Translocation and Complex Formation of Picloram and 2,4-D in Wheat Seedlings

Uptake, translocation and metabolism of ¹⁴C-labelled 4-amino-3,5,6-trichloropicolinic acid (picloram) and 2,4-dichlorophenoxyacetic acid (2,4-D) in seedlings of wheat (Triticum aestivum L.) were studied. The uptake of the herbicides through the upper surface of the first leaf was slow but was almost complete after nine days. Picloram was absorbed faster than 2,4-D. Picloram was also translocated into the stem and the untreated leaves to a greater extent than 2,4-D. Only small fractions of the activity were recovered from the roots and from the nutrient solution. Picloram and 2,4-D formed water-soluble conjugates in the tissues. These conjugates were very labile and hydrolyzed under release of the unchanged herbicides. The isotope from 2,4-D was also incorporated in an insoluble fraction, containing cell walls and proteins. Also from this fraction biologically active 2,4-D could be released by hydrolysis. The formation of the complexes was partly prevented by cycloheximide. It is suggested that herbicide detoxification through complex formation is of importance for the relatively low sensitivity of wheat to auxin herbicides.     

Translocation and Complex Formation of Root-applied 2,4-D and Picloram in Susceptible and Tolerant Species

Translocation and complex formation of ¹⁴C-labelled 2,4-dichlorophenoxyacetic acid (2,4-D) and 4-amino-3,5,6-trichloropicolinic acid (picloram) in seedlings of sunflower (Helianthus annuus L. var. uniflorus), rape (Brassica napus L. cv. Nilla), wheat (Triticum aestivum L. cv. Starke), and Norway spruce (Picea abies (L.) H. Karst.) were studied. The herbicides were absorbed through the roots from the nutrient solution, Picloram was well translocated to the shoots of the four species; while the acropetal translocation of 2,4-D was small except in rape. In 2,4-D-susceptible sunflower and rape and in picloramsusceptible sunflower and spruce the herbicides were recovered mainly in the uncomplexed form. In 2,4-D tolerant wheat and spruce most of the absorbed 2,4-D was converted into water-soluble or TCA-insoluble complexes. In picloram-tolerant wheat and in relatively picloram-tolerant rape, the absorbed picloram was also converted into complexes recovered predominantly in the water-soluble fraction. Most of the complexes released free herbicides by hydrolyzing in NaOH or HCI. The results further support the hypothesis that complex formation counteracts herbicide toxicity.     

Translocation and Metabolism of Picloram and 2,4-D in Populus tremula

Translocation and metabolism of 4-amino-3,5,6-trichloropicolinic acid (picloram) and 2,4-dichlorophenoxyacetic acid (2,4-D) in small plants of aspen (Populus tremula L.) were studied. In most experiments ¹⁴C-carboxyl-labelled herbicides were used. Considerable quantities of both herbicides were retained in the treated leaf. Translocation was mainly upwards into the growing shoot tip. Only minute quantities were found in the roots. Injection of the herbicides through a cut stem surface increased the translocation into the roots very little. One of the reasons for the limited downward translocation is considered to be a ready transfer of the herbicides between the phloem and the xylem. Both herbicides were incorporated into complexes from which the active herbicides could be released. However, this complex formation can only partly account for the retention of the herbicides in the treated leaves. The differences in metabolism found between 2,4-D and picloram cannot explain the considerable difference in toxicity between the compounds.     

Boron Deficiency and Translocation Profiles in Sunflower

The distribution of carbon-14 down the stems of comparable boron-deficient and boron-sufficient sunflower plants after photosynthesis of ¹⁴CO₂ by a single exposed leaf was investigated. In boron-deficient plants the advancing front of radioactivity was always found less far down the stem than in boron-sufficient plants. The general shape of the profile is the same in the two sets of plants. We conclude that the velocity of translocation is reduced in the boron-deficient plants.     

