Nitrate transporter NRT1.1 and anion channel SLAH3 form a functional unit to regulate nitrate-dependent alleviation of ammonium toxicity.

Ammonium (NH4 + ) and nitrate (NO3 - ) are major inorganic nitrogen (N) sources for plants. When serving as the sole or dominant N supply, NH4 + often causes root inhibition and shoot chlorosis in plants, known as ammonium toxicity. NO3 - usually causes no toxicity and can mitigate ammonium toxicity even at low concentrations, referred to as nitrate-dependent alleviation of ammonium toxicity. Our previous studies indicated a NO3 - efflux channel SLAH3 is involved in this process. However, whether additional components contribute to NO3 - -mediated NH4 + detoxification is unknown. Previously, mutations in NO3 - transporter NRT1.1 were shown to cause enhanced resistance to high concentrations of NH4 + . Whereas, in this study, we found when the high-NH4 + medium was supplemented with low concentrations of NO3 - , nrt1.1 mutant plants showed hyper-sensitive phenotype instead. Furthermore, mutation in NRT1.1 caused enhanced medium acidification under high-NH4 + /low-NO3 - condition, suggesting NRT1.1 regulates ammonium toxicity by facilitating H+ uptake. Moreover, NRT1.1 was shown to interact with SLAH3 to form a transporter-channel complex. Interestingly, SLAH3 appeared to affect NO3 - influx while NRT1.1 influenced NO3 - efflux, suggesting NRT1.1 and SLAH3 regulate each other at protein and/or gene expression levels. Our study thus revealed NRT1.1 and SLAH3 form a functional unit to regulate nitrate-dependent alleviation of ammonium toxicity through regulating NO3 - transport and balancing rhizosphere acidification.

Plant tissue succulence engineering improves water-use efficiency, water-deficit stress attenuation and salinity tolerance in Arabidopsis.

Tissue succulence (ratio of tissue water/leaf area or dry mass) or the ability to store water within living tissues is among the most successful adaptations to drought in the plant kingdom. This taxonomically widespread adaptation helps plants avoid the damaging effects of drought, and is often associated with the occupancy of epiphytic, epilithic, semi-arid and arid environments. Tissue succulence was engineered in Arabidopsis thaliana by overexpression of a codon-optimized helix-loop-helix transcription factor (VvCEB1opt ) from wine grape involved in the cell expansion phase of berry development. VvCEB1opt -overexpressing lines displayed significant increases in cell size, succulence and decreased intercellular air space. VvCEB1opt -overexpressing lines showed increased instantaneous and integrated water-use efficiency (WUE) due to reduced stomatal conductance caused by reduced stomatal aperture and density resulting in increased attenuation of water-deficit stress. VvCEB1opt -overexpressing lines also showed increased salinity tolerance due to reduced salinity uptake and dilution of internal Na+ and Cl- as well as other ions. Alterations in transporter activities were further suggested by media and apoplastic acidification, hygromycin B tolerance and changes in relative transcript abundance patterns of various transporters with known functions in salinity tolerance. Engineered tissue succulence might provide an effective strategy for improving WUE, drought avoidance or attenuation, salinity tolerance, and for crassulacean acid metabolism biodesign.

Strigolactones affect phosphorus acquisition strategies in tomato plants.

Strigolactones (SLs) are plant hormones that modulate morphological, physiological and biochemical changes as part of the acclimation strategies to phosphorus (P) deficiency, but an in-depth description of their effects on tomato P-acquisition strategies under P shortage is missing. Therefore, in this study, we investigate how SLs impact on root exudation and P uptake, in qualitative and quantitative terms over time, in wild-type and SL-depleted tomato plants grown with or without P. Under P shortage, SL-depleted plants were unable to efficiently activate most mechanisms associated with the P starvation response (PSR), except for the up-regulation of P transporters and increased activity of P-solubilizing enzymes. The reduced SL biosynthesis had negative effects also under normal P provision, because plants over-activated high-affinity transporters and enzymatic activities (phytase, acidic phosphatase) to sustain elevated P uptake, at great carbon and nitrogen costs. A shift in the onset of PSR was also highlighted in these plants. We conclude that SLs are master kinetic regulators of the PSR in tomato and that their defective synthesis might lead both to suboptimal nutritional outcomes under P depletion and an unbalanced control of P uptake when P is available.

