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The study aims to investigate at a physiological and molecular level the root absorption of nitrogen (N) during the annual cycle of grapevine. The study was performed on potted Pinot Gris plants grafted on Kober 5BB and grown for two consecutive years in a semi-controlled environment (Northeast, Italy). The study compared the response of plants N-fertilized in spring (T1), or in spring and in post-harvest (T2) or no-N fertilized (C). Results showed that under our climate conditions nitrate was taken up by the grapevines when applied both in spring or in spring and post-harvest. The nitrate acquisition in T1 roots is mediated at molecular and physiological level by a higher activation of high-affinity nitrate-transport system to take up nitrate in comparison to no-fertilized plants. Comparing the two N fertilization managements, the dynamic of nitrate uptake rates showed different patterns with an overall late response of High-Affinity Transport System when the application of part of N-fertilization was delayed to post-harvest (T2) in comparison to T1. Nevertheless, during the 2 years of the trial the fractionation of N fertilization applied in spring and post-harvest did not negatively affect the yield and quality parameters of vines. An increase of N concentration in T2 roots after two consecutive growing seasons may suggest that the fractionation can lead to beneficial effects on long period. Results of this work contribute to improve the comprehension of N acquisition in grapevine in order to optimize the use of N inputs in vineyard.

The wide use of copper (Cu)-based fungicide has caused a stepwise accumulation of Cu in the environment increasing the occurrence of phytotoxicity in crops. To understand and alleviate this abiotic stress, maize seedlings were grown in hydroponic solution with different combinations of Cu and iron (Fe) forms. Results showed that maize Cu sensitivity is related to the nature of the form supplied and to the chelate-exchange processes that might involve other elements, such as Fe. The use of CuSO4 excess (100 µM) caused severe reduction of plant growth, over accumulation of Cu, high activity of antioxidant enzymes, and impairment of the acquisition of other nutrients. In presence of chelating agents (citrate and ethylenediaminetetraacetic acid, EDTA) the ability of plants to tolerate high Cu-levels depends on the Fe nutritional status. Copper phytotoxicity symptoms do not occur when Cu was supplied chelated by EDTA. The use of synthetic agent EDTA (as Cu-EDTA and Fe-EDTA) prevented the accumulation of toxic Cu-level in plants and allowed a better homeostasis among nutrients. In presence of citrate, high concentration of Cu occurred in plants but its phytotoxicity was limited when even EDTA was available in solution. Results suggest that maize plants can operate a good control of nutritional status when Cu-excess is present concomitantly with a synthetic chelator (as EDTA) even when supplied as a Fe-fertilizer. These results pave the way to provide guidelines for the fertilization managements on Cu-contaminated soils to alleviate phytotoxicity in crops.

Despite the wide use of urea and ammonium as N-fertilizers, no information is available about the proper ratio useful to maximize the efficiency of their acquisition by crops. Ionomic analyses of maize seedlings fed with five different mixes of urea and ammonium indicated that after 7 days of treatment, the elemental composition of plant tissues was more influenced by ammonium in the nutrient solution than by urea. Within 24 h, similar high affinity influx rates of ammonium were measured in ammonium-treated seedlings, independently from the amount of the cation present in the nutrient solution (from 0.5 to 2.0 mM N), and it was confirmed by the similar accumulation of 15N derived from ammonium source. After 7 days, some changes in ammonium acquisition occurred among treatments, with the highest ammonium uptake efficiency when the urea-to-ammonium ratio was 3:1. Gene expression analyses of enzymes and transporters involved in N nutrition highlight a preferential induction of the cytosolic N-assimilatory pathway (via GS, ASNS) when both urea and ammonium were supplied in conjunction, this response might explain the higher N-acquisition efficiency when both sources are applied. In conclusion, this study provides new insights on plant responses to mixes of N sources that maximize the N-uptake efficiency by crops and thus could allow to adapt agronomic practices in order to limit the economic and environmental impact of N-fertilization.

The low bioavailability of nutrients, especially nitrogen (N) and phosphorus (P), is one of the most limiting factors for crop production. In this study, under N- and P-free nutrient solution (-N-P), nodulating white lupin plants developed some nodules and analogous cluster root structures characterized by different morphological, physiological, and molecular responses than those observed upon single nutrient deficiency (strong acidification of external media, a better nutritional status than -N+P and +N-P plants). The multi-elemental analysis highlighted that the concentrations of nutrients in white lupin plants were mainly affected by P availability. Gene-expression analyses provided evidence of interconnections between N and P nutritional pathways that are active to promote N and P balance in plants. The root exudome was mainly characterized by N availability in nutrient solution, and, in particular, the absence of N and P in the nutrient solution triggered a high release of phenolic compounds, nucleosides monophosphate and saponines by roots. These morphological, physiological, and molecular responses result from a close interplay between N and P nutritional pathways. They contribute to the good development of nodulating white lupin plants when grown on N- and P-free media. This study provides evidence that limited N and P availability in the nutrient solution can promote white lupin-Bradyrhizobium symbiosis, which is favourable for the sustainability of legume production.

Nitrogen (N) as well as Phosphorus (P) are key nutrients determining crop productivity. Legumes have developed strategies to overcome nutrient limitation by, for example, forming a symbiotic relationship with N-fixing rhizobia and the release of P-mobilizing exudates and are thus able to grow without supply of N or P fertilizers. The legume-rhizobial symbiosis starts with root release of isoflavonoids that act as signaling molecules perceived by compatible bacteria. Subsequently, bacteria release nod factors, which induce signaling cascades allowing the formation of functional N-fixing nodules. We report here the identification and functional characterization of a plasma membrane-localized MATE-type transporter (LaMATE2) involved in the release of genistein from white lupin roots. The LaMATE2 expression in the root is upregulated under N deficiency as well as low phosphate availability, two nutritional deficiencies that induce the release of this isoflavonoid. LaMATE2 silencing reduced genistein efflux and even more the formation of symbiotic nodules, supporting the crucial role of LaMATE2 in isoflavonoid release and nodulation. Furthermore, silencing of LaMATE2 limited the P-solubilization activity of lupin root exudates. Transport assays in yeast vesicles demonstrated that LaMATE2 acts as a proton-driven isoflavonoid transporter.

Nitrogen (N) as well as Phosphorus (P) are key nutrients determining crop productivity. Legumes have developed strategies to overcome nutrient limitation by, for example, forming a symbiotic relationship with N-fixing rhizobia and the release of P-mobilizing exudates and are thus able to grow without supply of N or P fertilizers. The legume-rhizobial symbiosis starts with root release of isoflavonoids that act as signaling molecules perceived by compatible bacteria. Subsequently, bacteria release nod factors, which induce signaling cascades allowing the formation of functional N-fixing nodules. We report here the identification and functional characterization of a plasma membrane-localized MATE-type transporter (LaMATE2) involved in the release of genistein from white lupin roots. The LaMATE2 expression in the root is upregulated under N deficiency as well as low phosphate availability, two nutritional deficiencies that induce the release of this isoflavonoid. LaMATE2 silencing reduced genistein efflux and even more the formation of symbiotic nodules, supporting the crucial role of LaMATE2 in isoflavonoid release and nodulation. Furthermore, silencing of LaMATE2 limited the P-solubilization activity of lupin root exudates. Transport assays in yeast vesicles demonstrated that LaMATE2 acts as a proton-driven isoflavonoid transporter.

