Chlorsulfuron modifies biosynthesis of acyl Acid substituents of sucrose esters secreted by tobacco trichomes

Sucrose esters and duvatrienediol diterpenes are principal constituents formed in and secreted outside head cells of trichomes occurring on surfaces of Nicotiana tabacum. Using trichome-bearing epidermal peels prepared from midveins of N. tabacum cv T.I. 1068 leaves, we found that chlorsulfuron reduced and modified radiolabeling of sucrose ester acyl acids derived from branched-chain amino acid metabolism. The herbicide did not effect formation and exudation of diterpenes which are products of isoprenoid metabolism. Treatment with 1.0 micromolar chlorsulfuron affected 8.5- and 6.3-fold reductions in radiolabeling of methylvaleryl and methylbutyryl groups of sucrose esters, respectively, and concomitant increases of 9- and 9.8-fold in radiolabeling of straight chain valeryl and butyryl groups, respectively. These results and others indicate that inhibition of acetolactate synthase causes an accumulation of 2-oxo-butyric acid that is utilized by enzymes common to Leu biosynthesis to form 2-oxo-valeric acid. Coenzyme A (CoA) activation of this keto acid gives rise to butyryl CoA, which is utilized to form butyryl containing sucrose esters. Alternatively, reutilization of 2-oxo-valeric acid by the same enzymes followed by CoA activation leads to valeryl containing sucrose esters. We propose that in trichome secretory cells synthase, isomerase and dehydrogenase enzymes which catalyze Leu synthesis/degredation in most tissues, convert iso-branched, anteiso-branched and straight-chain keto acids in the formation of sucrose ester acyl groups.     

Site of action of chlorsulfuron: inhibition of valine and isoleucine biosynthesis in plants

The sulfonylurea herbicide chlorsulfuron blocks the biosynthesis of the amino acids valine and isoleucine in plants. Addition of these two amino acids to excised pea root (Pisum sativum L. var Alaska) cultures incubated in the presence of chlorsulfuron completely alleviates herbicide-induced growth inhibition. The site of action of chlorsulfuron is the enzyme acetolactate synthase which catalyzes the first step in the biosynthesis of valine and isoleucine. This enzyme is extremely sensitive to inhibition by chlorsulfuron having I₅₀ values ranging from 18 to 36 nanomolar. In addition, acetolactate synthase from a wide variety of tolerant and sensitive plants species is highly sensitive to inhibition by chlorsulfuron.     

Reversal of chlorsulfuron-induced inhibition of mitotic entry by isoleucine and valine

Pea (Pisum sativum L. cv Alaska) root tips were excised and cultured aseptically in White's medium. Cultures were treated immediately or after a 24 hour equilibration time with 28 nanomolar chlorsulfuron plus isoleucine and valine (each 0.1 millimolar), isoleucine and valine, or untreated. The percentage of mitotic figures in untreated control roots sampled immediately after excision showed a transitory drop and recovery within 24 hours (an excision effect). In chlorsulfuron-treated roots, the percentage of mitotic figures did not recover. In roots treated with chlorsulfuron plus isoleucine and valine, a complete recovery did occur. If roots were treated with chlorsulfuron 24 hours after excision, the percentage of mitotic figures was reduced to near 0 by 8 hours. In roots treated with chlorsulfuron plus isoleucine and valine, no reduction in mitotic figures occurred. The complete reversal of chlorsulfuron-inhibited mitotic entry by isoleucine and valine implicates these amino acids, in some manner, with the control of cell cycles progression.     

Role of the Apoplast in the Control of Assimilate Transport,Photosynthesis, and Plant Productivity

