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Herbicides that inhibit branched chain amino acid biosynthesis produce a rapid carbohydrate increase in leaves of treated plants. The relationship between these processes is not known nor is the importance of carbohydrate accumulation in the growth inhibition caused by these herbicides. This work analyzes carbohydrate concentration in sources and sinks after herbicide treatments in pea (Pisum sativum L.), as well as photosynthetic carbon assimilation, using two classes of chemicals, chlorsulfuron and imazethapyr, applied to roots or leaves. The most remarkable result was that, in addition to carbohydrate accumulation in leaves, accumulation of sucrose and/or starch in roots was detected. This pattern of carbohydrate accumulation was similar for both herbicides and independent of whether the herbicides were applied to leaves or roots. This indicates that root growth inhibition was not caused by sugar starvation in sinks. Nevertheless, the results are consistent with a decrease in sink strength, leading to the inhibition of photoassimilate translocation.

Acetolactate synthase (ALS; EC 4.1.3.18) inhibition is the primary mechanism of action of imazethapyr (IM). However, the precise mechanisms that links ALS inhibition with plant death have not been elucidated. Supply of IM to pea (Pisum sativum L) plants produced an immediate cessation of growth, caused a 50% inhibition of the in vivo ALS activity within 1 day of treatment, and a remarkable accumulation (2.7-times) of free amino acids after 3 days. Carbohydrates (soluble and starch) were accumulated in both leaves and roots. Accumulation of soluble sugars in roots preceded that of starch in leaves, suggesting that the accumulation of carbohydrates in leaves is not the reason for the arrested root growth. A transient pyruvate accumulation was observed in roots, 1 day after the onset of IM supply. This was coincident with an increase in pyruvate decarboxylase (EC 4.1.1.1), and later increases in alcohol dehydrogenase (EC 1.1.1.1), lactate dehydrogenase (EC 1.1.1.27), and alanine amino transferase (EC 2.6.1.2) activities. This enhancement of fermentative activities was coincident with a slight decrease in aerobic respiration. The overall data suggest that the impairment of ALS activity may lead to a fermentative metabolism that may be involved in growth inhibition and plant death.

It has been suggested that semileafless varieties of pea (Pisum sativum L.), where leaflets are replaced by tendrils, are more tolerant to water deficit than conventional leafed varieties. This response to water stress has been assumed to be related to the reduced leaf area of the semileafless varieties. However, total leaf area of the varieties used in this study was not significantly different and, also, transpiration rate per plant did not differ under control conditions. Moreover, transpiration rates declined similarly in both varieties under water stress. In order to gain further insights into the better tolerance of semileafless plants to water stress, a detailed study was carried out both at tissue level and at single cell level in order to characterise the osmotic adjustment carried out in the different leaf structures. At tissue level, osmolarity did not differ among structures, and it increased similarly under water stress regardless of the leaf structure. While, at mesophyll vacuole level, most mineral elements analysed did not accumulate in response to water stress. At epidermal vacuole level, tendrils of the semileafless showed a higher osmolarity than the homologous structure of conventional pea. This might represent an advantage in order to face water restrictions. Since tendrils represent around a 40% of the compound leaf of the semileafless variety, a more efficient osmotic adjustment carried out by tendrils may represent an important improvement in the water use efficiency under water deficits.

Nitrogen fixation in legume nodules has been shown to be very sensitive to drought and other environmental constraints. It has been widely assumed that this decline in nitrogen fixation was a consequence of an increase in the so-called oxygen diffusion barrier and a subsequent impairment to bacteroid respiration. However, it has been recently shown that nitrogen fixation is highly correlated with nodule sucrose synthase (SS) activity under drought and other environmental stresses. Whether this correlation reflects a causative relationship or not has not been proven yet. The evidence presented here suggests that SS controls nitrogen fixation under mild drought conditions. However, nitrogen fixation cannot be enhanced only by increasing glycolytic flux, as under these conditions nodules become oxygen limited. Abscisic acid also induces a decline in nitrogen fixation that is independent of SS. The overall results suggest the occurrence of a complex regulation of nodule nitrogen fixation involving, at least, both carbohydrate and oxygen fluxes within the nodule. Loli Gálvez was the holder of a grant from the Spanish Ministry of Education (Plan F.P.U.). This work was supported by DGESIC (PB98-0545).

