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Horticultural Research Volume 8, Article Number: 80 (2021) Cite this article

The aluminum (Al) cation Al3 in acid soil shows severe rhizome toxicity and inhibits plant growth and development. Most woody plants adapted to acid soil have evolved specific strategies for Al3 toxicity, but the underlying mechanism is still elusive. The four-carbon amino acid gamma-aminobutyric acid (GABA) has been well studied as an inhibitory neurotransmitter in mammals. GABA also controls many physiological responses during environmental or biological stress. The woody plant hybrid Liriodendron chinense (L. chinense × tulipifera) is widely planted as a horticultural tree in China and provides high-quality wood; studying its adaptation to high-aluminum stress is very important to utilize its ecological and economic potential. Here, we performed quantitative iTRAQ (Isobaric Labeling for Relative and Absolute Quantification) to study how protein expression changes in hybrid Liriodendron leaf exposed to aluminum stress. Hybrid Liriodendron showed differential accumulation of several proteins related to cell wall biosynthesis, sugar and proline metabolism, antioxidant activity, autophagy, protein ubiquitination degradation, and anion transport in response to aluminum damage. We have observed that aluminum stress up-regulates glutamate decarboxylase (GAD) and its activity, leading to increased GABA biosynthesis. The additional GABA synergistically increases the Al-induced antioxidant enzyme activity to effectively eliminate ROS, enhance proline biosynthesis, and up-regulate the expression of MATE1/2, and then promote the efflux of citrate to chelate Al3. We also show a similar effect of GABA on the enhanced Al3 tolerance in Arabidopsis. Therefore, our results indicate that GABA signaling plays a role in enhancing the tolerance of hybrid Liriodendron tulipifera to aluminum stress by promoting organic acid transport and maintaining cellular redox and osmotic balance.

Acidic soils often appear on the earth; about 60% of tropical and subtropical regions suffer from soil acidification, severely limiting crop yields. Environmental pollution and acid rain may also increase soil acidity. Root toxic Al3 ions become soluble in acidic soils with a pH below 5 and significantly inhibit root growth. Therefore, aluminum poisoning has become a serious agronomic problem that restricts crop yield; enhancing the resistance of crops and biofuels to aluminum will be an important strategy to improve their productivity1,2,3. The anti-aluminum mechanism of plants can be divided into efflux or internal detoxification according to whether it occurs inside or outside plant cells. Several mechanisms have been proposed to explain the external repulsion mode of aluminum resistors4. The most well-known strategy is the mechanism by which organic acid ions (including citrate, oxalate or malate) flow out from the root tip, which can then directly chelate external aluminum to prevent aluminum poisoning4,5,6. For most crop plants, such as rice and wheat, or model plants, such as Arabidopsis, aluminum toxicity can be a powerful growth deterrent; however, most forest trees exhibit high tolerance to aluminum stress. For example, Norway spruce (Picea abies) or birch (Betula pendula) can tolerate aluminum concentrations up to 3 mM in the soil; however, aluminum concentrations below 50 µM can significantly inhibit root elongation in Arabidopsis. Most woody plants grow naturally in acidic soils and have evolved specific mechanisms to deal with high aluminum stress8. Therefore, deciphering the underlying mechanisms of woody plants' tolerance to aluminum stress can help us understand the same processes in crops and promote the use of genetic engineering strategies to improve crop tolerance to aluminum.