A concept is suggested, which supposes that assimilates are transferred within the plant downward through phloem sieve tubes and, after entering the stem apoplast, are carried up with the ascending flow of transpiration water. After entering the apoplast of fully expanded leaves, these solutes are reexported through the phloem. Thus, a common pool of assimilates with uniform concentration is formed in the plant apoplast. According to this concept, the mechanism of assimilate demand represents a response of photosynthetic apparatus to changes in the apoplastic level of metabolites consumed by sink organs. The ratios of labeled photoassimilates differ between the apoplast and mesophyll cells. Most of the apoplastic labeled carbon is contained in sucrose, less in amino acids, and even less in hexoses. The ¹⁴C-labeling of amino acids increases and the sucrose/hexose labeling ratio decreased under conditions of enhanced nitrate supply. The well-known effect of relative inhibition of assimilate export from leaves under conditions of enhanced nitrogen supply is explained by an enhanced hydrolysis of apoplast-derived sucrose due to the increase in invertase activity, rather than by diversion of primary photosynthetic products from sucrose synthesis to other pathways required for activated growth processes in leaves. This notion is based on observations that the sucrose/hexose ratio is reduced to a greater extent in the apoplast than in the symplast. The last assumption was supported by data obtained after artificial changes in the apoplastic pH. In these experiments intact plants were placed in the atmosphere of NH₃ or HCl vapors, which induced opposite changes in relative content of labeled assimilates in the apoplast and in the photosynthetic rate.     

Effect of Chlorsulfuron on Morphogenetic and Disordered Cell Division in Cultures of Passiflora edulis

We examined the effects of a sulfonylurea herbicide, chlorsulfuron, which is known as a potent inhibitor of plant cell division, on morphogenetic cell division and disorganized cell division using the culture system of multiple shoot primordia and callus of Passiflora edulis. The multiple shoot primordia tissue treated with chlorsulfuron failed to achieve shoot morphogenesis, and a large part of the tissue was necrotized during the posttreatment culture, even when it was washed and transferred to chlorsulfuron-free medium. The inhibition of Passiflora shoot morphogenesis by chlorsulfuron was not reversed by the simultaneous addition of branched amino acids, which are known to reverse the inhibitory effect of chlorsulfuron. In contrast, the same treatment of chlorsulfuron on the callus did not kill the cells, although the growth resumption was retarded by a prolonged lag period. The addition of branched amino acids enhanced the recovery growth of the chlorsulfuron-treated callus. These results suggest that the inhibition of disorganized cell division (callus growth) by chlorsulfuron is reversible, whereas morphogenetic cell division (shoot morphogenesis), which is under complex regulation, is inhibited irreversibly by chlorsulfuron. Qualitative differences between morphogenetic cell division and disordered simple proliferative cell division are discussed. Received November 17, 1997; accepted June 4, 1998     

Resistance to Acetolactate Synthase-Inhibiting Herbicides in Annual Ryegrass (Lolium rigidum) Involves at Least Two Mechanisms

WLR1, a biotype of Lolium rigidum Gaud. that had been treated with the sulfonylurea herbicide chlorsulfuron in 7 consecutive years, was found to be resistant to both the wheat-selective and the nonselective sulfonylurea and imidazolinone herbicides. Biotype SLR31, which became cross-resistant to chlorsulfuron following treatment with the aryloxyphenoxypropionate herbicide diclofop-methyl, was resistant to the wheat-selective, but not the nonselective, sulfonylurea and imidazolinone herbicides. The concentrations of herbicide required to reduce in vitro acetolactate synthase (ALs) activity 50% with respect to control assays minus herbicide for biotype WLR1 was greater than those for susceptible biotype VLR1 by a factor of >30, >30, 7,4, and 2 for the herbicides chlorsulfuron, sulfometuron-methyl, imazapyr, imazathapyr, and imazamethabenz, respectively. ALS activity from biotype SLR31 responded in a similar manner to that of the susceptible biotype VLR1. The resistant biotypes metabolized chlorsulfuron more rapidly than the susceptible biotype. Metabolism of 50% of [phenyl-U-¹⁴C]chlorsulfuron in the culms of two-leaf seedlings required 3.7 h in biotype SLR31, 5.1 h in biotype WLR1, and 7.1 h in biotype VLR1. In all biotypes the metabolism of chlorsulfuron in the culms was more rapid than that in the leaf lamina. Resistance to ALS inhibitors in L. rigidum may involve at least two mechanisms, increased metabolism of the herbicide and/or a herbicide-insensitive ALS.     