Role of TaALMT1 malate-GABA transporter in alkaline pH tolerance of wheat.

Malate exudation through wheat (Triticum aestivum L) aluminium-activated malate transporter 1 (TaALMT1) confers Al3+ tolerance at low pH, but is also activated by alkaline pH, and is regulated by and facilitates significant transport of gamma-aminobutyric acid (GABA, a zwitterionic buffer). Therefore, TaALMT1 may facilitate acidification of an alkaline rhizosphere by promoting exudation of both malate and GABA. Here, the performance of wheat near isogenic lines ET8 (Al+3 -tolerant, high TaALMT1 expression) and ES8 (Al+3 -sensitive, low TaALMT1 expression) are compared. Root growth (at 5 weeks) was higher for ET8 than ES8 at pH 9. ET8 roots exuded more malate and GABA at high pH and acidified the rhizosphere more rapidly. GABA and malate exudation was enhanced at high pH by the addition of aluminate in both ET8 and transgenic barley expressing TaALMT1. Xenopus laevis oocytes expressing TaALMT1 acidified an alkaline media more rapidly than controls corresponding to higher GABA efflux. TaALMT1 expression did not change under alkaline conditions but key genes involved in GABA turnover changed in accordance with a high rate of GABA synthesis. We propose that TaALMT1 plays a role in alkaline tolerance by exuding malate and GABA, possibly coupled to proton efflux.

The role of rhizosphere pH in regulating the rhizosphere priming effect and implications for the availability of soil-derived nitrogen to plants.

Background and Aims: A comprehensive understanding of the rhizosphere priming effect (RPE) on the decomposition of soil organic carbon (SOC) requires an integration of many factors. It is unclear how N form-induced change in soil pH affects the RPE and SOC sequestration. Methods: This study compared the change in the RPE under supply of NO3-N and NH4-N. The effect of the RPE on the mineralization of soil N and hence its availability to plant and microbes was also examined using a 15N-labelled N source. Key Results: The supply of NH4-N decreased rhizosphere pH by 0.16-0.38 units, and resulted in a decreased or negative RPE. In contrast, NO3-N nutrition increased rhizosphere pH by 0.19-0.78 units, and led to a persistently positive RPE. The amounts of rhizosphere-primed C were positively correlated with rhizosphere pH. Rhizosphere pH affected the RPE mainly through influencing microbial biomass, activity and utilization of root exudates, and the availability of SOC to microbes. Furthermore, the amount of rhizosphere primed C correlated negatively with microbial biomass atom% 15N (R2 0.77-0.98, n = 12), suggesting that microbes in the rhizosphere acted as the immediate sink for N released from enhanced SOC decomposition via the RPE. Conclusion: N form was an important factor affecting the magnitude and direction of the RPE via its effect on rhizosphere pH. Rhizosphere pH needs to be considered in SOC and RPE modelling.

H+ -pyrophosphatase IbVP1 promotes efficient iron use in sweet potato [Ipomoea batatas (L.) Lam.].

Iron (Fe) deficiency is one of the most common micronutrient deficiencies limiting crop production globally, especially in arid regions because of decreased availability of iron in alkaline soils. Sweet potato [Ipomoea batatas (L.) Lam.] grows well in arid regions and is tolerant to Fe deficiency. Here, we report that the transcription of type I H+ -pyrophosphatase (H+ -PPase) gene IbVP1 in sweet potato plants was strongly induced by Fe deficiency and auxin in hydroponics, improving Fe acquisition via increased rhizosphere acidification and auxin regulation. When overexpressed, transgenic plants show higher pyrophosphate hydrolysis and plasma membrane H+ -ATPase activity compared with the wild type, leading to increased rhizosphere acidification. The IbVP1-overexpressing plants showed better growth, including enlarged root systems, under Fe-sufficient or Fe-deficient conditions. Increased ferric precipitation and ferric chelate reductase activity in the roots of transgenic lines indicate improved iron uptake, which is also confirmed by increased Fe content and up-regulation of Fe uptake genes, e.g. FRO2, IRT1 and FIT. Carbohydrate metabolism is significantly affected in the transgenic lines, showing increased sugar and starch content associated with the increased expression of AGPase and SUT1 genes and the decrease in ß-amylase gene expression. Improved antioxidant capacities were also detected in the transgenic plants, which showed reduced H2 O2 accumulation associated with up-regulated ROS-scavenging activity. Therefore, H+ -PPase plays a key role in the response to Fe deficiency by sweet potato and effectively improves the Fe acquisition by overexpressing IbVP1 in crops cultivated in micronutrient-deficient soils.