The rate of nitrate (NO3 −) uptake and changes in rhizosphere properties were studied growing seedlings of two maize inbred lines differing in nitrogen use efficiency (NUE) in rhizoboxes. Changes in NO3 − uptake rates occurred in response to anion addition (induction) in seedlings grown both in hydroponic culture and in soil in rhizoboxes. The characterization of root exudate composition showed a line-specific metabolite profile, which was also affected by NO3 − availability. The induction affected respiration, nitrification, ammonification and enzyme activities of the rhizosphere. Furthermore, the composition of rhizosphere bacterial communities of the two maize lines differed suggesting the selective capacity of plants. Overall, results showed a strong and fast modification of rhizospheric soil properties in response to physiological changes in plants caused by fluctuating NO3 − availability.

Conversion of conventional farming (CF) to organic farming (OF) is claimed to allow a sustainable management of soil resources, but information on changes induced on dissolved organic matter (DOM) are scarce. Among DOM components, dissolved humic substances (DHS) were shown to possess stimulatory effects on plant growth. DHS were isolated from CF and OF soil leacheates collected from soil monolith columns: first in November (bare soils) and then in April and June (bare and planted soils). DHS caused an enhancement of nitrate uptake rates in maize roots and modulated several genes involved in nitrogen acquisition. The DHS from OF soil exerted a stronger biostimulant action on the nitrate uptake system, but the first assimilatory step of nitrate was mainly activated by DHS derived from CF soil. To validate the physiological response of plants to DHS exposure, real-time RT-PCR analyses were performed on those genes most involved in nitrate acquisition, such as ZmNRT2.1, ZmNRT2.2, ZmMHA2 (coding for two high-affinity nitrate transporters and a PM H+-proton pump), ZmNADH:NR, ZmNADPH:NR, and ZmNiR (coding for nitrate reductases and nitrite reductase). All tested DHS fractions induced the upregulation of nitrate reductase (NR), and in particular the OF2 DHS stimulated the expression of both tested transcripts encoding for two NR isoforms. Characteristics of DHS varied during the experiment in both OF and CF soils: a decrease of high molecular weight fractions in the OF soil, a general increase in the carboxylic groups content, as well as diverse structural modifications in OF vs. CF soils were observed. These changes were accelerated in planted soils. Similarity of chemical properties of DHS with the more easily obtainable water-soluble humic substance extracted from peat (WEHS) and the correspondence of their biostimulant actions confirm the validity of studies which employ WEHS as an easily available source of DHS to investigate biostimulant actions on agricultural crops.

Improvement of plant iron nutrition as a consequence of metal complexation by humic substances (HS) extracted from different sources has been widely reported. The presence of humified fractions of the organic matter in soil sediments and solutions would contribute, depending on the solubility and the molecular size of HS, to build up a reservoir of Fe available for plants which exude metal ligands and to provide Fe-HS complexes directly usable by plant Fe uptake mechanisms. It has also been shown that HS can promote the physiological mechanisms involved in Fe acquisition acting at the transcriptional and post-transcriptional level. Furthermore, the distribution and allocation of Fe within the plant could be modified when plants were supplied with water soluble Fe-HS complexes as compared with other natural or synthetic chelates. These effects are in line with previous observations showing that treatments with HS were able to induce changes in root morphology and modulate plant membrane activities related to nutrient acquisition, pathways of primary and secondary metabolism, hormonal and reactive oxygen balance. The multifaceted action of HS indicates that soluble Fe-HS complexes, either naturally present in the soil or exogenously supplied to the plants, can promote Fe acquisition in a complex way by providing a readily available iron form in the rhizosphere and by directly affecting plant physiology. Furthermore, the possibility to use Fe-HS of different sources, size and solubility may be considered as an environmental-friendly tool for Fe fertilization of crops.

Iron and phosphorus are abundant elements in soils but poorly available for plant nutrition. The availability of these two nutrients represents a major constraint for fruit tree cultivation such as apple (Malus × domestica) leading very often to a decrease of fruit productivity and quality worsening. Aim of this study was to characterize common and specific features of plant response to Fe and P deficiencies by ionomic, transcriptomic and exudation profiling of apple roots. Under P deficiency, the root release of oxalate and flavonoids increased. Genes encoding for transcription factors and transporters involved in the synthesis and release of root exudates were upregulated by P-deficient roots, as well as those directly related to P acquisition. In Fe-deficiency, plants showed an over-accumulation of P, Zn, Cu and Mn and induced the transcription of those genes involved in the mechanisms for the release of Fe-chelating compounds and Fe mobilization inside the plants. The intriguing modulation in roots of some transcription factors, might indicate that, in this condition, Fe homeostasis is regulated by a FIT-independent pathway. In the present work common and specific features of apple response to Fe and P deficiency has been reported. In particular, data indicate similar modulation of a. 230 genes, suggesting the occurrence of a crosstalk between the two nutritional responses involving the transcriptional regulation, shikimate pathway, and the root release of exudates.

Humic substances are known to positively influence plant growth and nutrition. In particular, the water-extractable fraction of humic substances (WEHS) has been shown to enhance nitrate acquisition, increasing the activity of high affinity nitrate uptake system. However, molecular bases of this physiological response are not clarified so far. Thus, in the present work, the physiological effect of WEHS on nitrate acquisition in maize roots was correlated with changes in the root transcriptomic profile. Results confirmed that WEHS caused a faster induction of a higher capacity to take up nitrate in maize roots. Comparing the root transcriptomic profile of Nitrate- and Nitrate + WEHS-treated plants with Control (-N) ones, more than 2000 transcripts appeared to be modulated only in the presence of WEHS. Among these, genes involved in nitrate transport and assimilation (NRT1s, NRT2s, NAR2.1, NR, GS, GOGAT, CNX, UPM) were strongly modulated by WEHS. Furthermore, also some genes known to be linked to the nitrogen limitation responses were affected by WEHS, as transcripts coding for transcription factors (as LBD37, NIN-like protein, NFY-A, GRF5) and enzymes of hormones’ metabolism. The modulation of these transcripts might play a crucial role in coordinating the induction to nitrate, favouring its uptake and assimilation in WEHS-treated plants. The overexpression of nitrogen assimilatory genes by WEHS might led to an early feedback regulation of the high affinity nitrate transport system, as being operated by N-metabolites. Results of the present work shed further light on the contribution of the organic soil component to the nitrogen use efficiency in crops.

In agricultural soil, the bioavailability of iron (Fe) and phosphorus (P) is often below the plant's requirement causing nutritional deficiency in crops. Under P-limiting conditions, white lupin (Lupinus albus L.) activates mechanisms that promote P solubility in the soil through morphological, physiological and molecular adaptations. Similar changes occur also in Fe-deficient white lupin roots; however, no information is available on the molecular bases of the response. In the present work, responses to Fe and P deficiency and their reciprocal interactions were studied. Transcriptomic analyses indicated that white lupin roots upregulated Fe-responsive genes ascribable to Strategy-I response, this behaviour was mainly evident in cluster roots. The upregulation of some components of Fe-acquisition mechanism occurred also in P-deficient cluster roots. Concerning P acquisition, some P-responsive genes (as phosphate transporters and transcription factors) were upregulated by P deficiency as well by Fe deficiency. These data indicate a strong cross-connection between the responses activated under Fe or P deficiency in white lupin. The activation of Fe- and P-acquisition mechanisms might play a crucial role to enhance the plant's capability to mobilize both nutrients in the rhizosphere, especially P from its associated metal cations.