Correspondingly, a series of transporter genes responsible for aluminum-activated malate or citric acid exudation have been reported. First, TaALM1 (a wheat aluminum-activated malate transporter) was identified as an aluminum-resistant malate anion efflux transporter9. In Arabidopsis and rape (Brassica napus), the homologue of TaALM1 was also identified as the Al3 enhanced malate transporter10,11. Through the map-based cloning method, another aluminum resistance gene family was identified in sorghum, called MATE (multidrug and toxic compound extrusion) transporter. Then several MATE orthologs that act as citrate transporters were isolated in Arabidopsis (AtMATE1), corn (ZmMATE), rice beans (VuMATE) and rice (OsFRD1)13. Recent evidence suggests that the abundance of these transporter families may also be transcriptionally regulated. Two zinc finger proteins, namely STOP1 from Arabidopsis and ART1 from rice, can directly regulate the aluminum-induced expression of ALMT1 and AtMATE14,15. In addition, WRKY transcription factor WRKY46 can bind to the ALMT1 promoter to inhibit its expression during aluminum stress, which may be part of a negative feedback loop. In addition to the use of organic acids to chelate aluminum, another effective aluminum elimination strategy is the absorption of aluminum by plant cell wall polysaccharides. In the case of high aluminum concentration in the environment, barley (Hordeum vulgare) may absorb about 85% of the outer aluminum into its root cell wall17, and the macroalga Chara coral can even absorb up to 99.9% of the total aluminum. Into its cell wall18.

The four-carbon amino acid gamma-aminobutyric acid (GABA) acts as an inhibitory neurotransmitter in animals19. In plants, it is found that GABA can regulate the response to various abiotic stresses (such as heat, cold, touch, or hypoxia) and biotic stresses (including herbivory, trauma, and pathogen infection). GABA signaling also regulates the balance of C:N or cytoplasmic p​​H20. GABA is biosynthesized from glutamate by the Ca2-calmodulin-related enzyme decarboxylase (GAD) in plants. GABA can also be degraded in mitochondria through a GABA shunt, which consists of two consecutive steps from the tricarboxylic acid cycle. In the GABA shunt cycle, GABA is converted to succinic semialdehyde by GABA transaminase (GABA-T), and then succinic semialdehyde dehydrogenase (SSADH) oxidizes succinic semialdehyde to succinic acid, and at the same time produces NADH 19,21. In Arabidopsis, salt stress induces the up-regulation of GAD and GAD2 transcription, resulting in high levels of GABA. Consistent with this, the pop2 mutant lacking GABA-T is very sensitive to ionic stress (such as salt stress) but not to osmotic stress22. It has been reported that GABA regulates malate transport plasma membrane channels 23, 24 during wheat aluminum stress, but it is necessary to study the mechanism by which GABA enhances the tolerance of plants to aluminum.

Liriodendron is a genus of Magnoliaceae, with two species, namely L. chinense and L. tulipifera. They are all widely planted horticultural trees in China and can produce high-quality wood25. Therefore, studying how to deal with abiotic stresses of Liriodendron tulipifera, such as aluminum toxicity, has important ecological and economic value. In this study, we used quantitative iTRAQ proteomics to study the response of hybrid Liriodendron tulipifera to aluminum stress. Using our existing L. chinense transcriptome data as a reference, we successfully isolated 198 proteins that showed significant differential expression after exposure to aluminum stress; this group includes energy metabolism, antioxidant activity, and defense response required protein. Among these proteins, we detected the up-regulation of putative GAD homologues after Al stress, accompanied by an increase in the accumulation of AlMT channel proteins. After aluminum stress, the content of malic acid and GAD-dependent GABA also increased; physiological analysis showed that inhibition of GABA biosynthesis increased, while the application of exogenous GABA weakened, and the vigor of hybrid Liriodendron chinensis mediated by aluminum toxicity was impaired. In addition, we found that the role of GABA in mediating Al stress resistance is conserved in both poplar and Arabidopsis. Therefore, our results indicate a new and conservative mechanism through which GABA enhances the tolerance of hybrid Liriodendron to aluminum stress through the outflow of malic acid that depends on the AlMT channel.