In Vitro Culture of Kodo Millet: Influence of 2,4-D and Picloram in Combination with Kinetin on Callus Initiation and Regeneration

Immature inflorescences of kodo millet (Paspalum scrobiculatum L. cv. GPUK-3) were cultured on MS medium. Induction of embryogenic callus and subsequent somatic embryogenesis was possible on both 2,4-D and Picloram alone or with kinetin from spikelets as well as rachis. Immature inflorescence cultured on medium supplemented with lower levels of Picloram in combination with kinetin developed organogenic callus with shoot buds. Direct somatic embryo formation on rachis was observed at higher levels of Picloram in combination with kinetin. Plant regeneration was observed when calluses were transferred to α-napthaleneacetic acid (NAA) plus 6-benzylaminopurine (BA) supplemented MS medium. Histological observations provided a clear evidence for both somatic embryogenesis and shoot organogenesis. Profuse rooting was induced on phenylacetic acid (PAA) supplemented medium. Regenerated plants were successfully transferred to pots under field conditions where most of the plants survived and set normal seeds.     

Sink-Regulation of Photosynthesis in Relation to Temperature in Sunflower and Rape

Stimulation of the rate of photosynthesis at 2·0 kPaO₂ in comparison with 21 kPa O₂ and carbohydrate accumulationover 4h were measured during exposure of sunflower (Helianthusannuus L.) and rape (Brassica napus L.), grown at 30 °Cand 13 °C, to temperatures between 7 °C and 35 °C.The effect of reducing source: sink ratio by shading on theresponse of photosynthetic rate to temperature was also determined.Stimulation of photosynthesis by 2·0 kPa O₂ in comparisonwith 21 kPa O₂ decreased over 4 h at cool temperatures in sunflowerplants grown at 30 °C but not in rape grown at 30 °C.Stimulation did not decrease over 4 h in plants grown at 13CC. Sucrose was the main carbohydrate accumulated over 4 h;its accumulation increased with decreasing temperature. Starchaccumulation either decreased or remained the same with decreasingtemperature. In plants grown at 30 °C more carbohydrateaccumulated between 8 °C and 21 °C in sunflower thanin rape, but more carbohydrate accumulated at 30 °C in rapethan in sunflower. In plants grown at 13 °C much less carbohydrateaccumulated between 13 °C and 23 °C than in plants grownat 30 °C. Photosynthetic rate in plants grown at 30 °Cexposed to between 20 °C and 35 °C over 32 h (14 h light-10h dark-8 h light), declined over 32 h at 20 °C and 25 °Cin sunflower and at 20 °C in rape. This fall over 32 h,especially at 20 °C in sunflower, was significantly reducedby shading the rest of the plant. Shading had little effecton photosynthetic rate above 25 °C. The work confirms thatlow temperature imposes a sink-limitation on photosynthesiswhich occurs at higher temperatures in sunflower than in rape.This limitation may be relieved by decreasing the source:sinkratio. Key words: Sunflower, rape, photosynthesis, carbohydrates, sink demand, temperature     

Translocation of Organic Compounds in Sunflower IV. Effect of Temperature upon Translocation of Sugars

Apical halves of sunflower leaves were fed a solution of either¹⁴C-glucose or ^l4C-glucose and oligomycin at 5 and 25°C,respectively, and temperature, metabolism and translocationof ¹⁴C were measured at 10- to 30-min intervals. ¹⁴C-Labeledcompounds produced by control group leaves were compared tothose produced at 5°C and in the presence of oligomycin,at 25°C. In both experiments, similar small amounts of ¹⁴C-glucosemigrated into the basal half of the leaf. Sucrose translocationfrom the apical half was apparently affected by low temperatureand oligomycin. At least two types (affected and unaffectedby low temperature and oligomycin) of translocation seem tooccur simultaneously in sunflower. (Received July 4, 1980; Accepted November 27, 1980)     

Stem Anoxia Temporarily Interrupts Translocation of 11C-Photosynthate in Sunflower