To limit nitrogen (N) losses from the soil, it has been suggested to provide urea to crops in conjunction with the urease inhibitor N-(n-butyl) thiophosphoric triamide (NBPT). However, recent studies reported that NBPT affects urea uptake and urease activity in plants. To shed light on these latter aspects, the effects of NBPT were studied analysing transcriptomic and metabolic changes occurring in urea-fed maize seedlings after a short-term exposure to the inhibitor. We provide evidence that NBPT treatment led to a wide reprogramming of plant metabolism. NBPT inhibited the activity of endogenous urease limiting the release and assimilation of ureic-ammonium, with a simultaneous accumulation of urea in plant tissues. Furthermore, NBPT determined changes in the glutamine, glutamate, and asparagine contents. Microarray data indicate that NBPT affects ureic-N assimilation and primary metabolism, such as glycolysis, TCA cycle, and electron transport chain, while activates the phenylalanine/tyrosine-derivative pathway. Moreover, the expression of genes relating to the transport and complexation of divalent metals was strongly modulated by NBPT. Data here presented suggest that when NBPT is provided in conjunction with urea an imbalance between C and N compounds might occur in plant cells. Under this condition, root cells also seem to activate a response to maintain the homeostasis of some micronutrients.

Soil-less cultivation of horticultural crops represents a fairly recent innovation to traditional agriculture which has several advantages including higher water-use efficiency. When plants are grown with this system, their roots come in contact with nutrients solely via the hydroponic solution. Although its beneficial effects have been widely demonstrated, silicon (Si) is mostly omitted from the composition of nutrient solutions. Therefore, the objective of this study was to assess the beneficial effect of Si addition to hydroponic solution on quali-quantitative aspects of edible production of two cultivars of corn salad (Valerianella locusta (L.) Laterr.) grown in soil-less floating system. Impacts on shelf life of this food were also studied. Results show that the supply of Si increased the edible yield and the quality level reducing the nitrate concentration in edible tissues. This result might be attributed to changes either in the metabolism (such as the nitrate assimilation process) or to the functionality of root mechanisms involved in the nutrient acquisition from the outer medium. In fact, our results show for the first time the ability of Si to modulate the root activity of nitrate and Fe uptake through, at least in part, a regulation of gene expression levels of the proteins involved in this phenomenon. In addition, the presence of Si decreased the levels of polyphenoloxidase gene expression at harvest and, in post-harvest, slowed down the chlorophyll degradation delaying leaf senescence and thus prolonging the shelf life of these edible tissues. In conclusion, data showed that the addition of Si to the nutrient solution can be a useful tool for improving quali-quantitatively the yield of baby leaf vegetable corn salad as well as its shelf life. Since the amelioration due to the Si has been achieved only with one cultivar, the recommendation of its inclusion in the nutrient solution does not exclude the identification of cultivars suitable for this cultivation system and the comprehension of agronomical and environmental factors which could limit the Si benefits.

Despite the widespread use of urease inhibitors in agriculture, little information is available on their effect on nitrogen (N) uptake and assimilation. Aim of this work was to study, at physiological and transcriptional level, the effects of N-(n-butyl) thiophosphoric triamide (NBPT) on urea nutrition in hydroponically grown maize plants. Presence of NBPT in the nutrient solution limited the capacity of plants to utilize urea as a N-source; this was shown by a decrease in urea uptake rate and (15)N accumulation. Noteworthy, these negative effects were evident only when plants were fed with urea, as NBPT did not alter (15)N accumulation in nitrate-fed plants. NBPT also impaired the growth of Arabidopsis plants when urea was used as N-source, while having no effect on plants grown with nitrate or ammonium. This response was related, at least in part, to a direct effect of NBPT on the high affinity urea transport system. Impact of NBPT on urea uptake was further evaluated using lines of Arabidopsis overexpressing ZmDUR3 and dur3-knockout; results suggest that not only transport but also urea assimilation could be compromised by the inhibitor. This hypothesis was reinforced by an over-accumulation of urea and a decrease in ammonium concentration in NBPT-treated plants. Furthermore, transcriptional analyses showed that in maize roots NBPT treatment severely impaired the expression of genes involved in the cytosolic pathway of ureic-N assimilation and ammonium transport. NBPT also limited the expression of a gene coding for a transcription factor highly induced by urea and possibly playing a crucial role in the regulation of its acquisition. This work provides evidence that NBPT can heavily interfere with urea nutrition in maize plants, limiting influx as well as the following assimilation pathway.

While nitrate acquisition has been extensively studied, less information is available on transport systems of urea. Furthermore, the reciprocal influence of the two sources has not been clarified, so far. In this review, we will discuss recent developments on plant response to urea and nitrate nutrition. Experimental evidence suggests that, when urea and nitrate are available in the external solution, the induction of the uptake systems of each nitrogen (N) source is limited, while plant growth and N utilization is promoted. This physiological behavior might reflect cooperation among acquisition processes, where the activation of different N assimilatory pathways (cytosolic and plastidic pathways), allow a better control on the nutrient uptake. Based on physiological and molecular evidence, plants might increase (N) metabolism promoting a more efficient assimilation of taken-up nitrogen. The beneficial effect of urea and nitrate nutrition might contribute to develop new agronomical approaches to increase the (N) use efficiency in crops.

Chlorosis-susceptible fruit trees growing on calcareous soils have been observed to recover in the presence of grass cover species. However, the physiological mechanisms behind this phenomenon are only scarcely understood. An investigation was carried out to verify whether citrus plants can use 59Fe solubilized from a sparingly soluble source by the phytosiderophores (PS) released from graminaceous species. Experiments were performed in hydroponics, using two citrus rootstocks differing in their sensitivity to Fe-deficiency in the field (Poncirus trifoliata · Citrus paradisi, citrumelo ‘‘Swingle’’, highly susceptible, and Citrus aurantium L., moderately tolerant). Barley (Hordeum vulgare L., cv Europa) was used as a model species for PS-releasing graminaceous plants. Fe-deficient citrus plants increased 59Fe uptake from 59Fe-hydroxide supplied inside a dialysis tube, when Fe-deficient barley plants or PS-containing barley root exudates were present in the uptake solution, this effect being particularly evident for the susceptible rootstock. 59Fe-uptake from 59Fe-hydroxide was also enhanced in Fe-deficient citrumelo ‘‘Swingle’’ in the presence of Fe-deficient Poa pratensis L. and Festuca rubra L., two perennial grasses normally grown in association with fruit trees; no effect was found when Fe-sufficient grasses were employed. The uptake of 59Fe by the susceptible citrus rootstock increased in proportion to the amount of 2¢-deoxymugineic acid (DMA), the major PS released by Fe-deficient F. rubra, present in the uptake solution. The beneficial effect of F. rubra or P. pratensis was evident from the leaf re-greening observed when Fe-deficient citrumelo ‘‘Swingle’’ plants were grown in association with the grasses in pots filled with a calcareous soil. Leaf regreening was not observed when citrumelo ‘‘Swingle’’ plants and yellow stripe 3 (ys3) maize (Zea mays L.) mutant plants, unable to release PS, were co-cultivated in pots filled with calcareous soil, unless exogenous PS were added to the soil. Results indicate that graminaceous cover species can improve the Fe-nutrition of fruit trees grown on calcareous soils by enhancing Fe-availability.

Recent research has evidenced a relationship between Fe nutrition and S nutrition. Aim of the present work was to investigate the effect of Fe deficiency on the capacity of maize roots to take up and metabolize S. Maize (Zea mays L. cv. Cecilia) plants were grown for 10 d in nutrient solution (NS) with (+S) or without (−S) sulphate and Fe was added as FeIII-EDTA at 80 μm. After removing the extraplasmatic Fe pool, half of the plants of each treatment (+S and −S) were transferred to a new Fe-free NS. Roots were collected 4 and 24 h from the beginning of Fe deprivation. Fe deprivation slightly increased root thiols content in both nutritive conditions (+S and −S). ATP sulphurylase activity was enhanced by sulphur deprivation, but greatly depressed when Fe and S were both omitted from the nutrient solution. O-Acetylserine sulphydrylase activity was also enhanced by S deprivation; this activity was increased by Fe starvation in +S plants, while it was unaffected by Fe nutrition in −S plants. S depriv...

