Roberto Pinton

Release of plant-borne flavonoids into the rhizosphere and their role in plant nutrition

Plants release a multitude of organic compounds into the rhizosphere, some of which are flavonoids. These products of secondary metabolism are mainly studied for their antioxidant properties and for their role in the establishment of rhizobium-legume symbiosis; however, it has been recently demonstrated that flavonoids can also affect nutrient availability through soil chemical changes. This review will give an overview of the types and amounts of flavonoids released by roots of different plant species, as well as summarize the available knowledge on root exudation mechanisms. Subsequently, factors influencing their release will be reported, and the methodological approaches used in the literature will be critically described. Finally, the direct contribution of plant-borne flavonoids on the nitrogen, phosphorous and iron availability into the rhizosphere will be discussed.

Plant-borne flavonoids released into the rhizosphere: impact on soil bio-activities related to plant nutrition. A review

Plants produce and release in the surrounding soil, the so-called rhizosphere, a vast variety of secondary metabolites. Among them, flavonoids are the most studied, mainly for their role in the establishment of rhizobium–legume symbiosis; on the other hand, some studies highlight that they are also important in the plant strategies to acquire nutrients from the soil, for example, by acting on its chemistry. The scope of this review is to give a quick overview on the types and amounts of plant-released flavonoids in order to focus on their effects on soil activities that in turn can influence nutrient availability and so plant mineral nutrition; emphasis is given to the different nutrient cycles, soil enzyme, and soil bacteria activities, and their influence on soil macrofauna and roots of other plants. Finally, the possible outcome of the climate change on these processes is discussed.

Review on iron availability in soil: interaction of Fe minerals, plants, and microbes

PurposeThe rationale of this paper is to review the state of the art regarding the biotic and abiotic reactions that can influence Fe availability in soils. In soil, the management-induced change from oxic to anoxic environment results in temporal and spatial variations of redox reactions, which, in turn, affect the Fe dynamics and Fe mineral constituents. Measuring the Fe forms in organic complexes and the interaction between bacteria and Fe is a major challenge in getting a better quantitative understanding of the dynamics of Fe in complex soil ecosystems.Materials and methodsWe review the existing literature on chemical and biochemical processes in soils related with the availability of Fe that influences plant nutrition. We describe Fe acquisition by plant and bacteria, and the different Fe–organic complexes in order to understand their relationships and the role of Fe in the soil carbon cycle.Results and discussionAlthough total Fe is generally high in soil, the magnitude of its available fraction is generally very low and is governed by very low solubility of Fe oxides. Plants and microorganisms can have different strategies in order to improve Fe uptake including the release of organic molecules and metabolites able to form complexes with FeIII. Microorganisms appear to be highly competitive for Fe compared with plant roots. Crystalline Fe and poorly crystalline (hydro)oxides are also able to influence the carbon storage in soil.ConclusionThe solubility of crystalline Fe minerals in soil is usually very low; however, the interaction with plant, microbes, and organic substance can improve the formation of soluble FeIII complexes and increase the availability of Fe for plant growth. Microbes release siderophores and plant exudates (e.g., phytosiderophores, organic acids, and flavonoids), which can bind and solubilize the Fe present in minerals. The improved understanding of this topic can enable the identification of effective solutions for remedying Fe deficiency or, alternatively, restricting the onset of its symptoms and yield’s limitations in crops. Therefore, development and testing of new analytical techniques and an integrated approach between soil biology and soil chemistry are important prerequisites.

Water-extractable humic substances enhance iron deficiency responses by Fe-deficient cucumber plants

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.

Modulation of NO-3 uptake by water-extractable humic substances : involvement of root plasma membrane H+ATPase

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.

Plasma membrane H+-ATPase-dependent citrate exudation from cluster roots of phosphate-deficient white lupin.

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.

Uptake of 59Fe from soluble 59Fe-humate complexes by cucumber and barley plants

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.

Response of barley plants to Fe deficiency and Cd contamination as affected by S starvation

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.

Solubilization of iron by water‐extractable humic substances

The capability of water-extractable humic substances (WEHS) to solubilize Fe from sparingly soluble Fe-hydroxide was studied. Addition of WEHS (1.7 mmol organic C l—1) to a dialysis tube containing labeled insoluble Fe-hydroxide caused an increase in the amount of 59Fe measured in the external solution. The humic fraction was also able to solubilize Fe from soil samples, with levels comparable to those obtained using a solution containing 100�μM DTPA. By measuring the amount of 59Fe eluted from soil columns pre-loaded with 59Fe-WEHS it was possible to evaluate the mobility of Fe complexed to the humic molecules. The recovery of 59Fe varied from 2% to 25% in respect to the soil type used. The ability of Fe-WEHS to serve as an Fe source for the phytosiderophore hydroxy-mugineic acid (HMA) was also analyzed. The removal of 59Fe from the Fe-WEHS complex by HMA was demonstrated by adding the phytosiderophore to a dialysis tube containing the 59Fe-WEHS complex. The observations suggested a ligand exchange between the phytosiderophore and the humic fraction. The results indicate that WEHS is able to increase the amount of Fe present in the soil solution, possibly by forming mobile complexes with the micronutrient. These complexes could act as easily available Fe sources in Fe acquisition processes by both monocot and dicot plants, playing an important role particularly in soils with low available Fe. Die Losung von Eisen mit wasserextrahierbaren Huminstoffen Es wurde die Fahigkeit von wasserextrahierbaren Huminstoffen (WEHS) untersucht, Fe aus schwerloslichen Fe-Hydroxiden zu losen. Die Zugabe von WEHS (1.7 mmol organic C l—1) in einen Dialyseschlauch mit 59Fe-Hydroxid fuhrte zu einem Ansteigen der 59Fe-Konzentration in der auseren Losung. Die WEHS waren im gleichen Mas wie eine 100 μM DTPA-Losung in der Lage, Fe aus Bodenproben zu mobilisieren. Die Mobilitat des mit Huminstoffen komplexierten Fe wurde durch die Eluierung von 59Fe aus einem mit 59Fe-WEHS beladenen Boden bestimmt. Dabei wurden in Abhangigkeit von dem verwendeten Bodentyp 2 bis 25% des nach der Beladung gebundenen 59Fe aus dem Boden eluiert. Ebenfalls wurde untersucht, ob Fe-WEHS als Fe-Quelle fur Phytosiderophore, z.B. Hydroxy-Mugineinsaure (HMA) dienen kann. Die Entfernung von 59Fe aus einem Dialysierschlauch mit 59Fe-WEHS nach einer Zugabe von HMA weist auf einen Ligandenaustausch zwischen Phytosiderophoren und Huminstoffen hin. Die Ergebnisse zeigen, dass WEHS moglicherweise wegen der Bildung von mobilen Komplexen in der Lage sind, die Fe-Konzentration in der Bodenlosung zu erhohen. Die Bedeutung dieser Eigenschaft fur die Fe-Ernahrung von Pflanzen wird diskutiert. Diese Komplexe konnten als leicht verfugbare Fe-Quelle bei monocotylen und dicotylen Pflanzen vor allem in Boden mit wenig verfugbarem Fe eine wichtige Rolle fur die Fe-Aneignung spielen.

Sulphur deprivation limits Fe-deficiency responses in tomato plants

The 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 in 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.