Non-invasive Micro-test Technique Research Articles

Insights into the antagonistic effects of calcium on cadmium accumulation in peanuts (Arachis hypogaea L.)

Peanut (Arachis hypogaea L.) plant has a high requirement for calcium (Ca) during its growth and development, and possesses the ability to accumulate cadmium (Cd) from soil. However, the precise mechanisms underlying the antagonistic effects between Ca and Cd remain unclear. This study aimed to explore the dynamic changes in Cd accumulation in peanut seedlings by varying the Ca-to-Cd concentration ratio (CRCa/Cd) from 250 to 3500. Additionally, the influence of ion channel competition and cell wall fixation in the root on Cd accumulation in peanuts was explored by analyzing Cd chemical forms, subcellular distribution, pectin content, and Cd2+ fluxes using a non-invasive micro-test technique (NMT). The findings revealed that Cd accumulation in peanut seedlings was significantly lower when the CRCa/Cd was higher than 2000. In the Ca-pretreated seedlings (cell wall fixation treatment), Cd content in the shoots and roots decreased by 18.9% and 25.0%, respectively, compared with the simultaneous exposure to Ca and Cd (ion channel competition treatment). Cd2+ influx in peanut roots decreased by 55.8% in the Ca-pretreated group. However, increasing the competitive strength of Ca2+ and Cd2+ did not affect Cd2+ influx under normal Ca conditions (>2 mM Ca). Meanwhile, Ca pretreatment significantly increased Cd distribution in the root cell wall, pectate, and protein-binding forms, while significantly reducing Cd distribution in root soluble components and inorganic Cd forms. The pectin content in the roots increased by 128% and 226% in the Ca and Cd simultaneous exposure treatment and Ca pretreatment, respectively. These results suggest that Ca pretreatment enhanced Cd retention in the root cell wall. Overall, exogenous Ca effectively mitigated Cd accumulation in peanut plants when the CRCa/Cd was below 2000, and Ca2+ channels partially facilitate the entry of Cd2+ into peanut roots. Under normal Ca supply conditions, exogenous Ca reduced Cd accumulation in peanuts primarily through root cell wall fixation rather than ion channel competition. Our findings provide insights into the mechanism by which Ca alleviates the uptake and transfer of Cd in peanuts.

Nitrogen reduces calcium availability by promoting oxalate biosynthesis in apple leaves.

N and Ca are essential nutrients for apple growth and development. Studies have found that Ca content was not low under high N conditions but was poorly available. However, the underlying physiological mechanism through which N regulates Ca availability remains unclear. In this study, apple plants were supplied with N and Ca to analyse the content, in situ distribution, and forms of Ca using noninvasive micro-test technique, electron probe microanalysis, Fourier transform infrared spectroscopy, and transcriptome analysis. A potential interaction was observed between N and Ca in apple leaves. The application of high N and Ca concentration led to a CaOx content of 12.51g/kg, representing 93.54% of the total Ca in the apple leaves. Electron probe microanalysis revealed that Ca deposited in the phloem primarily existed as CaOx rhombus-shaped crystals. Additionally, high N positively regulated oxalate accumulation in the leaves, increasing it by 40.79 times compared with low N concentration. Specifically, N induced oxalate synthesis in apple leaves by upregulating the MdICL, MdOXAC, and MdMDH genes, while simultaneously inhibiting degradation through downregulation of the MdAAE3 gene. Transcriptome and correlation analyses further confirmed oxaloacetate as the precursor for the synthesis of CaOx crystals in the apple leaves, which were produced via the 'photosynthesis/glycolysis -oxaloacetate -oxalate -CaOx' pathway. WGCNA identified potential regulators of the CaOx biosynthesis pathway triggered by N. Overall, the results provide insights into the regulation of Ca availability by N in apple leaves and support the development of Ca efficient cultivation technique.