Modulation of the Phosphate-Deficient Responses by MicroRNA156 and its Targeted SQUAMOSA PROMOTER BINDING PROTEIN-LIKE 3 in Arabidopsis.

The microRNA156 (miR156)-modulated SQUAMOSA PROMOTER BINDING PROTEIN-LIKE (SPL) is involved in diverse biological processes that include growth, development and metabolism. Here, we report that the Arabidopsis miR156 and SPL3 as regulators play important roles in phosphate (Pi) deficiency response. MiR156 was induced during Pi starvation whereas SPL3 expression was repressed. Phenotypes of reduced rhizosphere acidification and decreased anthocyanin accumulation were observed in 35S:MIM156 (via target mimicry) transgenic plants under Pi deficiency. The content and uptake of Pi in 35S:MIM156 Arabidopsis plants were increased compared with wild-type (Col-0 ecotype) plants. 35S:rSPL3 seedlings showed similar anthocyanin accumulation and Pi content phenotypes to those of 35S:MIM156 plants. Chromatin immunoprecipitation and an electrophoretic mobility shift assay indicated that the SPL3 protein directly bound to GTAC motifs in the PLDZ2, Pht1;5 and miR399f promoters. The expression of several Pi starvation-induced genes was increased in 35S:MIM156 and 35S:rSPL3 plants, including high-affinity Pi transporters, Mt4/TPS1-like genes and phosphatases. Collectively, our results suggest that the miR156-SPL3-Pht1;5 (-PLDZ2 and -miR399f) pathways constitute a component of the Pi deficiency-induced regulatory mechanism of Arabidopsis.

Rhizosphere priming effect on soil organic carbon decomposition under plant species differing in soil acidification and root exudation.

Effects of rhizosphere properties on the rhizosphere priming effect (RPE) are unknown. This study aimed to link species variation in RPE with plant traits and rhizosphere properties. Four C3 species (chickpea, Cicer arietinum; field pea, Pisum sativum; wheat, Triticum aestivum; and white lupin, Lupinus albus) differing in soil acidification and root exudation, were grown in a C4 soil. The CO2 released from soil was trapped using a newly developed NaOH-trapping system. White lupin and wheat showed greater positive RPEs, in contrast to the negative RPE produced by chickpea. The greatest RPE of white lupin was in line with its capacity to release root exudates, whereas the negative RPE of chickpea was attributed to its great ability to acidify rhizosphere soil. The enhanced RPE of field pea at maturity might result from high nitrogen deposition and release of structural root carbon components following root senescence. Root biomass and length played a minor role in the species variation in RPE. Rhizosphere acidification was shown to be an important factor affecting the magnitude and direction of RPE. Future studies on RPE modelling and mechanistic understanding of the processes that regulate RPE should consider the effect of rhizosphere pH.

miR156 modulates rhizosphere acidification in response to phosphate limitation in Arabidopsis.

Rhizosphere acidification is a general response to Pi deficiency, especially in dicotyledonous plants. However, the signaling pathway underlying this process is still unclear. Here, we demonstrate that miR156 is induced in the shoots and roots of wild type Arabidopsis plants during Pi starvation. The rhizosphere acidification capacity was increased in 35S:MIR156 (miR156 overexpression) plants, but was completely inhibited in 35S:MIM156 (target mimicry) plants. Both 35S:MIR156 and 35S:MIM156 plants showed altered proton efflux and H(+)-ATPase activity. In addition, significant up-regulation of H(+)-ATPase activity in 35S:MIR156 roots coupled with increased citric acid and malic acid exudates was observed. qRT-PCR results showed that most H(+)-ATPase and PPCK gene transcript levels were decreased in 35S:MIM156 plants, which may account for the decreased H(+)-ATPase activity in 35S:MIM156 plants. MiR156 also affect the root architecture system. Collectively, our results suggest that miR156 regulates the process of rhizosphere acidification in plants.