Loss of recovery capacity of plasmalemma k influx after cutting in chlorsulfuron pretreated maize roots

Active K⁺ influx was studied in apical segments from maize (Zea mays L., hybrid lines XL 342) and pea (Pisum sativum L. var Laxton superbo) seedlings pretreated with the herbicide chlorsulfuron (2-chloro-N-[(4-methoxy-6-methyl-1,3,5-triazin-2-yl) aminocarbonyl]benzenesulfonamide).

Even though both plants were sensitive to chlorsulfuron, a strong inhibition of K⁺ uptake only was evident in maize root segments after 12 hours pretreatment with 10 micromolar chlorsulfuron. The inhibition was revealed only when maize root segments were washed for 2 hours before uptake measurements. This was done in order to recover K⁺ influx inhibited by cutting injury. Consequently, we demonstrated that roots from chlorsulfuron pretreated maize seedlings lost the capacity to recover from cutting injury by washing. By contrast, K⁺ influx in pea roots was not inhibited by chlorsulfuron because pea roots notoriously do not exhibit the `washing' effect.

     

The influence of new herbicide ZJ0273 on the total- and branched-chain amino acids in oilseed rape (Brassica napus L.) leaves as revealed by near-infrared spectroscopy

Propyl 4-(2-(4,6-dimethoxypyrimidin-2-yloxy) benzylamino)benzoate (ZJ0273) is a new herbicide which inhibits acetolactate synthase (ALS). The ZJ0273 is considered as safe for the environment and exhibits a satisfactory effect on weed control in the rapeseed field. ALS is the key enzyme of reactions in the biosynthesis of total amino acids (TAAs) especially branched-chain amino acids (BCAAs). This study reports the effect of ZJ0273 on BCAAs and TAAs in rapeseed leaves using near-infrared spectroscopy (NIRS) techniques. A decrease in TAAs and BCAAs contents was observed as the herbicide dosages were increased along with leaf senescence. The wavelengths 2,416 and 1,340 nm were selected to develop the NIRS model for detecting BCAAs and TAAs, and correlation coefficients of model’s prediction set were 0.9823, 0.9764, 0.9831, and 0.9968 for valine, isoleucine, leucine, and TAAs, respectively. The results indicated that 100 mg/L ZJ0273 was a safe dosage for oilseed rape as it did not show a significant effect on the contents of amino acids compared to other higher dosages (500 and 1,000 mg/L).     

Effect of chlorsulfuron on ethylene evolution from sunflower seedlings

The effect of the herbicide chlorsulfuron (2-chloro-N-[(4-methoxy - 6 - methyl -1, 3,5 - triazin - 2 - yl)aminocarbonyl]benzenesulfonamide) on ethylene production in light-grown sunflower (Helianthus annuus L.) seedlings was examined. Application of chlorsulfuron to the apex stimulated ethylene production in all tissues examined: cotyledons, hypocotyls, and roots. The greatest stimulation occurred in the upper portion of the hypocotyl adjacent to, and including, the cotyledonary node. Ethylene evolution from hypocotyls excised from treated seedlings was stimulated over control levels 1 day after herbicide application and reached a maximum (approx. 75 x control or 17 nl/g f wt/h) 2 to 3 days after treatment. Labeling and inhibitor studies indicated that the ethylene produced was derived primarily from methionine. Chlorsulfuron treatment stimulated the rate of accumulation of the ethylene precursor, 1-aminocyclopropane-1-carboxylic acid (ACC), as well as the ability of the tissue to convert exogenous ACC to ethylene. Chlorsulfuron had little effect on ethylene production when administered to the hypocotylsin vitro. Removal of the cotyledons from treated seedlings reduced the rate of ethylene evolution from the hypocotyls. These results suggest that stimulation of ethylene production in sunflower hypocotyls by chlorsulfuron is not a wound response but rather is dependent on factors derived from the cotyledons.     