Carbon Nanoparticles Promoted the Absorption of Potassium Ions by Tobacco Roots via Regulation of K+ Flux and Ion Channel Gene Expression

Background: The regulatory effects of carbon nanomaterials (CNMs) on plant growth and their potential applications in agriculture have attracted a great deal of attention from researchers. CNMs have been shown to promote nutrient absorption and increase plant growth. However, the mechanisms by which CNMs affect plant growth and nutrient absorption are still unknown. Methods: The tobacco seedling biomass, potassium (K+) concentration, and accumulation in hydroponic were investigated to exposure of carbon nanoparticles (CNPs). To directly observe the effect of CNPs on K+ uptake by roots, we employed a noninvasive micro-test technique (NMT) to detect the net flux of K+ on the surface of tobacco roots. The K+-depletion experiment was carried out to explore the kinetic characteristics of K+ absorption, and qRT-PCR was used to monitor the expression levels of the K+ channel gene. Results: The results showed that tobacco seedling biomass significantly improved at 10 mg·L-1 CNP treatments, and K+ concentration and accumulation both in roots and shoots increased with 10 and 20 mg·L-1 CNPs. CNP treatments changed the flow rate of K+ from efflux to influx in tobacco roots; this was observed both in plants cultivated in a CNP-containing medium and after the addition of CNPs to previously untreated plants. A depletion test also showed that CNPs improved the K+ absorption capacity and low-K+ tolerance of tobacco seedlings. Conclusion: CNPs enhanced the K+ absorption capacity and low-K+ tolerance of tobacco seedlings. The promotion of K+ absorption by CNPs was closely related to the activation of K+ influx channel genes and inhibition of the K+ outflow channel gene. The K+ flux response and ion channel gene expression to CNPs in plants reveal the mechanism whereby CNPs promote plant nutrient absorption.

Early signaling enhance heat tolerance in Arabidopsis through modulating jasmonic acid synthesis mediated by HSFA2

Given the detrimental impact of global warming on crop production, it is particularly important to understand how plants respond and adapt to higher temperatures. Using the non-invasive micro-test technique and laser confocal microscopy, we found that the cascade process of early signals (K+, H2O2, H+, and Ca2+) ultimately resulted in an increase in the cytoplasmic Ca2+ concentration when Arabidopsis was exposed to heat stress. Quantitative real-time PCR demonstrated that heat stress significantly up-regulated the expression of CAM1, CAM3 and HSFA2; however, after CAM1 and CAM3 mutation, the upregulation of HSFA2 was reduced. In addition, heat stress affected the expression of LOX3 and OPR3, which was not observed when HSFA2 was mutated. Luciferase reporter gene expression assay and electrophoretic mobility shift assay showed that HSFA2 regulated the expression of both genes. Determination of jasmonic acid (JA) content showed that JA synthesis was promoted by heat stress, but was damaged when HSFA2 and OPR3 were mutated. Finally, physiological experiments showed that JA reduced the relative electrical conductivity of leaves, enhanced chlorophyll content and relative water content, and improved the survival rate of Arabidopsis under heat stress. Together, our results reveal a new pathway for Arabidopsis to sense and transmit heat signals; HSFA2 is involved in the JA synthesis, which can act as a defensive compound improving Arabidopsis heat tolerance.

Photosynthesis promotion mechanisms of artificial humic acid depend on plant types: A hydroponic study on C3 and C4 plants

