction and the B to A conversion is a thermal reaction. Rate constants for these reactions were estimated from single- and double-pulse experiments (at a fluence rate of 250 μmol·m -2 ·sec -1 , k 1 = 0.075 sec -1 ; thermal rate constant k d = 0.0014 sec -1 ), allowing the calculation of steady-state concentration of B under continuous irradiation. The calculated values accurately predicted the steady-state stomatal conductances under continuous blue light.

The effectiveness of diuron, atrazine, procyazine, and cyanazine were compared in controlling growth of redroot pigweed ( Amaranthus retroflexus L.) in hydroponic culture. A very marked differential inhibition response was observed for atrazine between resistant and susceptible biotypes. Procyazine and cyanazine exhibited less dramatic differential responses, whereas diuron was equally effective in controlling growth in both biotypes. Photosystem II activity of chloroplasts from both triazine-resistant and triazine-susceptible biotypes was inhibited by diuron but only the chloroplasts from triazine-susceptible biotypes were inhibited significantly by atrazine. The photochemical activity of chloroplasts from triazine-resistant biotypes was partially resistant to procyazine or cyanazine inhibition. The parallel lack of diuron differential effects, partial procyazine and cyanazine differential response, and very marked atrazine differential response in both whole plant and chloroplast assays indicates that the chloroplast is the site of selective herbicide tolerance in these triazine-resistant redroot pigweed biotypes. Photosystem II photochemical properties were characterized by analysis of chlorophyll fluorescence transients in the presence or absence of herbicides. Data with susceptible chloroplasts indicated that both diuron and atrazine inhibit electron flow very near the primary electron acceptor of photosystem II. Only diuron altered the fluorescence transient in resistant chloroplasts. In untreated preparations there were marked differences in the fast phases of the fluorescence increase in resistant vs. susceptible chloroplasts; these data are interpreted as showing that the resistant plastids have an alteration in the rate of reoxidation of the primary photosystem II electron acceptor. Electrophoretic analysis of chloroplast membrane proteins of the two biotypes showed small changes in the electrophoretic mobilities of two polypeptide species. The data provide evidence for the following herbicide resistance mechanism: genetically controlled modification of the herbicide target site.