When nitrogen fixing legumes are subjected to drought, it is not obvious which function of the stressed plant is actually affecting the nodule. There is a direct correlation between the decreased nodule water potential and the decline of nitrogenase activity (Pankhurst, Sprent, 1975). Recently, the idea that metabolic potential of nodules is reduced during drought has been confirmed (Diaz del Castillo et al., 1994). We have suggested a key role for sucrose synthase in nodules of soybean subjected to drought (Gonzalez et al., 1995). Our results suggest that this observation can be also assumed for temperate legumes with indeterminate nodules.

Legumes are intolerant of flooding and symbiotic nitrogen fixation is particularly sensitive to waterlogging. Morphologically, the most obvious features of nodules subjected to flooding are the enhancement of lenticel- like structures on their surfaces and increased nodule cortication. These anatomical features increase the supply of oxygen to the bacteroid- containing tissue. Nevertheless, nitrogen fixation is severely impaired under waterlogged conditions. Exposure to subambient oxygen concentrations decreases the nitrogenase activity of detached root nodules. However, some recovery may occur in intact nodulated legumes after exposure to short-term moderate anoxygenic conditions. Tolerance of long-term moderate oxygen deficiency by grain legumes with determinate nodules is achieved by several structural adaptive mechanisms (e.g., the nodule surface is covered with loosely packed cells or the nodules have an increased number of unoccluded intercellular spaces, enhancing gaseous permeability) and biochemical adaptations (increased expression of ureide-forming enzymes and leghemoglobin content). Nevertheless, the tolerance mechanisms displayed by these plants under conditions of moderate hypoxia fail to provide an adequate environment for nitrogen fixation under severe hypoxia. This adaptation involves changes in photosynthate allocation to underground organs, aerenchyma formation in both roots and nodules to help maintain high rates of respiration (and hence mineral uptake), a less compact cortex with an increased nodule gas permeability, increased recycling of CO2, a less demanding nitrogenase activity and a more efficient nodule structure.

Summary Chlorella pyrenoidosa Pringsheim (211/8 a) was grown photoautotrophically using the herbicide, glyphosate [N-(phosphonomethyl)-gycine], in concentrations ranging from 0.1 mM to 1 mM. Chlorophyll and carotenoid content, greening process, photosynthetic and respiration rates, and photosynthetic electron transport activities were investigated. Glyphosate decreased cell density and photosynthetic pigment content; 1.0 mM did not allow growth. Bleached cells were used for the greening process. Glyphosate had two different effects upon photosynthetic pigments: inhibition of chlorophyll synthesis and a decrease in carotenoids. Oxygen uptake was not affected, but oxygen evolution was strongly inhibited. The results suggest that glyphosate acts as an electron transport inhibitor, acting on both photosystems, but its effect was greater on PS II than PS I.

Summary The effect of the source of nitrogen nutrition (nitrogen fixation or nitrate assimilation) on the response of pea plants to a gradual and moderate water stress was studied. Growth declined under water deficit, but nodulated plants were less sensitive to drought than nitrate-fed plants. Stomatal conductance and internal CO2 concentration also decreased, but both were higher in nitrogen-fixing plants throughout the drought period, leading to better maintenance of carbon assimilation rates under water deficit. Glycolate oxidase, a key enzyme in the photorespiratory cycle, declined by 50% in nitrogen-fixing plants under water deficit, although it was not affected in nitrate-fed plants. Nitrogen assimilation declined during the drought period and was independent of nitrogen source. Free amino acid content declined in leaves of plants grown under both nutrition regimes, reflecting the decrease in nitrogen assimilation. Water stress led to carbohydrate accumulation in pea plants grown with either nitrogen source, but it was higher in nitrogen-fixing plants. Roots showed the greatest carbohydrate and amino acid accumulation in both nutritions regimes, with significantly greater increases in free amino acids in nitrate-fed plants. It is concluded that the nitrogen source is a major factor affecting pea responses to water stress, although the difference in sensitivity seems to be related not to the nitrogen assimilation process but to complex interactions with photorespiratory flux and stomatal conductance.