It is feasible to improve plant photosynthesis to address the global climate goals of carbon neutrality. The application of artificial humic acid (AHA) is a promising approach to promote plant photosynthesis, however, the associated mechanisms for C3 and C4 plants are still unclear. In this study, the real-time chlorophyll synthesis and microscopic physiological changes in plant leave cells with the application of AHA were first revealed using the real-time chlorophyll fluorescence parameters and Non-invasive Micro-test Technique. The transcriptomics suggested that the AHA application up-regulated the genes in photosynthesis, especially related to chlorophyll synthesis and light energy capture, in maize and the genes in photosynthetic vitality and carbohydrate metabolic process in lettuce. Structural equation model suggested that the photodegradable substances and growth hormones in AHA directly contributes to photosynthesis of C4 plants (0.37). AHA indirectly promotes the photosynthesis in the C4 plants by upregulating functional genes (e.g., Mg-CHLI and Chlorophyllase) involved in light capture and transformation (0.96). In contrast, AHA mainly indirectly promotes C3 plants photosynthesis by increasing chlorophyll synthesis, and the Rubisco activity and the ZmRbcS expression in the dark reaction of lettuce (0.55). In addition, Mg2+ transfer and flux in C3 plant leaves was significantly improved by AHA, indirectly contributes to plant photosynthesis (0.24). Finally, the AHA increased the net photosynthetic rate of maize by 46.50 % and that of lettuce by 88.00 %. Application of the nutrients- and hormone-rich AHA improves plant growth and photosynthesis even better than traditional Hoagland solution. The revelation of the different photosynthetic promotion mechanisms on C3 and C4 plant in this work guides the synthesis and efficient application of AHA in green agriculture and will propose the development of AHA technology to against climate change resulting from CO2 emissions in near future.

Selenite reduced cadmium uptake, interfered signal transduction of endogenous phytohormones, and stimulated secretion of tartaric acid based on a combined analysis of non-invasive micro-test technique, transcriptome and metabolome

Selenium (Se) can reduce uptake and translocation of cadmium (Cd) in plants via plenty of ways, including regulation of root morphology. However, the underlying mechanisms on how Se will regulate root morphology under metal(loid) stresses are not fully illustrated. To fill up this knowledge gap, we investigated the effects of 0.5 mg L−1 selenite (Se(IV)) on root exudates, root morphology, root endogenous hormones, and Cd uptake efficiency of rice under the 1 mg L−1 Cd stress condition. The results showed that Se(IV) significantly reduced shoot and root Cd concentrations, and decreased Cd uptake efficiency via root hairs determined by a non–invasive micro-test (NMT) technology. When compared to the 1 mg L−1 Cd (Cd1) treatment, addition of 0.5 mg L−1 Se(IV) (1) significantly reduced root surface area and tip numbers, and non–significantly reduced root length, but significantly enhanced root diameter and root volume; (2) significantly enhanced concentrations of tartaric acid in the root exudate solution, root auxin (IAA) and root jasmonic acid (JA) via a UHPLC or a HPLC analysis; (3) significantly up-regulated metabolites correlated with synthesis of IAA, JA, gibberellin (GA), and salicylic acid, such as GA53, M–SA, (+/−)7–epi–JA, and derivatives of tryptophan and indole in the metabolome analysis. However, results of transcriptome analysis showed that (1) no upregulated differentially expressed genes (DEGs) were enriched in IAA synthesis; (2) some upregulated DEGs were found to be enriched in JA and GA53 synthesis pathways. In summary, although Se(IV) stimulated the synthesis of IAA, JA, and GA53, it significantly inhibited root growth mainly by 1) affecting signal transduction of IAA and GA; 2) altering IAA polar transport and homeostasis; and 3) regulating DEGs including SAUR32, SAUR36, SAUR76, OsSub33, OsEXPA8, OsEXPA18, and Os6bglu24.

The aquaporin MePIP2;7 improves MeMGT9-mediated Mg2+ acquisition in cassava.

Aquaporins are important transmembrane water transport proteins that transport water and several neutral molecules. However, how aquaporins are involved in the synergistic transport of Mg2+ and water remains poorly understood. Here, we found that the cassava aquaporin MePIP2;7 was involved in Mg2+ transport through interaction with MeMGT9, a lower affinity magnesium transporter protein. Knockdown of MePIP2;7 in cassava led to magnesium deficiency in basal mature leaves with chlorosis and necrotic spots on their edges and starch over-accumulation. Mg2+ content was significantly decreased in leaves and roots of MePIP2;7-RNAi plants grown in both field and hydroponic solution with Mg2+ -free. Xenopus oocyte injection analysis verified that MePIP2;7 possessed the ability to transport water only and MeMGT9 was responsible for Mg2+ efflux. More importantly, MePIP2;7 improved the transportability of Mg2+ via MeMGT9 as verified using the CM66 mutant complementation assay and Xenopus oocytes expressing system. Yeast-two-hybrid, BiFC, co-localization and Co-IP assays demonstrated the direct protein-protein interaction between MePIP2;7 and MeMGT9 in vivo. Mg2+ flux was significantly elevated in MePIP2;7-overexpressing lines in hydroponic solution through Non-invasive Micro-test Technique analysis. Under Mg2+ -free condition, the retarded growth of MePIP2;7-RNAi transgenic plants could be recovered with Mg2+ supplementation. Taken together, our results demonstrated the synergistic effect of the MePIP2;7 and MeMGT9 interaction in regulating water and Mg2+ absorption and transport in cassava. This article is protected by copyright. All rights reserved.

