Transcriptional and physiological analyses uncover the mineralization and uptake mechanisms of phytic acid in symbiotically grown Vicia faba plants.

Legume-rhizobia symbiosis requires high phosphorus (P) in the form of ATP to convert atmospheric nitrogen (N) into ammonia. The fixed ammonia is converted to NH4+ by H+-ATPase via protonation. To the best of our knowledge, most of these research works resort to using only inorganic P (Pi) to the neglect of the organic P (Po) counterpart. As it stands, the potential regulating roles of plasma membrane (PM) H+-ATPases during legume-rhizobia symbiosis in response to phytic acid supply and how it alters and modulates the regulation of PM H+-ATPases remain obscure. To contribute to the above hypothesis, we investigate the mechanisms that coordinately facilitate the growth, uptake, and transcript expression of PM H+-ATPase gene isoforms in response to different P sources when hydroponically grown Vicia faba plants were exposed to three P treatments, viz., low- and high-Pi (2.0 and 200 µM KH2PO4; LPi and HPi), and phytic acid (200 µM; Po) and inoculated with Rhizobium leguminosarum bv. viciae 384 for 30 days. The results consistently reveal that the supply of Po improved not only the growth and biomass, but also enhanced photosynthetic parameters, P uptake and phosphatase activities in symbiotically grown Vicia faba relative to Pi. The supply of Po induced higher transcriptional expression of all PM H+-ATPase gene isoforms, with possible interactions between phosphatases and H+-ATPase genes in Vicia faba plants when exclusively reliant on N derived from nodule symbiosis. Overall, preliminary results suggest that Po could be used as an alternative nutrition in symbiotic crops to improve plant growth.

A method to experimentally clamp leaf water content to defined values to assess its effects on apoplastic pH.

BACKGROUND: Leaf hydration is controlled by feedback mechanisms, e.g. stomatal responses, adjustments of osmotic potential and hydraulic conductivity. Leaf water content thus is an input into related feedback-loops controlling the balance of water uptake and loss. Apoplastic alkalisation upon leaf dehydration is hypothesized to be involved together and in interaction with abscisic acid (ABA) in water stress related signaling on tissue level. However, important questions are still unresolved, e.g. the mechanisms leading to pH changes and the exact nature of its interaction with ABA. When studying these mechanisms and their intermediate signaling steps, an experimenter has only poor means to actually control the central experimental variable, leaf water content (LWC), because it is not only dependent on external variables (e.g. air humidity), which are under experimental control, but is also governed by the biological influences controlling transpiration and water uptake. Those are often unknown in their magnitude, unpredictable and fluctuating throughout an experiment and will prevent true repetitions of an experiment. The goal of the method presented here is to experimentally control and manipulate leaf water content (LWC) of attached intact leaves enclosed in a cuvette while simultaneously measuring physiological parameters like, in this case, apoplastic pH. RESULTS: An experimental setup was developed where LWC is measured by a sensor based on IR-transmission and its signal processed to control a pump which circulates air from the cuvette through a cold trap. Hereby a feedback-loop is formed, which by adjusting vapour pressure deficit (VPD) and consequently leaf transpiration can precisely control LWC. This technique is demonstrated here in a combination with microscopic fluorescence imaging of apoplastic pH (pHapo) as indicated by the excitation ratio of the pH sensitive dye OregonGreen. Initial results indicate that pHapo of the adaxial epidermis of Vicia faba is linearly related to reductions in LWC. CONCLUSIONS: Using this setup, constant LWC levels, step changes or ramps can be experimentally applied while simultaneously measuring physiological responses. The example experiments demonstrate that bringing LWC under experimental control in this way allows better controlled and more repeatable experiments to probe quantitative relationships between LWC and signaling and regulatory processes.

Alkali salt stress causes fast leaf apoplastic alkalinization together with shifts in ion and metabolite composition and transcription of key genes during the early adaptive response of Vicia faba L.

The mechanisms by which plants respond to alkali salt stress are still obscure, and the relevance of alkaline pH under combined alkali salt stress. Early stress responses can indicate mechanisms leading to damage and plant resistance. The apoplast contains essential determinants for plant growth, specifically early apoplastic pH fluctuations are induced by many stressors and hypothesized to be involved in stress signalling. Hence, this study aims to identify fast responses specific to alkaline pH and alkali salt stress by exposing the root of hydroponically grown Vicia faba L. plants to 150 min of either 50 mM NaHCO3 (pH 9) treatment or alkaline pH 9 alone. Apoplastic pH was monitored in real-time by ratiometric fluorescence microscopy simultaneously with SWIR transmission-based measurements of leaf water content (LWC). Moreover, we examined the effect of these stresses on apoplastic, symplastic and xylem ion and metabolite composition together with transcriptions of certain stress-responsive genes. Physiological and transcriptional changes were observed in response to NaHCO3 but not to alkaline pH alone. NaHCO3 elicited a transient reduction in LWC, followed by a transient alkalinization of the apoplast and stomatal closure. Simultaneously, organic acids and sugars accumulated. Fast upregulation of stress-responsive genes showed the significance of gene regulation for early plant adaptation to alkali salt stress.