Patterns of Assimilate Production and Translocation in Muskmelon (Cucumis melo L.) : II. Low Temperature Effects

Muskmelon (Cucumis melo L.) plants were exposed to a 10°C chilling treatment for 72 hours, which induced leaf chilling injury symptoms (wilting, appearance of water-soaked areas, necrosis). Chilling caused an accumulation of starch, sucrose, hexoses (glucose and fructose), and certain amino acids (glutamate, aspartate, and citrulline) in source leaf tissues, but no accumulation of stachyose or other galactosyl-oligosaccharides occurred. Chilling also caused a general increase in sugar (stachyose, raffinose, sucrose) and amino acid content of the phloem sap, although rates of phloem transport were apparently reduced. Pretreatment of the leaves with a 20-milligram per liter abscisic acid (ABA) spray before chilling prevented the appearance of chilling injury symptoms. ABA pretreatment had little or no affect on sugar accumulation in leaf tissues but greatly reduced or eliminated the chilling-induced amino acid accumulation. Higher levels of aspartate and particularly of arginine were found in phloem saps from ABA-pretreated plants. The data indicate that changes in leaf metabolism caused by environmental stresses such as chilling may change the composition of cucurbit phloem sap. This raises the possibility that some of the deleterious effects of stress on sink tissues may, in part, be due to alterations in the nature of the assimilate supply.     

Amino Acid Metabolism of Lemna minor L. : II. Responses to Chlorsulfuron

Chlorsulfuron, an inhibitor of acetolactate synthase (EC 4.1.3.18) (TB Ray 1984 Plant Physiol 75: 827-831), markedly inhibited the growth of Lemna minor at concentrations of 10⁻⁸ molar and above, but had no inhibitory effects on growth at 10⁻⁹ molar. At growth inhibitory concentrations, chlorsulfuron caused a pronounced increase in total free amino acid levels within 24 hours. Valine, leucine, and isoleucine, however, became smaller percentages of the total free amino acid pool as the concentration of chlorsulfuron was increased. At concentrations of chlorsulfuron of 10⁻⁸ molar and above, a new amino acid was accumulated in the free pool. This amino acid was identified as α-amino-n-butyrate by chemical ionization and electron impact gas chromatography-mass spectrometry. The amount of α-amino-n-butyrate increased from undetectable levels in untreated plants, to as high as 840 nanomoles per gram fresh weight (2.44% of the total free pool) in plants treated with 10⁻⁴ molar chlorsulfuron for 24 hours. The accumulation of this amino acid was completely inhibited by methionine sulfoximine. Chlorsulfuron did not inhibit the methionine sulfoximine induced accumulations of valine, leucine, and isoleucine, supporting the idea that the accumulation of the branched-chain amino acids in methionine sulfoximine treated plants is the result of protein turnover rather than enhanced synthesis. Protein turnover may be primarily responsible for the failure to achieve complete depletion of valine, leucine, and isoleucine even at concentrations of chlorsulfuron some 10⁴ times greater than that required to inhibit growth. Tracer studies with ¹⁵N demonstrate that chlorsulfuron inhibits the incorporation of ¹⁵N into valine, leucine, and isoleucine. The α-amino-n-butyrate accumulated in the presence of chlorsulfuron and [¹⁵N]H₄⁺ was heavily labeled with ¹⁵N at early time points and appeared to be derived by transamination from a rapidly labeled amino acid such as glutamate or alanine. We propose that chlorsulfuron inhibition of acetolactate synthase may lead to accumulation of 2-oxobutyrate in the isoleucine branch of the pathway, and transamination of 2-oxobutyrate to α-amino-n-butyrate by a constitutive transaminase utilizing either glutamate or alanine as α-amino-N donors.     

Cessation of assimilate uptake in maturing soybean seeds

In vitro assimilate uptake and metabolism were evaluated in embryos of known age isolated from seeds at mid-podfilling through physiological maturity. The capacity of isolated Wye soybean embryos to take up exogenous [¹⁴C]sucrose dropped nearly 4-fold in less than 1 week at incipient cotyledon yellowing. This drop in rate of sucrose uptake coincided with cessation of seed growth as well as rapid decline in leaf photosynthetic rate that preceded leaf yellowing. Conversely, the rate of [³H]glutamine uptake by cotyledons increased as they yellowed. Yellow cotyledons also rapidly converted exogenous [³H]glutamine to ethanolinsoluble components, but converted little exogenous [¹⁴C]sucrose to ethanol-insoluble components, primarily because of greatly reduced sucrose uptake. Sustained import and metabolism of amino acids remobilized from senescing leaves may prolong seed growth beyond loss of photosynthetic competency and sucrose availability.     