Even though urea and nitrate are the two major nitrogen (N) forms applied as fertilizers in agriculture and occur concomitantly in soils, the reciprocal influence of these two N sources on the mechanisms of their acquisition are poorly understood. Therefore, molecular and physiological aspects of urea and nitrate uptake were investigated in maize (Zea mays), a crop plant consuming high amounts of N. In roots, urea uptake was stimulated by the presence of urea in the external solution, indicating the presence of an inducible transport system. On the other hand, the presence of nitrate depressed the induction of urea uptake and, at the same time, the induction of nitrate uptake was depressed by the presence of urea. The expression of about 60,000 transcripts of maize in roots was monitored by microarray analyses and the transcriptional patterns of those genes involved in nitrogen acquisition were analyzed by real-time reverse transcription-PCR (RT-PCR). In comparison with the treatment without added N, the exposure of maize roots to urea modulated the expression of only very few genes, such as asparagine synthase. On the other hand, the concomitant presence of urea and nitrate enhanced the overexpression of genes involved in nitrate transport (NRT2) and assimilation (nitrate and nitrite reductase, glutamine synthetase 2), and a specific response of 41 transcripts was determined, including glutamine synthetase 1-5, glutamine oxoglutarate aminotransferase, shikimate kinase and arogenate dehydrogenase. Also based on the real-time RT-PCR analysis, the transcriptional modulation induced by both sources might determine an increase in N metabolism promoting a more efficient assimilation of the N that is taken up.

The capability of cucumber (Cucumis sativus L., cv. Serpente cinese), a Strategy I plant and barley (Hordeum vulgaris L., cv. Europa), a Strategy II plant to use Fe complexed by a water-soluble humic fraction (WEHS) extracted from a peat, was studied. Uptake of 59Fe from 59Fe-WEHS by cucumber plants was higher at pH 6.0 than at pH 7.5. Roots of intact cucumber plants were able to reduce the FeIII-WEHS complex either at pH 6.0 or 7.5, rates being higher in the assay medium buffered at pH 6.0. After supply of 59Fe-WEHS, a large pool of root extraplasmatic 59Fe was formed, which could be used to a large extent by Fe-deficient plants, particularly under acidic conditions. Uptake of 59Fe from 59Fe-WEHS by Fe-sufficient and Fe-deficient barley plants was examined during periods of high (morning) and low (evening) PS release. Uptake paralleled the diurnal rhythm of PS release. Furthermore, 59Fe uptake was strongly enhanced by addition of PS to the uptake solution in both Fe-sufficient and Fe-deficient plants. High amount of root extraplasmatic 59Fe was formed upon supply of Fe-WEHS, particularly in the evening experiment. Fe-deficient barley plants were able to utilize Fe from the root extraplasmatic pool, conceivably as a result of high rates of PS release. The results of the present work together with previous observations indicate that cucumber plants (Strategy I) utilize Fe complexed to WEHS, presumably via reduction of FeIII-WEHS by the plasma membrane-bound reductase, while barley plants (Strategy II) use an indirect mechanism involving ligand exchange between WEHS and PS.

Development of the coordinated response to decreasing Fe availability was studied in cucumber plants grown in nutrient solution (NS) over a range of FeIII-EDTA concentrations (from 0.1 to 80 μM). Physiological and biochemical parameters were evaluated in intact roots, root extracts and plasma membrane (pm) vesicles. Acidification of the NS was evident in plants grown at ≤ 1 μM FeIII-EDTA and inversely related to the external Fe concentration. FeIII-EDTA reduction by intact roots was also gradually depressed by increasing Fe supply. The rate of net nitrate uptake by the roots was directly related to the amount of FeIII-EDTA added to the NS. Activity of pmH+-ATPase was significantly higher in plants grown without added Fe as compared to those grown at 80 μM Fe. A lower increase, dependent on Fe concentration, was observed at 0.1, 1, 5 or 10 μM FeIII-EDTA. Activity of pmFeIII-EDTA reductase was also increased by Fe deprivation and strongly correlated with pmH+-ATPase activity. PEP-carboxylase activity gradually increased with decreasing Fe concentration in the NS. Changes in activity and amount of the enzyme showed a close correlation with parameters measured in intact roots (nitrate uptake, FeIII-EDTA reduction). Results show that the development of the Fe-deficiency response in cucumber roots can be finely tuned by the level of Fe supply. Adjustments to different levels of available Fe involve a correlated modulation of pm-associated enzymes. PEP-carboxylase activity appeared to be a suitable metabolic marker of the Fe nutritional status of the plant.

The effect of a water extractable humic substances fraction (WEHS) on nitrate uptake and plasma membrane (pm) H+-ATPase activity of maize roots was investigated. Four days old maize root seedlings were exposed for 4 to 24 h to a nutrient solution containing 200 μ M nitrate in the absence or presence of 5 mg org. C { L -1 WEHS. Plants exposed to nitrate developed a higher capacity to absorb the anion (induction): the net uptake rate progressively increased up to 12 h of contact with the solution; thereafter, a decline was observed. When WEHS was present together with nitrate in the nutrient solution, the induction of nitrate uptake was evident and maximal already 4 h after starting the treatment. The rate of net nitrate uptake decreased only slightly during the remaining period (4-24 h). Stimulation of net nitrate uptake rate was also observed when WEHS was added to a nitrogen- or nitrate-free nutrient solution or to a 5 mM CaSO4 solution. The activity of pmH+-ATPase raised upon exposure of the roots to nitrate with the same pattern observed for nitrate uptake. The contemporary presence of nitrate and WEHS caused a further stimulation of the pmH+-ATPase activity after 4 h treatment. An increase in the enzyme activity was also observed when plants were treated for 4 h in the presence of WEHS in CaSO4, nitrogen- or nitrate-free solutions. However, when nitrate was present the enhancement was even greater. Results support the idea that the plasma membrane proton pump might be one of the primary targets of the action of humic substances on plant nutrient acquisition. A role of WEHS in the modulation of nitrate uptake via an interaction with the pm H+-ATPase is also discussed.

The ability of Fe-deficient cucumber plants to use iron complexed to a water-extractable humic substances fraction (WEHS), was investigated. Seven-day-old Fe-deficient plants were transferred to a nutrient solution supplemented daily for 5 days with 0.2 μM Fe as Fe-WEHS (5 μg org. C mL-1), Fe-EDTA, Fe-citrate or FeCl3. These treatments all allowed re-greening of the leaf tissue, and partial recovery of dry matter accumulation, chlorophyll and iron contents. However, the recovery was faster in plants supplied with Fe-WEHS and was already evident 48 h after Fe supply. The addition of 0.2 μM Fe to the nutrient solution caused also a partial recovery of the dry matter and iron accumulation in roots of Fe-deficient cucumber plants, particularly in those supplied with Fe-WEHS. The addition of WEHS alone (5 μg org. C mL-1, 0.04 μM Fe) to the nutrient solution slightly but significantly increased iron and chlorophyll contents in leaves of Fe-deficient plants; in these plants, dry matter accumulation in leaves and roots was comparable or even higher than that measured in plants treated with Fe-citrate or FeCl3. After addition of the different iron sources for 5 days to Fe-deficient roots, morphological modifications (proliferation of lateral roots, increase in the diameter of the sub-apical zones and amplified root-hair formation) and physiological responses (enhanced Fe(III)-chelate reductase and acidification of the nutrient solution) induced by Fe deficiency, were still evident, particularly in plants treated with the humic molecules. The presence of WEHS caused also a further acidification of the nutrient medium by Fe-deficient plants. The Fe-WEHS complex (1 μM Fe) could be reduced by intact cucumber roots, at rates of reduction higher than those measured for Fe-EDTA at equimolar iron concentration. Plasma membrane vesicles, purified by two-phase partition from root microsomes of Fe-deficient plants, were also able to reduce Fe-WEHS. Results show that Fe-deficient cucumber plants can use iron complexed to water soluble humic substances, at least in part via reduction of complexed Fe(III) by the plasma membrane Fe(III)-chelate reductase of root cells. In addition, the stimulating effect of humic substances on H+ release might be of relevance for the overall response of the plants to iron shortage.

