Begoña González-Moro

Temporal study of the effect of phosphinothricin on the activity of glutamine synthetase, glutamate dehydrogenase and nitrate reductase in Medicago sativa L.

Summary In a preliminary work we showed that when plants were sprayed with several doses of glufosinate, GS activity of the leaf was reduced by 50% after 48 h of application of 250 µM herbicide. Enzyme inhibition was accompained by a dramatic accumulation of ammonia. In this paper we analyze the time-course effect of phosphinothricin on nitrogen metabolism. Our results show that GS activity is the first process affected; more than 50% activity reduction is observed after 2 h of a 1,000 µM treatment, whereby ammonia values 400% higher than control were reached. NRase did not change until 24 h of assay, whereas protein content was reduced after 48 h. GDH activity was enhanced, but only 24 h after starting the treatment. These results indicate that the main target of PPT action is on GS activity, and other processes are long-term modified. GDH could, perhaps, reassimilate some of the ammonia produced but its activity is not able to completely prevent, the injury caused by phosphinothricin.

Effect of Phosphinothricin (Glufosinate) on Activities of Glutamine Synthetase and Glutamate Dehydrogenase in Medicago sativa L.

Summary The effect of the glutamate analogue phosphinothricin on enzymes of nitrogen assimilation was studied as well as the accumulation of ammonia and nitrate after treatment of lucerne plants with this herbicide. Glutamine synthetase (GS) was assayed with several phosphinothricin concentrations. When plants were sprayed with 250, 500 and 1,000 μM of the herbicide, GS activity of the leaf was reduced by 50% at 250 μM. Phosphinothricin caused a high ammonia accumulation in the tissues, reaching values of 70 μmol ammonia/g dry weight after treatment with 1,000 μM phosphinothricin. Glutamate dehydrogenase (GDH) activity increased ca. 40 % and nitrate reductase activity (NRase) was strongly decreased. Neither nitrate content nor proteolytic activity were altered.

Boric acid and salinity effects on maize roots. Response of aquaporins ZmPIP1 and ZmPIP2, and plasma membrane H+‐ATPase, in relation to water and nutrient uptake

Under saline conditions, an optimal cell water balance, possibly mediated by aquaporins, is important to maintain the whole-plant water status. Furthermore, excessive accumulation of boric acid in the soil solution can be observed in saline soils. In this work, the interaction between salinity and excess boron with respect to the root hydraulic conductance (L(0)), abundance of aquaporins (ZmPIP1 and ZmPIP2), ATPase activity and root sap nutrient content, in the highly boron- and salt-tolerant Zea mays L. cv. amylacea, was evaluated. A downregulation of root ZmPIP1 and ZmPIP2 aquaporin contents were observed in NaCl-treated plants in agreement with the L(0) measurements. However, in the H3BO3-treated plants differences in the ZmPIP1 and ZmPIP2 abundance were observed. The ATPase activity was related directly to the amount of ATPase protein and Na+ concentration in the roots, for which an increase in NaCl- and H3BO3+ NaCl-treated plants was observed with respect to untreated and H3BO3-treated plants. Although nutrient imbalance may result from the effect of salinity or H3BO3 alone, an ameliorative effect was observed when both treatments were applied together. In conclusion, our results suggest that under salt stress, the activity of specific membrane components can be influenced directly by boric acid, regulating the functions of certain aquaporin isoforms and ATPase as possible components of the salinity tolerance mechanism.

Glycolate accumulation causes a decrease of photosynthesis by inhibiting RUBISCO activity in maize

Summary The consequences of interruption of the photorespiratory cycle by phosphinothricin (PPT) and amino-oxyacetic acid (AOA) in several aspects of maize photosynthesis were investigated. Both compounds inhibited the photorespiratory pathway, causing glycolate accumulation and photosynthesis inhibition. PEPCase activity was not affected by PPT or by AOA, but NADP-ME activity was slightly increased. The inhibition of RUBISCO activity was not due to depletion of its substrates, neither CO 2 nor RuBP. An inverse relationship was found between glycolate accumulation and RUBISCO activity when photorespiration is blocked by PPT or AOA. We suggest that the accumulation of the photorespiratory metabolite, glycolate, provokes the inhibition of RUBISCO activity, and consequendy diminution of CO 2 assimilation.