In this study, as previously reported, plantlets derived from the Chinese plum somatic embryogenesis system were used. In short, embryogenic callus is induced from immature seeds and grown on induction medium, and then the induced callus is transferred into liquid culture to promote its growth for 2 or 3 weeks. The embryogenic cells were transferred to solid medium for 4-5 weeks to induce seedling production. Under greenhouse conditions (relative humidity 50-70%, 25°C, 800 µmol photon m-2 s-1 of white light), transplant the regenerated seedlings into 0.5-L fine earthen pots. Each pot is watered with 300 mL nutrient solution, which contains KNO3 (1 mM), Ca(NO3)2 (1 mM), KH2PO4 (0.1 mM), MgSO4 (0.5 mM), H3BO3 (20 μM), MnCl2 (2 μM), ZnSO4 (2 μM), CuSO4 (0.5 μM), (NH4)6Mo7O24 (0.065 μM) and Fe-EDTA (20 μM), once every 5 days. The nutrient solution containing the specified AlCl3, GABA or AlCl3 GABA concentration is used for aluminum stress treatment.

A chlorophyll fluorometer (Heinz Walz GmbH, Effeltrich, Germany) 27 was used to collect plant leaves after different treatments for chlorophyll fluorescence intensity analysis. Each fallen leaf was placed in the dark for 30 minutes to adapt to the darkness. Then, the maximum quantum yield of PSII was monitored as Fv/Fm. The maximum fluorescence (Fm) is measured with 4000 µmol s-1 m-1 light and 0.8-s pulse. Each analysis is repeated at least 3 times.

Xylenol orange was used to determine the H2O2 content28. In short, hybrid Liriodendron leaf tissue (1 g) was collected after different treatments and homogenized in 5 mL HClO4 solution (0.2 M) in a cold room at 4 °C. After standing for 5 minutes, centrifuge at 10,000×g for 10 minutes, take the supernatant, add 100 μl of the supernatant to 1 mL of reaction buffer, and analyze the H2O2 content; the reaction is treated at room temperature for 1 hour. The H2O2 level is measured by calculating the absorbance at 560 nm based on the H2O2 standard curve.

The O2- content is determined according to the previously published method. The supernatant (100 μl) was incubated with 1 mL of a reaction mixture containing 50 mM potassium phosphate buffer (pH 7.0), 10 mM hydroxylamine hydrochloride, 17 mM p-aminobenzenesulfonic acid, and 7 mM α-naphthyl. Monitor the absorbance at 530 nm and measure the O2- level according to the O2- standard curve.

The thiobarbituric acid reactive substance (TBARS) method was used to analyze lipid peroxidation. The leaf samples of hybrid tulip tree (1 g) were quickly frozen in liquid nitrogen and homogenized with 10 mL extraction buffer containing 10% trichloroacetic acid (TCA). The extract was centrifuged at 10,000 × g for 20 minutes, and then the supernatant was used for further analysis. Add 1 mL of supernatant to 4 mL of reaction buffer containing 0.6% thiobarbituric acid and 20% TCA, and incubate the mixture at 95 °C for 30 min. Then, the reaction was stopped by rapid cooling in an ice bath. After the reaction was cooled to room temperature, the mixture was centrifuged at 10,000 × g for 10 minutes, and the absorbance of the supernatant was monitored at 532 and 600 nm. Measure the difference between the absorbance of the supernatant at 532 nm and 600 nm, and calculate the MDA level as described previously.

For quantitative RT-PCR analysis, TRIzol reagent (Chinese Tiangen) was used to extract the total RNA from hybrid Liriodendron leaf after different treatments. The first-strand cDNA synthesis and quantitative RT-PCR were performed using the previously reported method. The primers used for RT-qPCR are listed in Supplementary Table 2. For each sample, the statistical analysis was repeated using three separate biological experiments.

Collect leaf tissue (~1 g) for enzyme activity analysis. Different antioxidant enzyme activities were analyzed as described above, including ascorbate peroxidase (APX), glutathione reductase (GR), monodehydroascorbate reductase (MDHAR) and dehydroascorbate reductase (DHAR) ) Activity. The Bradford method was used to calculate the protein content.