This study addresses the question of the interference between iron (Fe) nutrition and cadmium (Cd) toxicity at the level of growth performance, phytosiderophores (PS) release, micronutrient accumulation and expression of genes involved in Fe homeostasis in barley seedlings, a plant with strategy II-based response to Fe shortage. Cd exposure induced responses similar to those of genuine Fe deficiency also in Fe-sufficient plants. Most genes involved in PS biosynthesis and secretion (HvNAS3, HvNAS4, HvNAS6, HvNAS7, HvNAAT-A, HvDMAS1 and HvTOM1) induced by Fe deprivation were also significantly upregulated in the presence of Cd under Fe sufficient conditions. Accordingly, the enhanced expression of these genes in roots under Cd exposure was accompanied by an increase of PS release. However, induced expression of HvIRO2 and the downregulation of HvIDEF1 and HvIRT1, after Cd exposure, suggested the presence of a pathway that induces HvIRO2-mediated PS biosynthesis under Cd stress, which probably is not simply caused by Fe deficiency. The downregulation of HvIRT1 and HvNramp5 may represent a protective mechanism at transcriptional level against further Cd uptake by these transporters. These results likely indicate that Cd itself may be able to activate Fe acquisition mechanism in an Fe-independent manner.

Background Despite its extensive use as a nitrogen fertilizer, the role of urea as a directly accessible nitrogen source for crop plants is still poorly understood. So far, the physiological and molecular aspects of urea acquisition have been investigated only in few plant species highlighting the importance of a high-affinity transport system. With respect to maize, a worldwide-cultivated crop requiring high amounts of nitrogen fertilizer, the mechanisms involved in the transport of urea have not yet been identified. The aim of the present work was to characterize the high-affinity urea transport system in maize roots and to identify the high affinity urea transporter. Results Kinetic characterization of urea uptake (

Uptake of 14C-sugars and activities of sucrose metabolizing enzymes were determined in order to study the mechanism(s) of sucrose accumulation in developing peach fruit. Mesocarp of young peach fruit contained glucose and fructose but little sucrose. Starting 88 days after anthesis (DAA) the sucrose concentration increased greatly. The mechanism of sucrose accumulation was studied by measuring 14C-sucrose and 14C-glucose uptake rates at three different stages of fruit development, and by assaying weekly the activity of enzymes involved in the hydrolysis and/or synthesis of the soluble sugars. Uptake of 0.5–100 mM14C-sucrose and 14C-glucose by mesocarp tissue slices showed a complex pattern at the first stage of fruit development (62 DAA). During the subsequent growth stages the pattern of sugar uptake changed and was approximately monophasic at the third stage of fruit development. At 10 mM, glucose was taken up more rapidly than sucrose at the first and second stage of fruit development. Uptake was partially inhibited by the uncoupler carbonylcyanide m-chlorophenylhydrazone (CCCP) at 25 μM. These results, together with the presence of a putative extracellular invertase, suggest an apoplastic route for sucrose uptake which is dependent, at least in part, on energy supply. Activities of sucrose hydrolyzing enzymes (insoluble acid invertase, soluble acid invertase, neutral invertase, sucrose synthase) were high in young fruits and declined sharply with fruit development concomitantly with accumulation of sucrose. The storage of the sugar was not accompanied by a rise in synthetic activities (sucrose synthase, sucrose phosphate synthase), suggesting that sucrose could, at least in part enter the carbohydrate pool directly.

Summary The effect of nitrate, chloride and 4, 4' -diisothiocyano-2, 2'-disulfonic stilbene acid (DIDS) on H + -ATPase of plasma membrane vesicles isolated from corn roots was investigated in order to acquire information on mechanism(s) of anion transport. The initial rate of ATP-dependent intravesicular H + accumulation in inside-out plasma membrane vesicles was stimulated both in the presence of NO 3 − and Cl − . However, the kinetics of stimulation were different, NO 3 − showing a clear saturable profile and Cla complex apparently biphasic pattern with no saturation even at concentrations as high as 200 mM. The stimulation was not related to a direct effect of the anions on H + -ATPase as indicated by the lack of effect on the phosphohydrolyzing activity measured in the presence of Brij 58 or Gramicidin D. DIDS at 1-2 μM strongly inhibited the anion-stimulated H + accumulation without a simultaneous effect on passive membrane permeability or on phosphate hydrolysis, which was almost unaffected even at a DIDS concentration of 20 μM. The results suggest that transport of NO 3 − and Cl − , in a direction that would correspond in vivo to the efflux from the cell, is mediated by proteinaceous structure(s) possessing different kinetic properties with respect to these anions.

The relationship between the plasma membrane-bound NAD(P)H-nitrate reductase (NR) and a plasma membrane (PM)-bound peroxidase was investigated using highly purified PM vesicles isolated from corn roots. The PM-bound NR activity was strongly enhanced by MnCl 2 and SHAM, which stimulated peroxidase activity. Since both activities, the NAD(P)H-dependent NR and the peroxidase compete for NAD(P)H as electron donor, we propose a model in which a product of peroxidation is able to offer electrons to the nitrate reductase in a more reactive form with respect to NAD(P)H. Out hypothesis was confirmed by experiments in which the effects of inhibitors of peroxidative reactions, catalase, superoxide dismutase, and ascorbate on the PM-bound NR were studied. Results indicate that the putative electron donor for nitrate reduction could be a radicalic species, possibly NAD . . Furthermore, since cytochrome c decreased the activity of the plasma membrane-bound NAD(P)H-dependent NR, cytochrome b 557 might be the site of the enzyme accepting electrons from NAD . . Out results indicate that the PM environment of the NR may be involved in the extent of the membrane-associated nitrate reduction and that redox enzymes at the PM, the NAD(P)H-NR and a peroxidase-like NADH-oxidase, can interact.