Treatment of 5.0 cm of stem or petiole of sunflower with nitrogengas for 5 min or longer reduced phloem translocation of ¹¹C-photosynthateor stopped it entirely for a 15–20 min period after whichtime it slowly resumed When the N₂ was replaced by air the transportrestarted immediately and soon reached speeds higher than pre-anoxiarates. Thus anoxia, which we know inhibits export of photosynthateby preventing dark respiration in the leaf (Grodzinski, Jahnke,and Thompson, 1984), also inhibited transport in veins. It reducedthe speed of photosynthate movement eventually stopping it,probably by inhibiting the loading-unloading mechanisms alongthe sieve-tubes. These mechanisms are, apparently, themselves under control ofa metabolic respiratory mechanism (Fensom, 1981). Short-termanoxia of the stem did not appreciably affect the loading andtransport in the leaf; it probably did not destroy the integrityof membranes at the point of treatment nor cause a physicalblockage of sieve-plate pores nor change the viscosity of sieve-tubesap. A metabolic component which assists transport is indicatedin the mechanism of short-term translocation in sunflower. Key words: Sunflower, Petiole, Nitrogen gas, Phloem transport     

Mineralization of 2,4-Dichlorophenoxyacetic Acid (2,4-D) and Mixtures of 2,4-D and 2,4,5-Trichlorophenoxyacetic Acid by Phanerochaete chrysosporium

Evidence is presented for mineralization of 2,4-dichlorophenoxyacetic acid (2,4-D) in nutrient-rich media (high-nitrogen and malt extract media) by wild-type Phanerochaete chrysosporium and by a peroxidase-negative mutant of this organism. Mass balance analysis of [U-ring-¹⁴C]2,4-D mineralization in malt extract cultures showed 82.7% recovery of radioactivity. Of this, 38.6% was released as ¹⁴CO₂ and 27.0, 11.2, and 5.9% were present in the aqueous, methylene chloride, and mycelial fractions, respectively. 2,4-D and 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) were simultaneously mineralized when presented as a mixture, and mutual inhibition of degradation was not observed. In contrast, a relatively higher rate of mineralization of 2,4-D and 2,4,5-T was observed when these compounds were tested as mixtures than when they were tested alone.     

Metabolism of 2,4-Dichlorophenoxyacetic Acid (2,4-D) in Soybean Root Callus : EVIDENCE FOR THE CONVERSION OF 2,4-D AMINO ACID CONJUGATES TO FREE 2,4-D

An auxin-requiring soybean root callus metabolized [1-¹⁴C]-2,4-dichlorophenoxyacetic acid (2,4-D) to diethyl ether-soluble amino acid conjugates and water-soluble metabolites. The uptake in tissue varied with incubation time, concentration, and amount of tissue. Uptake was essentially complete (80%) after a 24-hour incubation and the percentage of free 2,4-D in the tissue fell to its lowest point at this time. At later times, the percentage of free 2,4-D increased and the percentage of amino acid conjugates decreased, whereas the percentage of water-soluble metabolites increased only slightly. Similar trends were seen if the tissue was incubated for 24 hours in radioactive 2,4-D, followed by incubation in media without 2,4-D for 24 hours. Inclusion of nonlabeled 2,4-D during the 24-hour chase period did not reduce amino acid conjugate disappearance but did reduce the percentage of free [1-¹⁴C]2,4-D. Thus, an external supply of 2,4-D does not directly prevent amino acid conjugate metabolism in this tissue. It is concluded that 2,4-D amino acid conjugates were actively metabolized by this tissue to free 2,4-D and water-soluble metabolites.     

Ammonia Gas Temporarily Interrupts Translocation of 11C Photosynthate in Sunflower

Exposure of a leaf of sunflower to ammonia gas for 1 to 2 sreduced translocation of ¹¹C-photosynthate or stopped it entirelyfor a 13 min period, after which time it slowly resumed. Treatmentof a stem of sunflower with NH₃ gas for 30 s to 90 s had a similareffect on ¹¹C translocation in the veins of the stem, by inhibitingloading from adjacent tissue. Unloading of ¹¹C from stem tissuecould, at times, be detected before normal transport resumed.Small applications of NH3 gas did not appear to damage the tissueand are thought to act by inhibiting transport primarily inplasmodesmata. Key words: Translocation, ammonia gas, ¹¹C photosynthate, sunflower     

The Effect of Temperature on Photosynthesis and Carbon Fluxes in Sunflower and Rape