STOP1-regulated SMALL AUXIN UP RNA55 (SAUR55) is involved in proton/malate co-secretion for Al tolerance in Arabidopsis.

Proton (H+) release is linked to aluminum (Al)-enhanced organic acids (OAs) excretion from the roots under Al rhizotoxicity in plants. It is well-reported that the Al-enhanced organic acid excretion mechanism is regulated by SENSITIVE TO PROTON RHIZOTOXICITY1 (STOP1), a zinc-finger TF that regulates major Al tolerance genes. However, the mechanism of H+ release linked to OAs excretion under Al stress has not been fully elucidated. Recent physiological and molecular-genetic studies have implicated the involvement of SMALL AUXIN UP RNAs (SAURs) in the activation of plasma membrane H+-ATPases for stress responses in plants. We hypothesized that STOP1 is involved in the regulation of Al-responsive SAURs, which may contribute to the co-secretion of protons and malate under Al stress conditions. In our transcriptome analysis of the roots of the stop1 (sensitive to proton rhizotoxicity1) mutant, we found that STOP1 regulates the transcription of one of the SAURs, namely SAUR55. Furthermore, we observed that the expression of SAUR55 was induced by Al and repressed in the STOP1 T-DNA insertion knockout (KO) mutant (STOP1-KO). Through in silico analysis, we identified a functional STOP1-binding site in the promoter of SAUR55. Subsequent in vitro and in vivo studies confirmed that STOP1 directly binds to the promoter of SAUR55. This suggests that STOP1 directly regulates the expression of SAUR55 under Al stress. We next examined proton release in the rhizosphere and malate excretion in the T-DNA insertion KO mutant of SAUR55 (saur55), in conjunction with STOP1-KO. Both saur55 and STOP1-KO suppressed rhizosphere acidification and malate release under Al stress. Additionally, the root growth of saur55 was sensitive to Al-containing media. In contrast, the overexpressed line of SAUR55 enhanced rhizosphere acidification and malate release, leading to increased Al tolerance. These associations with Al tolerance were also observed in natural variations of Arabidopsis. These findings demonstrate that transcriptional regulation of SAUR55 by STOP1 positively regulates H+ excretion via PM H+-ATPase 2 which enhances Al tolerance by malate secretion from the roots of Arabidopsis. The activation of PM H+-ATPase 2 by SAUR55 was suggested to be due to PP2C.D2/D5 inhibition by interaction on the plasma membrane with its phosphatase. Furthermore, RNAi-suppression of NtSTOP1 in tobacco shows suppression of rhizosphere acidification under Al stress, which was associated with the suppression of SAUR55 orthologs, which are inducible by Al in tobacco. It suggests that transcriptional regulation of Al-inducible SAURs by STOP1 plays a critical role in OAs excretion in several plant species as an Al tolerance mechanism.

Plasma Membrane (PM) H+-ATPase Mediates Rhizosphere Acidification and Regulates Herbicide Imazethapyr Toxicity in Wheat.

The plasma membrane (PM) H+-ATPase is crucial for a plant defense system. However, there is currently no consensus on whether the PM H+-ATPase plays a role in alleviating the toxic effects of herbicides on nontarget plants. We found that under the herbicide imazethapyr (IM) exposure, PM H+-ATPase activity in wheat roots increased by approximately 69.53%, leading to rhizosphere acidification. When PM H+-ATPase activity is inhibited, the toxicity of IM significantly increases: When exposed to IM alone, the total Fe content of wheat roots decreased by 29.07%, the relative Fe2+ content increased by 27.75%, and the ROS content increased by 27.74%. When the PM H+-ATPase activity was inhibited, the corresponding data under IM exposure were 37.36%, 215%, and 57.68%, respectively. This work delves into the role of PM H+-ATPase in mediating the detoxification mechanism in plants exposed to herbicides, offering new insights into enhancing crop resistance against herbicides.

Urea reduces the sustainability of soil Cd immobilization by upregulating the expression of AmSTOP1 and AmMATE genes in edible amaranth roots.