Cellular Cd2+ fluxes in roots confirm increased Cd availability to rice (Oryza sativa L.) induced by soil acidifications

Soil acidifications become one of the main causes restricting the sustainable development of agriculture and causing issues of agricultural product safety. In order to explore the effect of different acidification on soil cadmium (Cd) availability, soil pot culture and hydroponic (soil potting solution extraction) were applied, and non-invasive micro-test technique (NMT) was combined. Here three different soil acidification processes were simulated, including direct acidification by adding sulfuric acid (AP1), acid rain acidification (AP2) by adding artificial simulated acid rain and excessive fertilization acidification by adding (NH4)2SO4 (AP3). The results showed that for direct acidification (AP1), DTPA-Cd concentration in field soils in Liaoning (S1) and Zhejiang (S2) increased by 0.167 - 0.217 mg/kg and 0.181 - 0.346 mg/kg, respectively, compared with control group. When soil pH decreased by 0.45 units in S1, the Cd content of rice stems, leaves and roots increased by 0.48 to 6.04 mg/kg and 2.58 to 12.84 mg/kg, respectively, When the pH value of soil S1 and S2 decreased by 0.20 units, the average velocity of Cd2+ at 200 µm increased by 10.03 - 33.11 pmol/cm2/sec and 21.33 -52.86 pmol/cm2/sec, respectively, and followed the order of AP3 > AP2 > AP1. In summary, different acidification measures would improve the effectiveness of Cd, under the same pH reduction condition, fertilization acidification increased Cd availability most significantly.

Silicon facilitated the physical barrier and adsorption of cadmium of iron plaque by changing the biochemical composition to reduce cadmium absorption of rice roots

Silicon effectively inhibits cadmium (Cd) uptake in rice, iron plaque on root surface was the primary link and first interface of Cd entering into rice root. To elucidate the mechanism of iron plaque under silicon treatment on root Cd uptake, the morphological characteristics of iron plaque, mechanisms of Cd adsorption of iron plaque and effect of iron plaque on Cd uptake by rice roots of Yuzhenxiang (YZX) and Xiangwanxian (XWX) rice varieties were studied by employing energy spectrum analysis technique, non-invasive micro-test technique, and isothermal-kinetic adsorption method. Scanning electron microscopy-X-ray energy dispersive (SEM-EDX) analysis showed that denser crystal structure of iron plaque was observed at Si treatment, silicon promoted the thickening of iron plaque and strengthened the isolation of iron plaque to Cd, which reduced the Cd content of white roots of YZX and XWX varieties by 30.2% and 20.9% respectively. However, the blocking effect of iron plaque on Cd was weakened under silicon treatment with iron plaque removed, Cd content in iron plaque of YZX and XWX cultivars was significantly decreased by 36.3% and 18.4%, Cd concentrations in white root and shoot was significantly increased, and the influxes of Cd2+ at elongation and maturation zone of root were increased in multiples. The results of adsorption test showed that the adsorption process of iron plaque was mainly a monolayer adsorption completed by boundary diffusion. The X-ray photoelectron spectroscopy (XPS) results demonstrated that silicon changed the biochemical composition of iron plaque and increased the density of the carbon-oxygen bound groups on iron plaque, which is the most likely reasons for the higher affinity of Cd adsorption ability of iron plaque observed in the silicon treated iron plaque. This study suggested the silicon-facilitated iron plaque have played critical effects in controlling the Cd accumulation in rice roots by changing the morphology and chemical composition of iron plaque.