Uptake, subcellular distribution, and translocation of foliar-applied phosphorus: Short-term effects on ion relations in deficient young maize plants.

One crucial aspect for successful foliar application is the uptake of the nutrient into the symplast for metabolization by the plant. Our aim was to determine the subcellular distribution of foliar-applied P in leaves, the translocation of this element within the whole plant, and its impact on the ion status of P-deficient maize plants within the first 48 h of treatment. Maize plants with P deficiency were sprayed with 200 mM KH2PO4. After 6, 24, and 48 h, the 5th leaf of each plant was harvested for the isolation of apoplastic washing fluid, cell sap, and vascular bundle sap and for the examination of transporter gene expression. The remaining tissues were divided into 4th leaf, older and younger shoots, and root for total P determination. No accumulation of foliar-applied P was measured in the apoplast. P was mostly taken up into the cytosol within the first 6 h and was associated with increased mRNA levels of PHT1 transporters. A strong tendency towards rapid translocation into the younger shoot and an increase in NO3- uptake or a decrease in organic acid translocation were observed. The apoplast seems to exert no effect on the uptake of foliar-applied P into the epidermal and mesophyll cells of intact leaves. Instead, the plant responds with the rapid translocation of P and changes in ion status to generate further growth. The effect of the absorbed foliar-applied P is assumed to be a rapid process with no transient storage in the leaf apoplast.

One-Time Foliar Application and Continuous Resupply via Roots Equally Improved the Growth and Physiological Response of B-Deficient Oilseed Rape.

Oilseed rape (Brassica napus L.) is a high-boron (B)-demanding crop, and initially, normal growing plants might show B deficiency at advanced growth stages on soils with marginal B availability. Hence, we compared the effects of B resupply via roots and leaves on growth and physiological response, and relative expression of B transporters in B-deficient oilseed rape plants. Four-week-old plants initially grown with inadequate B (1 µM B for the first two weeks and 0.25 µM B for the next two weeks) were later grown either as such with 0.25 µM B, with 25 µM B in nutrient solution or foliar sprayed with 7 mL of 30, 60 and 150 mM B solution plant-1 as boric acid. Plants grown with 25 µM B in the nutrient solution from the beginning were included as adequate B treatment. Results showed that B resupply to B-deficient plants via roots and leaves (60 mM B) equally improved root and shoot dry matter, but not to the level of plants grown with adequate B supply. Foliar-applied 150 mM B proved toxic, causing leaf burn but not affecting dry matter. Resupply of B via roots increased B concentration in roots and leaves, while leaf-applied B did so only in leaves. Net carbon assimilation had a positive relationship with dry matter accumulation. Except for the highest foliar B level, B resupply via roots and leaves increased the accumulation of glucose, fructose and sucrose in leaves. Boron-deficient plants showed significant upregulation of BnaNIP5;1 in leaves and roots and of BnaBOR1;2 in roots. Boron resupply via roots reversed the B-deficiency-induced upregulation of BnaNIP5;1 in roots, whereas the expression of BnaBOR1;2 was reversed by both root and foliar B resupply. In leaves, B resupply by both methods reversed the expression of BnaNIP5;1 to the level of B-adequate plants. It is concluded that B resupply to B-deficient plants via roots and leaves equally but partially corrected B deficiency in B. napus grown in hydroponics.

Boron uptake and distribution by oilseed rape (Brassica napus L.) as affected by different nitrogen forms under low and high boron supply.

Ammonium (NH4+) and nitrate (NO3-) conversely alter pH of the rooting medium, and thus differentially affect the equilibrium between boric acid and borate in soil solution. This can alter boron (B) uptake by plants, which is passive under high, but facilitated (boric acid) or active (borate) under low B supply. Therefore, the effect of NH4+ and NO3- forms was investigated on the growth, 10B uptake rate and accumulation, and expression of B transporters in Brassica napus grown with low (1 µM) or high (100 µM) 10B for five days in the nutrient solution. At the low 10B level, NO3--fed plants had the same specific 10B uptake rate, 10B accumulation and xylem 10B concentration as NH4NO3-fed plants but these attributes were reduced at the high 10B level. BnaBOR1;2 and BnaNIP5;1 were upregulated in roots of NO3-fed plants at low 10B supply. NH4+-fed plants had substantially lower dry matters; due to nutrient solution acidification (2.0 units)-induced deficiency of nitrogen, potassium, magnesium, and iron in plant shoots. Reduced transpiration rates resulted in lower 10B uptake rate and accumulation in the roots and shoots of NH4+-fed plants. BnaNIP5;1 in roots, while both BnaBOR1;2 and BnaNIP5;1 in shoots were upregulated in NH4+-fed plants at low 10B level. Collectively, NH4+-induced acidity and consequent lowering of 10B uptake induced the upregulation of B transport mechanisms, even at marginal 10B concentrations, while NO3--induced alkalinization resulted in altered B distribution between roots and shoots due to restricted B transport, especially at higher 10B supply.