After cadmium (Cd) immobilization remediation in contaminated farmland soil, which forms of nitrogen fertilizer should be implemented to keep its sustainability? Urea and nitrate were used to compare for their effects on the remobilization of stabilized Cd in the rhizosphere soil of edible amaranth at nitrogen concentrations of 60, 95, and 130 mg kg-1. The results showed that compared to nitrate nitrogen, the Cd content in shoots increased by 76.2%, 65.6%, and 148% after applying three different concentrations of urea, and the total remobilization amount of Cd also increased by 16.0%, 24.9%, and 14.0% respectively. Urea application promotes root secretion of citric acid, malic acid, pyruvate, and γ-aminobutyric acid, crucial in remobilizing stable Cd. The application of urea promoted the expression of genes involved in sucrose transport, glycolysis, the TCA cycle, amino acid secretion, citric acid efflux, and proton efflux. Arabidopsis heterologous expression and yeast one-hybrid assays identify critical roles of AmMATE42 and AmMATE43 in citric acid and fumaric acid efflux, with AmSTOP1 activating their transcription. Inhibition of SIZ1 expression in urea treatment reduce AmSTOP1 SUMOylation, leading to increased expression of AmMATE42 and AmMATE43 and enhanced organic acids efflux. Using edible amaranth as a model vegetable, we discovered that urea is not beneficial to preserving the sustainability of stabilized Cd during the reuse of remediated farmlands contaminated with Cd.

An Adapted Protocol for Quantitative Rhizosphere Acidification Assay.

Acidification of the rhizosphere is a key process in the homeostasis of multiple essential nutrients, including iron. Under iron deficiency, the release of protons from the roots helps solubilize and increase the accessibility of iron in the soil. Rhizosphere acidification has been widely examined in many iron homeostasis studies, generally using a qualitative method based on the color change of bromocresol purple, a pH indicator dye, near the roots. In this chapter, we introduce an adapted version of a rhizosphere acidification assay protocol that allows for the quantitative assessment of small pH changes in the rhizosphere. This colorimetric method also utilizes bromocresol purple, but the ratio of its absorbance at 434 nm and 588 nm is considered to quantify protons released into the assay solution. Furthermore, the assay is compatible with small sample volumes, such as those with young Arabidopsis seedlings.

Silicon-phosphorus pathway mitigates heavy metal stress by buffering rhizosphere acidification.

Heavy metal pollution threatens food security, and rhizosphere acidification will increase the bioavailability of heavy metals. As a beneficial element in plants, silicon can relieve heavy metal stress. However, less attention has been paid to its effects on plant rhizosphere processes. Here, we show that for Japonica (Nipponbare and Oochikara) and Indica (Jinzao 47) rice cultivars, the degree of root acidification was significantly reduced after silicon uptake, and the total organic carbon, citric acid, and malic acid concentrations in rice root exudates were significantly reduced. We further confirmed the results by q-PCR that the expressions of proton pump and organic acid secretion genes were down-regulated by 35-61 % after silicon treatment. Intriguingly, phosphorus allocation, an intensively studied mechanism of rhizosphere acidification, was altered by silicon treatment. Specifically, among total phosphorus in rice seedlings, the soluble proportion increased from 52.0 % to 61.7 %, while cell wall phosphorus decreased from 48.0 % to 32.3 %. Additionally, silicon-mediated alleviation of rhizosphere acidification has positive effects on relieving heavy metal stress. Simulation revealed that low acidification of the nutrient solution resulted in a decrease in bioavailable heavy metal concentrations, thereby reducing rice uptake. We further confirmed that the impediment of rhizosphere acidification led to free-state Cr3+ in solutions decreasing by 43 % and contributed up to 63 % of silicon's mitigation of Cr(III) stress. Overall, we propose a novel mechanism in which silicon reduces heavy metal absorption by increasing plant soluble phosphorus concentration and buffering rhizosphere acidification. This paper provides a unique insight into the role of silicon in plants and, more importantly, a theoretical reference for the rational application of silicon fertilizer to improve phosphorus utilization efficiency, alleviate heavy metal stress, and balance soil pH.

Mechanistic insights into trace metal mobilization at the micro-scale in the rhizosphere of Vallisneria spiralis.