TaBln1, a member of the Blufensin family, negatively regulates wheat resistance to stripe rust by reducing Ca2+ influx.

Blufensin1 (Bln1) has been identified as a susceptibility factor of basal defense mechanisms which is unique to the cereal grain crops barley (Hordeum vulgare), wheat (Triticum aestivum), rice (Oryza sativa), and rye (Secale cereale). However, the molecular mechanisms through which Bln1 regulates the wheat immune response are poorly understood. In this study, we found that TaBln1 was significantly induced by Puccinia striiformis f. sp. tritici (Pst) virulent race CYR31 infection. Knockdown of TaBln1 expression by virus-induced gene silencing reduced Pst growth and development, and enhanced the host defense response. In addition, TaBln1 was found to physically interact with a calmodulin, TaCaM3, on the plasma membrane. Silencing TaCaM3 with virus-induced gene silencing increased fungal infection areas and sporulation and reduced wheat resistance to the Pst avirulent race CYR23 (incompatible interaction) and virulent race CYR31 (compatible interaction). Moreover, we found that the accumulation of TaCaM3 transcripts could be induced by treatment with chitin but not flg22. Silencing TaCaM3 decreased the calcium (Ca2+) influx induced by chitin, but silencing TaBln1 increased the Ca2+ influx in vivo using a noninvasive micro-test technique. Taken together, we identified the wheat susceptibility factor TaBln1, which interacts with TaCaM3 to impair Ca2+ influx and inhibit plant defenses.

Ammonium has stronger Cd detoxification ability than nitrate by reducing Cd influx and increasing Cd fixation in Solanum nigrum L.

Cadmium (Cd) is a harmful heavy metal that affects the growth and development of plants. Nitrogen (N) is an essential nutrient for plants, and appropriate N management can improve Cd tolerance. The aim of our study was to explore the effects of different forms of N on the molecular and physiological responses of the hyperaccumulator Solanum nigrum to Cd toxicity. Measurement of biomass, photosynthetic parameters, and Cd2+ fluxes using non-invasive micro-test technique, Cd fluorescent dying, biochemical methods and quantitative real-time PCR analysis were performed in our study. Our results showed that ammonium (NH4+) has stronger Cd detoxification ability than nitrate (NO3-), which are likely attributed to the following three reasons: (1) NH4+ decreased the influx and accumulation of Cd2+ by regulating the transcription of Cd transport-related genes; (2) the ameliorative effects of NH4+ were accompanied by the increased retention of Cd in the cell walls of roots; and (3) NH4+ up-regulated SnExp expression.

Physiological characteristics and miRNA sequencing of two root zones with contrasting ammonium assimilation patterns in Populus.

The net ammonium fluxes differ among the different root zones of Populus, but the physiological and microRNA regulatory mechanisms are unclear. To elucidate the physiological and miRNA regulatory mechanisms, we investigated the two root zones displaying significant differences in net NH4+ effluxes of P. × canescens. Populus plantlets were cultivated with 500μM NH4Cl for 10days. Six plants were randomly selected to determine the net NH4+ fluxes using a noninvasive microtest technique. High-throughput sequencing were used to determine the dynamic expression profile of miRNA among the different root zones of Populus. Net NH4+ efflux in zone I (from 0 to 40mm from the root apex) was - 19.64pmolcm-2s-1 and in zone II (from 40 to 80mm) it was - 43.96pmolcm-2s-1. The expression of eleven miRNAs was significantly upregulated, whereas fifteen miRNAs were downregulated. Moreover, eighty-eight target genes of the significantly differentially expressed miRNAs were identified in root zone II compared with zone I. Particularly, ptc-miR171a/b/e and their target, SCL6, were found to be important for the difference in net NH4+ effluxes in the two root zones. Moreover, the expression of the target of ptc-miR169d, NFYA3 was upregulated in root zone II compared with root zone I, contributing to increased NH4+ efflux and decreased NH4+ assimilation in root zone II. These results indicate that miRNAs regulate the expression levels of their target genes and thus play key roles in net NH4+ fluxes and NH4+ assimilation in different poplar root zones.