The effect of the source of nitrogen (N) nutrition [N2 fixation and nitrate (NO3) assimilation] on plant productivity, gas exchange, N assimilation, water relations, and glycollate oxidase activity were studied in pea plants. Nitrogen‐fixing plants showed a higher RGR over the study period. This higher RGR was correlated with higher stomatal conductance and, consequently, a higher transpiration rate. Photosynthetic N‐use efficiency was also higher when plants were nodulated. We found glycollate oxidase activity, a key enzyme of the photorespiratory pathway, much lower in N2 fixing plants, what strongly suggests that energy losses due to photorespiration are lower in these plants, and therefore, photosynthetic efficiency was higher.

Summary Imazethapyr (IM) is a herbicide that inhibits the branched-chain amino acid (BCAA) biosynthesis through the specific inhibition of acetolactate synthase activity. This herbicide acts very slowly and several weeks are required for complete plant death. From the BCAA biosynthesis inhibition to the growth inhibition and plant death, the processes involved are not fully understood. Starvation for BCAAs and/or starvation for carbohydrates in sinks. have been proposed as part of the death mechanisms. In this study, a permanent acetolactate synthase inhibition is used in order to (1) determine whether the growth inhibition effects can be attributed to a reduction in BCAA content and/or to starvation of carbohydrates; and (2) to analyse the physiological changes induced. Sublethal doses of IM were continuously supplied in the nutrient solution of nodulated pea plants. These conditions led to a significant decline in plant growth. The herbicide also caused a decline in nodule initiation, but had little effect on nodule development. However, plants were not nitrogen-limited and net photosynthesis was only slightly affected at the higher herbicide concentration. Total soluble sugars and starch were accumulated in both leaves and roots following herbicide supply. These results were also found in non-nodulated, nitrate-fed plants. In relation with a likely BCAA starvation, a significant increase was observed in the free amino acid pool, with a marked imbalance among different amino acids, although among BCAAs, only valine pool declined as a consequence of IM supply. It is concluded that acetolactate synthase inhibition by continuous, sublethal IM supply does not induce carbohydrate or a specific BCAA starvation in pea plants.

Several ethyl 2,3-dihydro-3-oxoisothiazolo[5,4-b]pyridine-2-alkanoate derivatives were synthesized as herbicides. Only 5-methyl derivatives inhibited both hypocotyl and root growth in the lettuce (Lactuca sativa L.) seedling test at 100 mg litre -1 . Only ethyl propionate and valerate derivatives showed significant inhibition at 0.1 mg litre -1 , whereas ethyl acetate or butyrate derivatives were inactive. Contrary to unoxidized derivatives, the inhibitory effect of 1-oxide and 1,1-dioxide derivatives was strongly dependent on concentration; ethyl 2,3-dihydro-5-methyl-3-oxoisothiazolo[5,4-b]pyridine-2-propionate 1,1-dioxide inhibited 100% of germination at 100 mg litre -1 and 45% of lettuce seedling growth at 0.1 mg litre -1 . Quantitative structure-inhibition of growth relationship analysis carried out by adaptive least-squares (ALS) method gave a good correlation with small and hydrophobic 5-substituents as well as with odd carbon-chain ethyl alkanoates in position 2. Active compounds did not show auxin-like activity from 0.1 to 100 mg litre -1 .