Mobilization of trace metals in the rhizosphere of macrophytes is controlled by root-driven chemical changes, especially the steep gradients of O2 and pH from the rhizosphere to bulk sediments. Here, the O2 and pH dynamics, and the distribution of trace metal, in the rhizosphere of Vallisneria spiralis were obtained using planar optodes and diffusive gradients in thin films, respectively. Radial O2 loss (ROL) and acidification occurred on all visible roots of V. spiralis and exhibited highly spatiotemporal dynamics depending on the root growth and various environmental conditions. Trace metals showed different mobilization mechanisms in the rhizosphere. ROL and produced Fe(III) (oxyhydr)oxides decreased the mobility of Fe, As, Co, V and W in the rhizosphere. However, Mn, Ni and Cu exhibited greater mobility in the rhizosphere than bulk sediments as a result of the oxidation of metal sulfide and proton-induced dissolution of minerals. In particular, Co and Ni presented increased activity at the interface between rhizosphere and bulk sediment, which was attributed to the redox dissolution processes of Fe and Mn as a result of ROL and rhizosphere acidification. These results provide new insights into the roles of macrophyte root-induced O2 and pH changes in controlling trace metal mobility in sediments.

Impacts of oxalic acid-activated phosphate rock and root-induced changes on Pb bioavailability in the rhizosphere and its distribution in mung bean plant.

Rhizosphere acidification in leguminous plants can release P from the dissolution of phosphate compounds which can reduce Pb bioavailability to them via the formation of insoluble Pb compounds in their rhizosphere. A soil polluted from Pb-acid batteries effluent (SPBE), having total Pb = 639 mg kg-1, was amended with six different rates (0, 0.5, 1, 2, 4 and 6%) of oxalic acid-activated phosphate rock (OAPR) and their effects on pH, available P and bioavailable Pb concentrations in the rhizosphere and bulk soils of mung bean plant were evaluated. Furthermore, the effects of these variant OAPR rates on Pb concentrations in plant parts, bioaccumulation factor (BAF) and translocation factor (TF) for Pb in grain and traits like productivity, the activities of antioxidant enzymes, and grain biochemistry were investigated. Results revealed that increasing rates of OAPR significantly increased pH values and available P while decreased bioavailable Pb concentrations in the rhizosphere over control. The highest dissolution of P in the rhizosphere was with 4 and 6% OAPR rates. As a result, the formation of insoluble Pb compounds affected on reduced Pb concentrations in shoots, roots, and grain in addition to lower grain BAF and TF values for Pb over control. Likewise, the highest plant productivity, improved grain biochemistry, high Ca and Mg concentrations, least oxidative stress, and enhanced soil alkaline phosphatase activity were found with 4 and 6% OAPR rates. The OAPR 4% rate is suggested for reducing grain Pb concentration, cell oxidative injury, and improving grain biochemistry in mung bean.

Nitrogen fertilizer affects rhizosphere Cd re-mobilization by mediating gene AmALM2 and AmALMT7 expression in edible amaranth roots.

In-situ stabilization of Cd-contaminated farmland is a commonly used remediation technology. Yet, rhizosphere metabolites (e.g., organic acids) during crop cultivation may cause Cd re-mobilization and over-accumulation. Here, we identified four pivotal cytomembrane-localized genes underlying Cd accumulation difference between two contrasting edible amaranth cultivars based on root gene expression profile, studied their subcellular localization and functional characteristics, and then investigated effects of nitrogen fertilizer on their expression and rhizosphere Cd re-mobilization. Results showed that more Cd accumulated by edible amaranth was due to rhizosphere Cd mobilization by mediating high expression of AmALMT2 and AmALMT7 genes, not Cd transporters in roots. This was confirmed by heterologous expression of AmALMT2 and AmALMT7 genes in Arabidopsis thaliana, since they mediated malic, fumaric, succinic, and aspartic acids efflux. Furthermore, nitrogen influencing rhizosphere acidification might be closely associated with organic acids efflux genes. Compared with N-NO3- application, N-NH4+ was massively assimilated into glutamates and oxaloacetates through up-regulating glutamine synthetase and alanine-aspartate-glutamate metabolic pathways, thereby enhancing TCA cycle and organic acids efflux dominated by binary carboxylic acids via up-regulating AmALMT2 and AmALMT7 genes, which finally caused Cd re-mobilization. Therefore, N-NO3--dominated nitrogen retarded rhizosphere Cd re-mobilization via inhibiting organic acids efflux function of AmALMT2 and AmALMT7 proteins.