Foliar application of gibberellin inhibits the cadmium uptake and xylem transport in lettuce (Lactuca sativa L.)

Gibberellin (GA) has been shown to exert numerous beneficial physiological functions in the resistance of plant to various abiotic stresses, however, the mechanism of GA for the mitigation of Cd stress in vegetables has not been well established. This study was carried out to investigate the effect of GA on the growth and Cd uptake in lettuce plants. Result showed that 10 µmol/L GA treatment restored part of the root morphology and vigor of lettuce under Cd stress by increasing the total root length, surface area and total root tip number, which directly showed an increased biomass of lettuce. On the other hand, GA reduced Cd uptake by roots and Cd root-to-shoot translocation by down-regulated expression of IRT1, Nramp1, HMA2 and HMA4, which finally alleviated the toxic effects of Cd on lettuce leaves, as confirmed by Non-invasive micro-test Technique (NMT) results. Overall, these findings provide new insights into the mechanism of cadmium toxicity mitigation in vegetables by GA and provide a theoretical basis for the development of physiological blockers with gibberellin components.

Gibberellin biosynthesis inhibitor mepiquat chloride enhances root K+ uptake in cotton by modulating plasma membrane H+-ATPase.

Potassium deficiency causes severe losses in yield and quality in crops. Mepiquat chloride, a plant growth regulator, can increase K+ uptake in cotton (Gossypium hirsutum), but the underlying physiological mechanisms remain unclear. In this study, we used a non-invasive micro-test technique to measure K+ and H+ fluxes in the root apex with or without inhibitors of K+ channels, K+ transporters, non-selective cation channels, and plasma membrane H+-ATPases. We found that soaking seeds in mepiquat chloride solution increased the K+ influx mediated by K+ channels and reduced the K+ efflux mediated by non-selective cation channels in cotton seedlings. Mepiquat chloride also increased negative membrane potential (Em) and the activity of plasma membrane H+-ATPases in roots, due to higher levels of gene expression and protein accumulation of plasma membrane H+-ATPases as well as phosphorylation of H+-ATPase 11 (GhAHA11). Thus, plasma membrane hyperpolarization mediated by H+-ATPases was able to stimulate the activity of K+ channels in roots treated with mepiquat chloride. In addition, reduced K+ efflux under mepiquat chloride treatment was associated with reduced accumulation of H2O2 in roots. Our results provide important insights into the mechanisms of mepiquat chloride-induced K+ uptake in cotton and hence have the potential to help in improving K nutrition for enhancing cotton production.

How marine diatoms cope with metal challenge: Insights from the morphotype-dependent metal tolerance in Phaeodactylum tricornutum

Metal tolerance in marine diatoms vary between morphotypes, strains, and species due to their long-term adaptations to stochastic environments. The mechanisms underlying this highly variable trait remain a matter of interest in ecotoxicology. In this study, we used several cutting-edge techniques, including a non-invasive micro-test technique, atomic force microscopy, and X-ray photoelectron spectroscopy to examine cadmium (Cd) accumulation and tolerance in the three morphotypes of Phaeodactylum tricornutum. Subcellular Cd distribution, metal transporter expression, and glutathione and phytochelatin activity were also analyzed to characterize the morphology-dependent Cd homeostasis and detoxification. We found that the oval morphotype accumulated more Cd, but was also more Cd tolerant than the other morphotypes. The greater surface binding of Cd to the oval morphotype is attributable to its smaller spherical form, rougher cell surface, and lower surface potential. Moreover, the oval morphotype was less permeable to Cd ions and contained higher phytochelatin and glutathione levels, which explained its higher metal tolerance. Our study offers new explanations for diatom’s adaptations to changing environments that may contribute to its evolutionary success.