Cadmium (Cd) detoxification involves glutathione and phytochelatins biosynthesis: the higher need of nitrogen should require increased nitrate (NO(3)(-)) uptake and metabolism. We investigated inducible high-affinity NO(3)(-) uptake across the plasma membrane (PM) in maize seedlings roots upon short exposure (10 min to 24 h) to low Cd concentrations (0, 1 or 10 μM): the activity and gene transcript abundance of high-affinity NO(3)(-) transporters, NO(3)(-) reductases and PM H(+)-ATPases were analyzed. Exposure to 1 mM NO(3)(-) led to a peak in high-affinity (0.2 mM) NO(3)(-) uptake rate (induction), which was markedly lowered in Cd-treated roots. Plasma membrane H(+)-ATPase activity was also strongly limited, while internal NO(3)(-) accumulation and NO(3)(-) reductase activity in extracts of Cd treated roots were only slightly lowered. Kinetics of high- and low-affinity NO(3)(-) uptake showed that Cd rapidly (10 min) blocked the inducible high-affinity transport system; the constitutive high-affinity transport system appeared not vulnerable to Cd and the low-affinity transport system appeared to be less affected and only after a prolonged exposure (12 h). Cd-treatment also modified transcript levels of genes encoding high-affinity NO(3)(-) transporters (ZmNTR2.1, ZmNRT2.2), PM H(+)-ATPases (ZmMHA3, ZmMHA4) and NO(3)(-) reductases (ZmNR1, ZmNADH:NR). Despite an expectable increase in NO(3)(-) demand, a negative effect of Cd on NO(3)(-) nutrition is reported. Cd effect results in alterations at the physiological and transcriptional levels of NO(3)(-) uptake from the external solution and it is particularly severe on the inducible high-affinity anion transport system. Furthermore, Cd would limit the capacity of the plant to respond to changes in NO(3) (-) availability.

Background The mechanisms by which nitrate is transported into the roots have been characterized both at physiological and molecular levels. It has been demonstrated that nitrate is taken up in an energy-dependent way by a four-component uptake machinery involving high- and low- affinity transport systems. In contrast very little is known about the physiology of nitrate transport towards different plant tissues and in particular at the leaf level. Results The mechanism of nitrate uptake in leaves of cucumber (Cucumis sativus L. cv. Chinese long) plants was studied and compared with that of the root. Net nitrate uptake by roots of nitrate-depleted cucumber plants proved to be substrate-inducible and biphasic showing a saturable kinetics with a clear linear non saturable component at an anion concentration higher than 2 mM. Nitrate uptake by leaf discs of cucumber plants showed some similarities with that operating in the roots (e.g. electrogenic H+ dependence via involvement of proton pump, a certain degree of induction). However, it did not exhibit typical biphasic kinetics and was characterized by a higher K m with values out of the range usually recorded in roots of several different plant species. The quantity and activity of plasma membrane (PM) H+-ATPase of the vesicles isolated from leaf tissues of nitrate-treated plants for 12 h (peak of nitrate foliar uptake rate) increased with respect to that observed in the vesicles isolated from N-deprived control plants, thus suggesting an involvement of this enzyme in the leaf nitrate uptake process similar to that described in roots. Molecular analyses suggest the involvement of a specific isoform of PM H+-ATPase (CsHA1) and NRT2 transporter (CsNRT2) in root nitrate uptake. At the leaf level, nitrate treatment modulated the expression of CsHA2, highlighting a main putative role of this isogene in the process. Conclusions Obtained results provide for the first time evidence that a saturable and substrate-inducible nitrate uptake mechanism operates in cucumber leaves. Its activity appears to be related to that of PM H+-ATPase activity and in particular to the induction of CsHA2 isoform. However the question about the molecular entity responsible for the transport of nitrate into leaf cells therefore still remains unresolved.

Both Fe deficiency and Cd exposure induce rapid changes in the S nutritional requirement of plants. The aim of this work was to characterize the strategies adopted by plants to cope with both Fe deficiency (release of phytosiderophores) and Cd contamination [production of glutathione (GSH) and phytochelatins] when grown under conditions of limited S supply. Experiments were performed in hydroponics, using barley plants grown under S sufficiency (1.2 mM sulphate) and S deficiency (0 mM sulphate), with or without Fe(III)-EDTA at 0.08 mM for 11 d and subsequently exposed to 0.05 mM Cd for 24 h or 72 h. In S-sufficient plants, Fe deficiency enhanced both root and shoot Cd concentrations and increased GSH and phytochelatin levels. In S-deficient plants, Fe starvation caused a slight increase in Cd concentration, but this change was accompanied neither by an increase in GSH nor by an accumulation of phytochelatins. Release of phytosiderophores, only detectable in Fe-deficient plants, was strongly decreased by S deficiency and further reduced after Cd treatment. In roots Cd exposure increased the expression of the high affinity sulphate transporter gene (HvST1) regardless of the S supply, and the expression of the Fe deficiency-responsive genes, HvYS1 and HvIDS2, irrespective of Fe supply. In conclusion, adequate S availability is necessary to cope with Fe deficiency and Cd toxicity in barley plants. Moreover, it appears that in Fe-deficient plants grown in the presence of Cd with limited S supply, sulphur may be preferentially employed in the pathway for biosynthesis of phytosiderophores, rather than for phytochelatin production.

Main components of Strategy II mechanism for Fe uptake are secretion of chelating compounds, phytosiderophores, and specific uptake of Fe(III)-phytosiderophores complex. Since the amount of phytosiderophores secreted correlates positively with plant ability to cope with Fe shortage, a role of root hairs in enhancing root capability to store phytosiderophores under Fe stress might be envisaged. In this study the root-hairless mutant of barley (Hordeum vulgare L.) brb (bald root barley) and the wild-type genotype (cv. Pallas) were compared with respect to their capacity to respond to Fe shortage in nutrient solution. Plants were grown with FeIII-EDTA at 0, 0.02 and 0.08 mM, in order to reproduce severe or moderate Fe deficiency, and adequate Fe nutritional status, respectively. Analysis was performed after 11 and 14 days considering leaf Fe content, phytosiderophores release and accumulation in root tips, and 59Fe uptake. Biomass accumulation and chlorophyll content were not reduced in mutant plants as compared to wild-type ones; leaf Fe content was similar in both genotypes after 14 days of growth. Accumulation and release of phytosiderophores showed a similar trend in both genotypes when subjected to Fe limitation. Furthermore, no significant difference between the two genotypes was observed when 59Fe uptake was measured. Results seem to support the idea that the presence of root hairs and their increased production in response to low-Fe availability, while causing major modifications of root geometry, did not necessarily lead neither to an effect on growth nor on Fe uptake and accumulation in barley plants.

Plasma membrane-enriched vesicles were isolated by density gradient centrifugation from roots of zinc-sufficient and zinc-deficient bean (Phaseolus vulgaris L. cv. Prélude) plants. The two populations of vesicles had similar activities of specific membrane marker enzymes and ATP hydrolysis and were competent for proton transport. However, vesicles from zinc-deficient roots showed lower rates of ATP-dependent intravesicular acidification and increases in passive permeability to protons as well as in the rate, of dissipation of a non-metabolic transmembrane pH gradient. The decrease of the rate of proton accumulation in isolated vesicles closely paralleled the increase in potassium leakage from intact roots and the appearance of visual zinc deficiency symptoms in the shoots. Re-supply of zinc to deficient plants for 24 h promoted shoot growth, reduced potassium leakage from roots and led to partial recovery of the proton accumulation capacity and to a decrease in passive permeability to protons in isolated vesicles. The results obtained with isolated vesicles confirm the previously observed 'in vivo' effects of zinc deficiency and are consistent with the idea that an alteration of plasma membrane lipids leads to an increase in permeability and an impairment in trans-plasma membrane proton gradient. © 1993 Oxford University Press. National Council for Scientific Research ACKNOWLEDGEMENTS R. Pinton was a postdoctoral fellow of the Alexander von Humboldt-Stiftung, at the Institut fur Pflanzener- nahrung Universitat Hohenheim. Part of this research was supported by National Research Council of Italy, Special Project RAISA, Sub-project No. 2 paper No. 752. Authors thank Dr H. Frits Bienfait, Utrecht, and Dr M. Ida De Michelis, University of Messina, for helpful comments.