Efficacy of Organic Soil Amendments for Management of Heterodera glycines in Greenhouse Experiments

In a repeated greenhouse experiment, organic soil amendments were screened for effects on population density of soybean cyst nematode (SCN), Heterodera glycines, and soybean growth. Ten amendments at various rates were tested: fresh plant material of field pennycress, marigold, spring camelina, and Cuphea; condensed distiller’s solubles (CDS), ash of combusted CDS, ash of combusted turkey manure (TMA), marigold powder, canola meal, and pennycress seed powder. Soybeans were grown for 70 d in field soil with amendments and SCN eggs incorporated at planting. At 40 d after planting (DAP), many amendments reduced SCN egg population density, but some also reduced plant height. Cuphea plant at application rate of 2.9% (amendment:soil, w:w, same below), marigold plant at 2.9%, pennycress seed powder at 0.5%, canola meal at 1%, and CDS at 4.3% were effective against SCN with population reductions of 35.2%, 46.6%, 46.7%, 73.2%, and 73.3% compared with control, respectively. For Experiment 1 at 70 DAP, canola meal at 1% and pennycress seed powder at 0.5% reduced SCN population density 70% and 54%, respectively. CDS at 4.3%, ash of CDS at 0.2%, and TMA at 1% increased dry plant mass whereas CDS at 4.3% and pennycress seed powder at 0.1% reduced plant height. For Experiment 2 at 70 DAP, amendments did not affect SCN population nor plant growth. In summary, some amendments were effective for SCN management, but phytoxicity was a concern.     

Trends in carbohydrate depletion, respiratory carbon loss, and assimilate export from soybean leaves at night

To evaluate assimilate export from soybean (Glycine max [L.] Merrill) leaves at night, rates of respiratory CO₂ loss, specific leaf weight loss, starch mobilization, and changes in sucrose concentration were measured during a 10-hour dark period in leaves of pod-bearing `Amsoy 71' and `Wells II' plants in a controlled environment. Lateral leaflets were removed at various times between 2200 hours (beginning dark period) and 0800 hours (ending dark period) for dry weight determination and carbohydrate analyses. Respiratory CO₂ loss was measured throughout the 10-hour dark period. Rate of export was estimated from the rate of loss in specific leaf weight and rate of CO₂ efflux. Rate of assimilate export was not constant. Rate of export was relatively low during the beginning of the dark period, peaked during the middle of the dark period, and then decreased to near zero by the end of darkness. Rate of assimilate export was associated with rate of starch mobilization and amount of starch reserves available for export. Leaves of Amsoy 71 had a higher maximum export rate in conjunction with a greater total change in starch concentration than did leaves of Wells II. Sucrose concentration rapidly declined during the first hour of darkness and then remained constant throughout the rest of the night in leaves of both cultivars. Rate of assimilate export was not associated with leaf sucrose concentration.     

Acclimation of potato plants to polyethylene glycol-induced water deficit II. Contents and subcellular distribution of organic solutes

Potato plants (Solanum tuberosum cv. Désirée) were grown hydroponically and subjected to water deficit induced by addition of 10% (w/v) PEG 6000. The potato plants were able to grow under water deficit by accumulating organic solutes (osmoregulation). Osmoregulation occurred in two phases. During the initial 2d hexoses were accumulated, and after 7 d of PEG treatment osmotic adjustment was mostly due to the accumulation of amino acids, especially proline, which accumulated up to 150 times the control content. Sucrose contents remained unchanged in leaves of PEG-treated plants compared with controls, whereas the starch content decreased during PEG treatment.In control leaves, the hexoses and malate were compartmented in the vacuole and sucrose was found in the cytosol and vacuole. Amino acids were distributed between the cytosol and stroma, but only minor amounts of amino acids could be detected in the vacuole. Under water deficit the subcellular distribution of hexoses, malate and sucrose remained unchanged. Most amino acids showed a slight to moderate higher concentration in the vacuole under water deficit. Proline, the metabolite contributing mainly to osmoregulation, was concentrated mostly in the chloroplast and the cytosol. This underlines the important role of proline as the osmolyte under water deficit.     