The flux rate of Ca2+ into embryo can be used to evaluate the vigour level of maize seeds

Seed vigour is an important trait and is often used to evaluate seed quality. A rapid and accurate evaluation of seed vigour is very important for agricultural production. Ca2+ is an important secondary messenger in plants, responding to various biotic and abiotic stimuli by Ca2+ flux into cytoplasm. To the best of our knowledge, however, no report has been published about seed vigour and Ca2+ influx. In this study, we used two hybrid maize (Zea mays L.) lines and their corresponding female parent lines as materials, and performed ageing and ‘ageing + priming’ treatments to obtain seeds with different vigour levels. After seeds were imbibed for 24 h, the intact seeds or embryos were used as materials for determining the Ca2+ influx rate using non-invasive micro-test technique (NMT). Results showed that, with the intact embryos as materials, the Ca2+ influx rate showed higher stability and higher values. Correlation analysis indicated that there was a significant, positive linear correlation between the shoot dry weight vigour index and Ca2+ flux rate into embryo. The results demonstrated that the Ca2+ influx rate can be used to evaluate the vigour levels of maize seeds.

ABA-dependent K+ flux is one of the important features of the drought response that distinguishes Catalpa from two different habitats

ABSTRACTAbscisic acid (ABA)-induced stomatal closure can improve drought tolerance in higher plants. However, the relationship between ABA-related ion flux and improved drought resistance in the roots of woody plants is unclear. To investigate this relationship, we employed a noninvasive micro-test technique (NMT) to detect potassium (K+) flux in Catalpa fargesii and C. fargesii f. duclouxii after treatment with polyethylene glycol (PEG) and ABA. PEG treatment slightly increased the free proline content in both Catalpa species. However, simultaneous treatment with ABA and PEG resulted in a large increase in free proline content. Treatment with PEG led to a significant increase in K+ efflux, and both ABA and tetraethylammonium (TEA, a K+ channel inhibitor) blocked this efflux under short-term (1 d) and long-term (7 d) drought conditions. Furthermore, we detected SKOR (stelar K+ outward-rectifying channel) gene expression in roots, and the results showed that PEG significantly increased SKOR expression in C. fargesii f. duclouxii, but SKOR expression was inhibited by ABA in Catalpa fargesii. These findings indicate that ABA improves drought tolerance by inhibiting K+ efflux in Catalpa, but distinct ABA response patterns exist. Drought-tolerant species have better potassium retention are dependent on ABA, and can accumulate more proline than other species. SKOR is also ABA-dependent and sensitive to ABA, and K+ flux is a target of the ABA-mediated drought response.

A Glycine max sodium/hydrogen exchanger enhances salt tolerance through maintaining higher Na+ efflux rate and K+/Na+ ratio in Arabidopsis

BackgroundSoybean (Glycine max (L.)) is one the most important oil-yielding cash crops. However, the soybean production has been seriously restricted by salinization. It is therefore crucial to identify salt tolerance-related genes and reveal molecular mechanisms underlying salt tolerance in soybean crops. A better understanding of how plants resist salt stress provides insights in improving existing soybean varieties as well as cultivating novel salt tolerant varieties. In this study, the biological function of GmNHX1, a NHX-like gene, and the molecular basis underlying GmNHX1-mediated salt stress resistance have been revealed.ResultsWe found that the transcription level of GmNHX1 was up-regulated under salt stress condition in soybean, reaching its peak at 24 h after salt treatment. By employing the virus-induced gene silencing technique (VIGS), we also found that soybean plants became more susceptible to salt stress after silencing GmNHX1 than wild-type and more silenced plants wilted than wild-type under salt treatment. Furthermore, Arabidopsis thaliana expressing GmNHX1 grew taller and generated more rosette leaves under salt stress condition compared to wild-type. Exogenous expression of GmNHX1 resulted in an increase of Na+ transportation to leaves along with a reduction of Na+ absorption in roots, and the consequent maintenance of a high K+/Na+ ratio under salt stress condition. GmNHX1-GFP-transformed onion bulb endothelium cells showed fluorescent pattern in which GFP fluorescence signals enriched in vacuolar membranes. Using the non-invasive micro-test technique (NMT), we found that the Na+ efflux rate of both wild-type and transformed plants after salt treatment were significantly higher than that of before salt treatment. Additionally, the Na+ efflux rate of transformed plants after salt treatment were significantly higher than that of wild-type. Meanwhile, the transcription levels of three osmotic stress-related genes, SKOR, SOS1 and AKT1 were all up-regulated in GmNHX1-expressing plants under salt stress condition.ConclusionVacuolar membrane-localized GmNHX1 enhances plant salt tolerance through maintaining a high K+/Na+ ratio along with inducing the expression of SKOR, SOS1 and AKT1. Our findings provide molecular insights on the roles of GmNHX1 and similar sodium/hydrogen exchangers in regulating salt tolerance.