Sequential effects of acidic precipitation and drought on photosynthesis and chlorophyll fluorescence parameters of Pinus radiata D. Don seedlings

Summary We studied the sequential effect of simulated acid rain (SAR) and water stress on net photosynthesis (A), internal CO 2 concentration ( Ci ), stomatal conductance (g), and instantaneous water use efficiency (A/E), and the photochemical damage caused by these stressors by analysing chlorophyll fluorescence parameters such as Fv/Fm, Fv/Fo and Fm/Fo. SAR caused inhibition of photosynthesis. The depletion of CO 2 assimilation seems not to be solely a stomatal effect as effects on chloroplasts contribute to this photosynthetic inhibition. No noticeable damage seems to occur to the photochemical apparatus since well-watered plants treated with acid rain did not show any change in the chlorophyll fluorescence. Water stress produced a large effect on both gas exchange and chlorophyll fluorescence parameters, indicating structural and functional damage. Interactions among SAR and drought had a larger impact on all parameters studied. However, after rewatering, significant recovery in both gas exchange and chlorophyll fluorescence parameters occurred within 6 days, although the values measured prior to the imposition of treatments were not recovered. We conclude that Pinus radiata exposed to acid rain were affected in a wide range of components in the photosynthetic apparatus, thereby modifying its response to sequential drought stress.

Sequential Effects of Acidic Precipitation and Drought on Water Relations of Pinus radiata Seedlings

Summary In this work we investigated the sequential effects of simulated acid rain and drought on water relations of radiata pine ( Pinus radiata D. Don) seedlings. Whole seedlings were firstly subjected during 1 month to acid rain, pH 3.0, applied 5 times a week at 3 mm precipitation equivalent per day. Afterwards, drought treatment was applied by withholding water for 20 days and subsequent rewatering for 6 days to analyze the sequential effect of acid precipitation and drought. Water status parameters were determined before the initiation of acid rain treatment and within 3 weeks after the end of treatment. Acid rain treatment in well-watered plants did not affect either water potential or relative water content, whereas a marked effect on electrolyte leakage from the needles and on instantaneous transpiration was recorded. Drought treatment had great effects on leaf water potential (-2.5 MPa), RWC (50 % diminution), membrane permeability (340 % increase) and transpiration rate (25 % inhibition). Interactions involving acid precipitation and drought led to much greater impacts on all of the parameters analyzed. We conclude that although acid rain had slight, if any, direct effects on the water relations of well-watered radiata pine, it altered the drought tolerance of this tree species. Under conditions of soil-water deficit, plants exposed previously to acid rain are more sensitive to drought and desiccation.

Glutamine synthetase from mesophyll and bundle sheath maize cells: isoenzyme complements and different sensitivities to phosphinothricin.

Abstract Anion-exchange FPLC has been used to resolve the isoforms of glutamine synthetase (GS, EC 6.3.1.2) from Zea mays mesophyll (MC) and bundle sheath cells (BSC). Two different isoforms were detected in both types of photosynthetic cells. The predominantly active isoform was GS1 (61%) in MC and GS2 (67%) in BSC. The relative contribution of GS1 and GS2 to the overall GS activity in BSC in maize here reported resembles the proportion described for most C3 plants. Differences among these isoforms in terms of their susceptibility to phosphinothricin (PPT), an analogue of glutamate and known inhibitor of GS, were found. The GS1 isoenzyme from MC was the most sensitive form, being inhibited by 50% at approximately 2.0 μM DL-PPT, whereas the GS2 from BSC presented the highest tolerance to the inhibitor (I50=30 μM). The transferase-to-semibiosynthetic activity ratio for the MC isoforms, which was higher than the ratio for the BSC isoforms, and the differences shown by the isoforms in susceptibility to PPT predict important differences in the biochemical properties and regulation of GS isoenzymes. In this regard, the cytoplasmic isoenzymes, and especially the one in MC, due to its relatively high contribution to mesophyll cell GS activity, could play a vital role in nitrogen metabolism in maize.

Phosphinothricin Reverts the Ammonia-DependentEnhancement of Phosphoenolpyruvate Carboxylase Activity

Summary The effect of ammonium assimilation on phosphoenolpyruvate carboxylase (PEPCase) activity was investigatedin detached leaves from N-limited barley plants. The time-course induction of PEPCase was dependent on the rate of ammonia assimilation and not on ammonia uptake or accumulation. Treatment of N-deprived leaves with phosphinothricin (an inhibitor of glutamine synthetase activity) caused inhibition of ammonia assimilation, resulting in the reversion of ammonia-dependent enhancement of PEP-Case. The results indicate that glutamine level controls phosphoenolpyruvate carboxylase activation; consequently, if glutamine synthesis is inhibited, PEPCase activity is not enhanced. Determination of malate content showed that as PEPCase activity increased in response to increasingammonia assimilation, there was a linear decline in the level of that metabolite. We have also analyzed the in vivo effect of malate on ammonium-dependent activation of PEPCase.High malate uptake partially abolished PEPCase activation, but the induced PEPCase activity seems to be less sensitive to malate than the control one.