Sunflower (Helianthus annuusL.) and oilseed rape (Brassica napusL.) were grown at constant temperatures of 30 ?C (warm) and13 ?C (cold). Maximal rates of photosynthesis between 5 ?C and35 ?C were at higher temperatures in sunflower than rape. Photosyntheticrate over 4 h at the growth temperature declined in warm-andcold-grown rape and cold-grown sunflower, but remained constantin warm-grown sunflower. The stimulation of photosynthesis by2.0 kPa O₂ compared to 21 kPa O₂ declined with decreasing temperature.At 10 ?C in warm-grown rape photosynthesis was insensitive to2.0 kPa O₂. However, sensitivity to low O₂ continued at 10 ?Cin warm-grown sunflower. Carbohydrates accumulated in the cold,particularly fructose, glucose and sucrose in warm-grown sunflowertransferred to 13 ?C. By monitoring changes of ¹⁴C in leaves after the assimilationof ¹⁴CO₂, the rates of carbon export from leaves, pool sizesand carbon fluxes between them were estimated. The transferof warm- and cold-grown rape to 13 ?C and 30 ?C, respectively,had little effect on these parameters over 22 h. However, exportof carbon from sunflower leaves at 13 ?C was markedly less thanat 30 ?C, irrespective of the growth temperature, due to slowerexport from the transport pool. The rapid suppression of carbonexport at 13 ?C in warm-grown sunflower may be due to inhibitedtranslocation rather than reduced sink demand in the cold. It is concluded that assimilate utilisation is more depressedin the cold than is photosynthesis; this imposes a greater restrictionon biomass production in sunflower than in rape. Key words: Sunflower, rape, temperature, photosynthesis, carbon fluxes     

14CO2 Assimilation and 14C-photosynthate Translocation in Different Leaves of Sunflower

Chlorophyll and nitrogen contents were highest in leaves of middle position, similarly as photosynthetic efficiency represented by ¹⁴C fixation (maxima in leaf 5 from the top). All the leaves lost ¹⁴C after 2 weeks of ¹⁴CO₂ exposure. However, the reduction in radioactivity was less in young upper leaves than in the mature lower leaves. Leaves exported ¹⁴C-photosynthates to stem both above and below the exposed leaf. Very little radioactivity was recovered from the seeds of plants in which only first or second leaves were exposed to ¹⁴CO₂ implying thereby that the carbon contribution of first two leaves to seed filling was negligible. The contribution of leaves to seed filling increased with the leaf position up to the sixth leaf from the top and after the seventh leaf their contribution to seed filling declined gradually.     

Translocation and Pileus Formation in Polyporus brumalis

The already-known promotion of pileus formation in pure culturesof Poly-porus brumalis by heightened evaporating conditionsis shown to be associated with enhanced translocation ratesinto the fruit-body (as investigated by dyes). This does notresult in changed proportions of dry matter or potassium inthe tissues at the stipe apex, but accelerated growth (dry-weightbasis) occurs in this region. Reduction of concentration ofthe medium (and hence probably of the concentration of the translocationstream) diminishes pileus production. Light intensity has onlya minor effect on dye transport. Fruit-bodies arrested in growthor killed show a negligible capacity for dye transport.     

2,4-D impact on bacterial communities, and the activity and genetic potential of 2,4-D degrading communities in soil

The key role of telluric microorganisms in pesticide degradation is well recognized but the possible relationships between the biodiversity of soil microbial communities and their functions still remain poorly documented. If microorganisms influence the fate of pesticides, pesticide application may reciprocally affect soil microorganisms. The objective of our work was to estimate the impact of 2,4-D application on the genetic structure of bacterial communities and the 2,4-D-degrading genetic potential in relation to 2,4-D mineralization. Experiments combined isotope measurements with molecular analyses. The impact of 2,4-D on soil bacterial populations was followed with ribosomal intergenic spacer analysis. The 2,4-D degrading genetic potential was estimated by real-time PCR targeted on tfdA sequences coding an enzyme specifically involved in 2,4-D mineralization. The genetic structure of bacterial communities was significantly modified in response to 2,4-D application, but only during the intense phase of 2,4-D biodegradation. This effect disappeared 7 days after the treatment. The 2,4-D degrading genetic potential increased rapidly following 2,4-D application. There was a concomitant increase between the tfdA copy number and the 14C microbial biomass. The maximum of tfdA sequences corresponded to the maximum rate of 2,4-D mineralization. In this soil, 2,4-D degrading microbial communities seem preferentially to use the tfd pathway to degrade 2,4-D.     