Biochemical basis for effects of k-deficiency on assimilate export rate and accumulation of soluble sugars in soybean leaves

The effects of K-deficiency on carbon exchange rates (CER), photosynthate partitioning, export rate, and activities of key enzymes involved in sucrose metabolism were studied in soybean (Glycine max [L.] Merr.) leaves. The different parameters were monitored in mature leaves that had expanded prior to, or during, imposition of a complete K-deficiency (plants received K-free nutrition solution). In general, recently expanded leaves had the highest concentration of K, and imposition of K-stress at any stage of leaf expansion resulted in decreased K concentrations relative to control plants (10 millimolar K). A reduction in CER, relative to control plants, was only observed in leaves that expanded during the K-stress. Stomatal conductance also declined, but this was not the primary cause of the decrease in carbon fixation because internal CO₂ concentration was unaffected by K-stress. Assimilate export rate from K-deficient leaves was reduced but relative export, calculated as a percentage of CER, was similar to control leaves. Over all the data, export rate was correlated positively with both CER and activity of sucrose phosphate synthase in leaf extracts. K-deficient leaves had higher concentrations of sucrose and hexose sugars. Accumulation of hexose sugars was associated with increased activities of acid invertase. Neutral invertase activity was low and unaffected by K-nutrition. It is concluded that decreased rates of assimilate export are associated with decreased activities of sucrose phosphate synthase, a key enzyme involved in sucrose formation, and that accumulation of hexose sugars may occur because of increased hydrolysis of sucrose in K-deficient leaves.     

Effect of chlorsulfuron on phenylpropanoid metabolism in sunflower seedlings

The effect of the herbicide chlorsulfuron on phenylpropanoid titer and metabolism and the role of endogenous ethylene in this response was examined in light-grown sunflower (Helianthus annuus L.) seedlings. Application of chlorsulfuron to the apex resulted in large increases in both total phenolic and hydroxycinnamic acid content in hypocotyls isolated from the treated seedlings. Both of these parameters were increased within 24 h of herbicide treatment, and both reached a maximum level 3–4 days post-treatment. An increase in ethylene evolution was found to proceed in parallel with the alterations of phenolic content. The extractable activities of phenylalanine ammonia lyase,trans-cinnamic-4-hydroxylase, and soluble peroxidase were increased by chlorsulfuron treatment. Chlorsulfuron had little effect on total phenolic content when administered directly to isolated hypocotyl segments. Exogenous ethylene had no effect on the endogenous titer of phenolic compounds. Root-fed cobalt chloride (5 × 10^?4 M) inhibited chlorsulfuron-induced ethylene production by 92% and also inhibited the accumulation of phenolic materials by 56%. Exogenous ethylene was unable to reverse the inhibition caused by cobalt chloride. It was concluded that chlorsulfuron-induced increases in phenolic compounds were not mediated solely by endogenous ethylene.     

Apoplastic expression of yeast-derived invertase in potato : effects on photosynthesis, leaf solute composition, water relations, and tuber composition

In potato plants (Solanum tuberosum), a chimeric yeast-derived invertase gene fused to a 35S cauliflower mosaic virus promoter has been expressed. The protein was targeted to the cell wall by using the signal peptide of proteinase inhibitor II fused to the amino terminus of the yeast invertase. The transformed plants had crinkled leaves, showed a reduced growth rate, and produced fewer tubers. Although in the apoplast of the leaves of the transformed plants the content of glucose and fructose rose by a factor of 20, and that of sucrose declined 20-fold, 98% of the carbohydrate in the phloem sap consisted of sucrose, demonstrating the strong specificity of phloem loading. In the leaf cells of the transformed plants, glucose, fructose, and amino acids, especially proline, were accumulated. Consequently, the osmolality of the cell sap rose from 250 to 350 mosmol/kg. Our results show that the observed 75% decrease of photosynthesis is not caused by a feedback regulation of sucrose synthesis and is accompanied by an increase in the osmotic pressure in the leaf cells. In the transformed plants, not only the amino acid to sucrose ratio in the phloem sap, but also the amino acid and protein contents in the tubers were found to be elevated. In the tubers of the transformed plants, the protein to starch ratio increased.