Exploring the Potential of Overexpressed OsCIPK2 Rice as a Nitrogen Utilization Efficient Crop and Analysis of Its Associated Rhizo-Compartmental Microbial Communities.

Nitrogen (N) is one of the indispensable factors in rice growth and development. China holds a premier position in the production of rice and at the same time also faces higher N fertilizer costs along with serious damage to the environment. A better solution is much needed to address these issues, without disrupting the production of rice as an important cereal, while minimizing all the deleterious effects on the environment. Two isogenic lines Kitaake (WT) and its genetically modified line CIPK2 (RC), overexpressing the gene for Calcineurin B-like interacting protein kinase 2 (OsCIPK2) with better nitrogen use efficiency (NUE), were compared for their growth and development under low versus normal levels of N. NUE is a complex trait mainly related to a plant’s efficiency in extraction, assimilation, and recycling of N from soil. The microbial population was analyzed using high-throughput Illumina Miseq 16S rRNA sequencing and found that RC with CIPK2, specifically expressed in rice root, not only performed better without nitrogen fertilizer (LN) but also increased the diversity of bacterial communities in rice rhizosphere compartments (rhizosphere, rhizoplane, and endosphere). The relative abundance of beneficial bacteria phyla increased, which are known to promote the circulation and transformation of N in rhizosphere soil. To further explore the potential of RC regarding better performance under LN, the ion fluxes in root apical were detected by non-invasive micro-test technique (NMT). We found that RC can absorb more Ca2+ and NO3− under LN as compared to WT. Finally, compared to WT, RC plants exhibited better growth of root and shoot, and increased yield and N uptake under LN, whereas there was no significant difference in the growth of two rice lines under normal nitrogen (NN) treatment. We are able to get preliminary results, dealing with the OsCIPK2 overexpressed rice line, by studying the rice molecular, physiological, and chemical parameters related to NUE. The results laid the foundation for further research on N absorption and utilization in rice from the soil and the interaction with microbial communities.

High temperature and drought stress cause abscisic acid and reactive oxygen species accumulation and suppress seed germination growth in rice.

Seed germination is one of the most important biological processes in the life cycle of plants, and temperature and water are the two most critical environmental factors that influence seed germination. In the present study, we investigated the roles of the plant hormone abscisic acid (ABA) and reactive oxygen species (ROS) in high temperature (HT) and drought-induced inhibition of rice seed germination. HT and drought stress caused ABA accumulation in seeds and inhibited seed germination and seedling establishment. Quantitative real-time polymerase chain reaction analysis revealed that HT and drought stress induced the expression of OsNCED3, a key gene in ABA synthesis in rice seeds. In addition, ROS (O2•- and H2O2) and malondialdehyde contents were increased in germinating seeds under HT and drought stress. Moreover, we adopted the non-invasive micro-test technique to detect H2O2 and Ca2+ fluxes at the site of coleoptile emergence. HT and drought stress resulted in a H2O2 efflux, but only drought stress significantly induced Ca2+ influx. Antioxidant enzyme assays revealed that superoxide dismutase (SOD), peroxidase, catalase (CAT), and ascorbate peroxidase (APX) activity were reduced by HT and drought stress, consistent with the expression of OsCu/ZnSOD, OsCATc, and OsAPX2 during seed germination. Altogether, these results suggest that ABA and ROS accumulation under HT and drought conditions can inhibit rice seed germination and growth.