The effect of the S nutritional status on a plant’s capability to cope with Fe shortage was studied in solution cultivation experiments in barley (Hordeum vulgare L. cv. Europa). Barley is a Strategy II plant and responds to Fe deficiency by secretion of chelating compounds, phytosiderophores (PS). All PS are derived from nicotianamine whose precursor is methionine. This suggests that a long-term supply of an inadequate amount of S could reduce a plant’s capability to respond to Fe deficiency by limiting the rate of PS biosynthesis. The responses of barley (Hordeum vulgare L. cv. Europa) plants grown for 12 d on Fe-free nutrient solutions (NS) containing 0 or 1.2 mM SO22 4 , was examined after 24 h or 48 h from transfer to NS containing 1.2 mM SO22 4 . After the supply of S was restored to S-deprived plants, an increase in PS release in root exudates was evident after 24 h of growth in S-sufficient NS and the increment reached values up to 4-fold higher than the control 48 h after S resupply. When S was supplied to S-deficient plants, leaf ATPS (EC 2.7.7.4) and OASTL (EC 4.2.99.8) activities exhibited a progressive recovery. Furthermore, root HvST1 transcript abundance remained high for 48 h following S resupply and a significant increase in the level of root HvYS1 transcripts was also found after only 24 h of S resupply. Data support the idea that the extent to which the plant is able to cope with Fe starvation is strongly associated with its S nutritional status. In particular, our results are indicative that barley plants fully recover their capability to cope with Fe shortage after the supply of S is restored to S-deficient plants. L'articolo é disponibile sul sito dell'editore: http://www.jxb.oxfordjournals.org Research was supported by grants from Italian MIUR-PRIN 2006 and by funding from the Max-Planck-Society.

The effect of a low molecular size (

White lupin (Lupinus albus L.) is able to grow on soils with sparingly available phosphate (P) by producing specialized structures called cluster roots. To mobilize sparingly soluble P forms in soils, cluster roots release substantial amounts of carboxylates and concomitantly acidify the rhizosphere. The relationship between acidification and carboxylate exudation is still largely unknown. In the present work, we studied the linkage between organic acids (malate and citrate) and proton exudations in cluster roots of P-deficient white lupin. After the illumination started, citrate exudation increased transiently and reached a maximum after 5 h. This effect was accompanied by a strong acidification of the external medium and alkalinization of the cytosol, as evidenced by in vivo nuclear magnetic resonance (NMR) analysis. Fusicoccin, an activator of the plasma membrane (PM) H+-ATPase, stimulated citrate exudation, whereas vanadate, an inhibitor of the H+-ATPase, reduced citrate exudation. The burst of citrate exudation was associated with an increase in expression of the LHA1 PM H+-ATPase gene, an increased amount of H+-ATPase protein, a shift in pH optimum of the enzyme and post-translational modification of an H+-ATPase protein involving binding of activating 14-3-3 protein. Taken together, our results indicate a close link in cluster roots of P-deficient white lupin between the burst of citrate exudation and PM H+-ATPase-catalysed proton efflux.

Aim of this work was to clarify the role of S supply in the development of the response to Fe depletion in Strategy I plants. In S-sufficient plants, Fe-deficiency caused an increase in the Fe(III)-chelate reductase activity, 59Fe uptake rate and ethylene production at root level. This response was associated with increased expression of LeFRO1 (Fe(III)-chelate reductase) and LeIRT1 (Fe2+ transporter) genes. Instead, when S-deficient plants were transferred to a Fe-free solution, no induction of Fe(III)-chelate reductase activity and ethylene production was observed. The same held true for LeFRO1 gene expression, while the increase in 59Fe2+ uptake rate and LeIRT1 gene over-expression were limited. Sulphur deficiency caused a decrease in total sulphur and thiol content; a concomitant increase in 35SO42- uptake rate was observed, this behaviour being particularly evident in Fe-deficient plants. Sulphur deficiency also virtually abolished expression of the nicotianamine synthase gene (LeNAS), independently of the Fe growth conditions. Sulphur deficiency alone also caused a decrease in Fe content of tomato leaves and an increase in root ethylene production; however these events were not associated with either increased Fe(III)-chelate reductase activity, higher rates of 59Fe uptake, or over-expression of either LeFRO1 or LeIRT1 genes. Results show that S-deficiency could limit the capacity of tomato plants to cope with Fe-shortage by preventing the induction of the Fe(III)-chelate reductase and limiting the activity and expression of the Fe2+ transporter. Furthermore, the results support the idea that ethylene alone cannot trigger specific Fe-deficiency physiological responses in a Strategy I plant, such as tomato. L'articolo é disponibile sul sito dell'editore: http://www.springerlink.com Research was supported by grants from Italian M.I.U.R.-COFIN 2006.

It is well known that in the rhizosphere soluble Fe sources available for plants are mainly a mixture of complexes between the micronutrient and organic ligands such as organic acids and phytosiderophores (PS) released by roots, microbial siderophores as well as fractions of humified organic matter. In the present work, mechanisms of Fe acquisition operating at the leaf level of plants fed with different Fe-complexes were investigated at the micro-analytical, physiological and molecular levels. Fe-deficient tomato plants (Solanum Lycopersicum L., cv. ‘Marmande’) were fed for 24 h with a solution (pH 7.5) containing 1 µM Fe as Fe-PS, Fe-citrate or Fe-WEHS. Thereafter, leaf tissue was used for the visualization of Fe distribution, measurements of Fe content, reduction and uptake, and evaluation of expression of Fe-chelate reductase (LeFRO1), Fe-transporter (LeIRT1) and Ferritin (Ferritin2) genes. Leaf discs isolated from Fe-deficient plants treated for 24 h with Fe-WEHS developed higher rates of translocation, Fe-chelate reduction and 59Fe uptake as compared to plants supplied with Fe-citrate or Fe-PS. Leaves of plants treated with Fe-WEHS also showed higher transcript levels of LeFRO1, LeIRT1 and Ferritin2 genes with respect to plants fed with the other Fe-sources. Data obtained support the idea that the efficient use of Fe complexed to WEHS-like humic fractions involves, at least in part, also the activation of Fe-acquisition mechanisms operating at the leaf level.

Iron chlorosis is a wide-spread limiting factor of production in agriculture. To cope with this problem, synthetic chelates (like EDTA or EDDHA) of Fe are used in foliar-spray or in soil treatments; however, these products are very expensive. Therefore paper-production byproducts, like Lignosulfonates (LS), with varying content of carboxylate and sulfonate groups, were tested with respect to their ability to maintain Fe in the solution of soils and to feed plants grown in hydroponics with Fe through foliar sprays or application to the nutrient solution. Results show that LS had a low capability to solubilize 59Fe-hydroxide and that preformed 59Fe(III)-LS complexes had poor mobility through a soil column (pH 7.5) and scarce stability when interacting with soils compared to 59Fe(III)-EDDHA. However when 59Fe(III)-LS were supplied to roots in a hydroponic system, they demonstrated an even higher capability to fed Fe-deficient tomato plants than 59Fe(III)-EDDHA. Hence, data here presented indicate that the low Fe use efficiency from Fe-LS observed in soil-applications is due to interactions of these Fe-sources with soil colloids rather than to the low capability of roots to use them. Foliar application experiments of 59Fe(III)-LS or 59Fe(III)-EDTA to Fe-deficient cucumber plants show that uptake and reduction rates of Fe were similar between all these complexes; on the other hand, when 59Fe(III)-LS were sprayed on Fe-deficient tomato leaves, they showed a lower uptake rate, but a similar reduction rate, than 59Fe(III)-EDTA did. In conclusion, Fe-LS may be a valid, eco-compatible and cheap alternative to synthetic chelates in dealing with Fe chlorosis when applied foliarly or in the nutrient solution of hydroponically grown plants.