The Effect of 2,4-Dinitrophenol on Translocation in the Phloem

The effect of 2,4-dinitrophenol (DNP) on sucrose-¹⁴C transport in Soya seedlings has been analysed. The aim was to distinguish between an effect of the inhibitor on sugar movement within the phloem sieve tubes themselves, and on the prior steps of uptake and secretion of sugar into the conducting cells. DNP drastically inhibited sucrose-¹⁴C transport if it was applied to the ¹⁴C-treated leaf immediately before, or during, ¹⁴C supply. Transport was also strongly inhibited if DNP was applied along the translocation path while the ¹⁴C-treated leaflet was still in position on the plant. When, bowever, DNP was applied through the cut petioles of the primary leaves after removal of the ¹⁴C-treated terminal leaflet of the first trifoliate leaf, no inbibition was observed. On the contrary, transport appeared to have been promoted: significantly more ¹⁴C disappeared from the upper regions of DNP-treated plants as compared with controls, while in the lower plant parts more ¹⁴C accumulated. Different rates of synthesis of sucrose-¹⁴C into non-alcohol-soluble compounds could not account for this result. A similar stimulatory effect was observed when DNP was applied to the cut petiole of a primary leaf opposite that treated with ¹⁴C. Several indications were obtained that ¹⁴C which has reached the lower parts of the plant may circulate upwards again through the phloem within about 15 minutes. When sucrose-¹⁴C was introduced into the roots via the xylem, both DNP treatment and prior steam girdling resulted in the apparent accumulation of ¹⁴C in the lower plant parts. the results would be compatible with DNP inhibition of upwards movement in the phloem. DNP might also have affected sugar uptake processes in cells neighbouring the translocation path. It is concluded that the inhibitory effect of DNP on downwards phloem transport reported by earlier workers was probably due to an effect on uptake and/or secretion into the sieve tubes, not to an effect on the conducting cells themselves. Modern theories for phloem transport are discussed in the light of these findings.     

Impacts of 2,4-D application on soil microbial community structure and on populations associated with 2,4-D degradation

The effect of 2,4-dichlorophenoxyacetic acid (2,4-D) application rate on microbial community structure and on the diversity of dominant 2,4-D degrading bacteria in an agricultural soil was examined using cultivation-independent molecular techniques coupled with traditional isolation and enumeration methods. Fingerprints of microbial communities established under increasing concentrations of 2,4-D (0-500 mg kg-1) in batch soil microcosms were obtained using denaturing gradient gel electrophoresis (DGGE) of PCR-amplified 16S rRNA gene segments. While a 2,4-D concentration of at least 100 mg kg-1 was required to obtain an apparent change in the community structure as visualized by DGGE, the greatest impact of 2,4-D concentration occurred in the 500 mg kg-1 treatment, resulting in significantly reduced diversity of the dominant populations and enrichment by Burkholderia-like populations. The greatest diversity of 2,4-D degrading isolates was cultivated from the 10 mg kg-1 treatment, indicating that under these conditions, cultivation was more sensitive than DGGE for detecting changes in community structure. Most of these isolates harbored homologs of Ralstonia eutrophus JMP134 and Burkholderia cepacia tfdA catabolic genes. Results from this study revealed that agriculturally relevant application rates of 2,4-D may provide a temporary selective advantage for organisms capable of utilizing 2,4-D as a carbon and energy source.     

The localization of 2,4-D in leaf tissue

Summary Disruption of chloroplast structure is known to occur following applications of 2,4-dichlorophenoxyacetic acid (2,4-D) to the leaves of Phaseolus vulgaris. Although chloroplasts which had been extracted from treated tissue in an aqueous medium were free from 2,4-D and its metabolites, those extracted from freeze-dried tissue in organic solvents are the principal cell components to contain the herbicide.