One-time abscisic acid priming induces long-term salinity resistance in Vicia faba: Changes in key transcripts, metabolites, and ionic relations.

Abscisic acid (ABA) priming is known to enhance plant growth and survival under salinity. However, the mechanisms mediating this long-term acclimatization to salt stress are still obscure. Specifically, the long-term transcriptional changes and their effects on ion relations were never investigated. This motivated us to study the long-term (8 days) effect of one-time 24 h root priming treatment with 10 µM ABA on transcription levels of relevant regulated key genes, osmotically relevant metabolites, and ionic concentrations in Vicia faba grown under 50 mM NaCl salinity. The novelty of this study is that we could demonstrate long-term effects of a one-time ABA application. ABA-priming was found to prevent the salt-induced decline in root and shoot dry matter, improved photosynthesis, and inhibited terminal wilting of plants. It substantially increased the mRNA level of AAPK and 14-3-3 ABA inducible kinases and ion transporters (PM H+ -ATPase, VFK1, KUP7, SOS1, and CLC1). These ABA-induced transcriptional changes went along with altered tissue ion patterns. Primed plants accumulated less Na+ and Cl- but more K+ , Ca2+ , Zn2+ , Fe2+ , Mn2+ , NO3 - , and SO4 2- . Priming changed the composition pattern of organic osmolytes under salinity, with glucose and fructose being dominant in unprimed, whereas sucrose was dominant in the primed plants. We conclude that one-time ABA priming mitigates salt stress in Vicia faba by persistently changing transcription patterns of key genes, stabilizing the ionic and osmotic balance, and improving photosynthesis and growth.

Sulfate facilitates cadmium accumulation in leaves of Vicia faba L. at flowering stage.

Cadmium (Cd) is a toxic element, and is prevalent all over the world because of industrialization, mining, sewage sludge, or pesticide supply. Sulfur deficiency is also a frequent problem faced in agriculture. To date, information relating to effects of sulfate on Cd toxicity is still limited. To elucidate how sulfate affects Cd accumulation in faba bean, subcellular accumulation of Cd in leaves consisting of apoplastic washing fluid, symplastic fluid and the cell wall under deficient, sufficient and excess sulfate treatments were investigated in the present study. By using stable isotope of Cd (106Cd), we also traced Cd accumulation in young leaves at flowering stage from early and newly uptake of Cd in the same plants as affected by sulfate. We found that excess sulfate supply significantly increased newly uptake of Cd without affecting early uptake of Cd when compared with sufficient sulfate treatment, which resulted in enhanced total Cd in leaves by excess sulfate. Since newly uptake of Cd in leaves was from root uptake directly, we conclude that excess sulfate supply enhanced Cd originated from root uptake directly rather than re-translocation from old leaves, which is related to increased Cd accumulation in young leaves of faba bean. Subcellular analysis showed that the enhanced Cd by excess sulfate addition was a consequence of enhanced Cd in cell walls, while Cd accumulation in the apoplastic washing fluid and symplastic fluid were unchanged. The increased Cd by excess sulfate supply might be related to increased proportion of Cd speciation CdSO40 in the growth medium because of faster diffusion of CdSO40 than Cd2+. To test whether macronutrients, micronutrients, sulfate and non-protein thiol involved in cell wall-Cd accumulation as affected by sulfate, correlations of subcellular Cd with subcellular macronutrients, micronutrients, sulfate, and non-protein thiol were analyzed. We found that cell wall-Cd was negatively correlated with K and Ca concentrations, whereas cell wall-Cd was positively correlated with Cu and symplastic non-protein thiol concentrations. However, when compared with sufficient sulfate, excess sulfate decreased K concentration and increased symplastic non-protein thiol concentration without changing Ca and Cu concentrations. Based on those results, reduction of K concentration and enhancement of symplastic non-protein thiol concentration by sulfate supply might be a reason for increase of cell wall-Cd concentration. Taken together, increased Cd in cell walls of leaves by sulfate supply contributes to enhance Cd accumulation.