Uptake rates of (86Rb)K+ by seedling roots of six cultivars were measured and compared with K+ content of the root, K+ leakage, H+ efflux, and K+-ATPase activity of a partially purified plasmalemma fraction.

The effect of a low-molecular weight, water-extractable fraction of humic substances (WEHS) derived from sphagnum peat on post-embryonic plant development has been studied using Arabidopsis roots. Application of humic substances caused an array of changes in root morphology, such as an increase in root hair length and density, formation of ectopic root hairs, and an increase in cell proliferation in the root ground tissue. Application of WEHS affected genes involved in epidermal cell fate specification, suggesting that humic substances can alter developmental programs at an early stage of root cell differentiation. The WEREWOLF and GLABRA2 genes, encoding negative regulators of the root hair cell fate, were significantly down-regulated in the presence of WEHS. Thus, the presence of humic substances caused an ordered remodeling of the root morphology, leading to an increased absorptive surface of the root. Growth in the presence of WEHS did not rescue the phenotype of the root hair defective rhd6 mutant. Analyzing BA3:uidA and DR5:uidA transgenic plants, carrying auxin response elements, and monitoring the expression of the auxin-responsive GH3 gene by real-time RT-PCR did not provide evidence for a WEHS-induced expression of auxin-related genes. It is concluded that WEHS do not exert their effects in an auxin-like manner.

Cucumber (Cucumis sativus L.) plants were precultured for 7 days in either optimal (10 µm) or low (0.5 µm) Fe conditions and then grown for further 5 days in a N-free nutrient solution with (+Fe) or without (–Fe) 10 µm Fe. Thereafter NO3– (4 mm) was added to the nutrient solution for 24 h and, concomitantly, half of the –Fe plants were treated with 1 µm Fe complexed to water extractable humic substances (WEHS). Supply of NO3– to +Fe–N-deprived plants caused a large induction in the capacity to take up the anion by roots, which was accompanied by a rise in root–shoot NO3– concentration. The –Fe plants showed a lower level of induction of NO3– uptake and hence a lower accumulation of the anion in the tissues, these effects being reversed by supply of Fe-WEHS. Supply of either NO3–- or NH4+-N (+/– Fe-WEHS) to –Fe plants promoted the development of the root FeIII-chelate reductase activity, but the capacity of roots to take up the Fe2+ remained unaffected. Results show that an inadequate Fe supply can limit the acquisition of NO3–, whereas NO3– supply can affect Fe uptake by influencing the development and maintenance of a high FeIII-chelate reducing capacity.

We studied the possibility that the sulfur (S) assimilatory pathway might be modulated by iron (Fe) starvation in barley, as a consequence of plant requirement for an adequate amount of reduced S to maintain methionine and, in turn, phytosiderophore biosynthesis. Barley seedlings were grown with or without 100 µm FeIII–EDTA, at three S levels in the nutrient solution (S2 = 1200, S1 = 60, and S0 = 0 µm sulfate) in order to reproduce conditions of optimal supply, latent and severe deficiency, respectively. Fe deprivation increased root cysteine content irrespective of the S supply. However, this increase was not associated with either higher rates of 35SO42– uptake or increased expression of the gene for the high-affinity sulfate transporter, HvST1, and these roots failed to increase their activities of ATP sulfurylase (ATPS) and O-acetylserine(thiol) lyase (OASTL). We observed a significant increase in 35SO42– uptake rate (+76%) only in Fe-deficient S1 plants and we found an increase in root ATPS activity only in S0 plants. We observed an increase of ATPS enzyme activity in leaves of S1 and S2 plants, most likely suggesting increased S assimilation followed by translocation of thiols (Cys) to the root. Taken together, our results suggest that Fe deficiency affects the partitioning from the shoot to the root of the reduced S pool within the plant and can affect SO42– uptake under limited S supply.

Aim of the present work was to investigate the involvement of plasma membrane (PM) H(+)-ATPase (E.C. 3.6.3.6) isoforms of cucumber (Cucumis sativus L.) in the response to Fe deficiency. Two PM H(+)-ATPase cDNAs (CsHA1 and CsHA2) were isolated from cucumber and their expression analysed as a function of Fe nutritional status. Semi-quantitative reverse transcriptase (RT)-PCR and quantitative real-time RT-PCR revealed in Fe-deficient roots an enhanced accumulation of CsHA1 gene transcripts, which were hardly detectable in leaves. Supply of iron to deficient plants caused a decrease in the transcript level of CsHA1. In contrast, CsHA2 transcripts, detected both in roots and leaves, appeared to be unaffected by Fe. This work shows for the first time that a transcriptional regulation of PM H(+)-ATPase involving a specific isoform occurs in the response to Fe deficiency.

An investigation was carried out to assess the effect of nitrate supply on the root plasma membrane (PM) H + -ATPase of etiolated maize (Zea mays L.) seedlings grown in hydroponics. The treatment induced higher uptake rates of the anion and the expression of a putative high-affinity nitrate transporter gene (ZmNRT2.1), the first to be identified in maize. Root PM H + -ATPase activity displayed a similar time-course pattern as that of net nitrate uptake and investigations were carried out to determine which of the two isoforms reported to date in maize, MHA1 and 2, responded to the treatment. MHA1 was not expressed under the conditions analysed. Genome analysis revealed that MHA2, described as the most abundant form in all maize tissues, was not present in the maize hybrid investigated, but a similar form was found instead and named MHA3. A second gene (named MHA4) was also identified and partially sequenced. Both genes, classified as members of the PM H + -ATPase subfamily II, responded to nitrate supply, although to different degrees: MHA4, in particular, proved more sensitive than MHA3, with a greater up- and down-regulation in response to the treatment. Increased expression of subfamily II genes resulted in higher steady-state levels of the enzyme in the root tissues and enhanced ATP-hydrolysing activity. The results support the idea that greater proton-pumping activity is required when nitrate inflow increases and suggest that nitrate may be the signal triggering the expression of the two members of PM H + -ATPase subfamily II.

There is increasing interest in the hydroponic technology to produce leafy vegetables for ready-to-eat salads. Optimisation of the growing system can lead to higher yield and/or improved nutritional value of the product. Selenium (Se) is an essential element for animal and humans, with quite a narrow range between deficiency and toxicity, whereas it is assumed beneficial for plants. In the present study, two cultivars (Gala and Baron) of corn salad (Valerianella locusta (L.) Laterr.) were used to test the possibility to increase Se content in the edible parts (leaves). Effects on yield, nitrate content, and accumulation of sulfur (S) and S-containing amino acids and Se and Se-containing amino acids were studied. Results showed that corn salad tolerates selenate (Na2SeO4) concentrations ranging from 10 to 40 µm in the nutrient solution, with plants accumulating Se at levels compatible with the need in human diets at 10 µm selenate. Se-treated plants showed some benefits with respect to a decrease of nitrate concentration and increase of pigment contents (chlorophylls and carotenoids). At 10 µm selenate, Se-cysteine and Se-methionine were produced, without affecting non-protein thiols or cysteine and methionine contents. At the higher Se supply, sulfate accumulated in the leaves with a parallel decrease in the amount of S-amino acids and a rise in the relative amount of Se-amino acids. Based on the chemical analyses, cv. Gala showed better tolerance than cv. Baron to moderate selenate supply (40 µm).