The GABA content in the leaf extract was measured as described previously. In short, about 10 g of leaf samples were ground into powder in a Falcon tube and extracted with 10 mL of 80% (v/v) ethanol. Collect the extraction buffer and centrifuge at 1200 × g and 4°C for 10 minutes. Remove the supernatant and filter with Millipore filter paper. The filtration was repeated 3 times, and the filtrate was combined and dried on a rotary evaporator until the ethanol was completely evaporated. Then, the dried residue was dissolved in 1 mL of water, and 1 mL of methanol containing 2-hydroxynaphthaldehyde (2.5% w/v) was added to the dissolved GABA for derivatization. Then, add 0.5 mL of boric acid-NaOH (pH 8.5) to neutralize the solution. The resulting sample was dried at 85°C for 20 minutes and cooled to room temperature. The residue was dissolved in 5 mL methanol for further analysis. The Agilent 1200 HPLC instrument was used for HPLC analysis. Inject approximately 5 µl of the solution into a reversed-phase SB-C18 column with a methanol gradient system (60% methanol for 2 minutes, 70% methanol for 5 minutes, 80% methanol for 8 minutes, 90% methanol for 10 minutes) and 50% methanol for 12 minutes ), the flow rate is 0.8 ml/min. A 254 nm UV detector was used to monitor the GABA content. Analyze the GABA content by comparing with the retention time of the standard product.

GAD activity is calculated by measuring the conversion rate of the substrate glutamate to GABA22. About 1 g of plant leaf samples were extracted in 10 mL reaction buffer containing 80 mM sodium phosphate (pH 5.6) and 100 mM L-glutamic acid, and then kept at 40 °C for 60 minutes. Then, stop the reaction at 90°C. The reaction was cooled and centrifuged at 1200 × g for 10 minutes, and then the supernatant was taken for GAD enzyme activity analysis as described above.

The method 27 described previously was used to measure Pro accumulation in hybrid Liriodendron leaf, using l-Pro as the standard. In short, about 1 g of leaves are collected and extracted using 3% sulfosalicylic acid in a cold room. The supernatant was obtained after centrifugation at 12,000 × g for 10 minutes at 4°C. Take a portion (2 mL) of the supernatant and ninhydrin solution, which contains 2.5% [w/v] ninhydrin, 40% 6 M phosphoric acid and 60% [v/v] glacial acetic acid, at 100 °C React for 30 minutes, then add ice to quickly cool to terminate the reaction. Then, 5 mL of toluene was added, and the solution was incubated overnight at room temperature. The proline content was monitored by measuring the absorbance at 520 nm using a spectrophotometer.

The RRG value was determined as previously reported2. The seed surface was disinfected and sown in Murashige and Skoog (MS) medium under 60 µmol photon m-2 s-1 light for 3 days, and the initial root length was measured. Then, half of the seedlings were transferred to MS medium containing different concentrations of AlCl3, while the remaining half of the seedlings continued to grow on the same MS medium without Al stress. After growing for 5 days under the same light conditions, the root length was monitored with a ruler, and the degree of inhibition of root elongation was expressed as the percentage of RRG.

As mentioned earlier, total leaf protein was extracted from frozen samples using phenol extraction buffer. In short, use a mortar and pestle to quickly freeze about 10 g of leaf tissue in liquid nitrogen and grind it, then in 10 mL of ice-cold protein extraction buffer (100 mM Tris-HCl buffer (pH 7.8), 100 mM KCl, 1% v/v Triton X-100, 1% v/v β-mercaptoethanol, 50 mM L-ascorbic acid, 1 mM benzyl sulfonyl fluoride). The supernatant was obtained by centrifugation at 12,000 × g for 10 minutes and mixed with an equal volume of Tris-phenol buffer (100 mM Tris, pH 8.0). Vortex the mixture thoroughly and collect the upper phenol phase after centrifugation at 12,000 × g for 30 minutes at 4°C. Finally, add 5 volumes of methanol containing 10 mM ammonium acetate to the upper phenol phase. The mixture was left overnight at -20 °C, and the protein pellet was obtained by centrifugation at 12,000 × g for 15 minutes. The wet particles were washed 3 times with cold acetone and 0.1% β-mercaptoethanol, and the washed particles were dried in the air. Store the dried pellets at -80 °C for further use or dissolve them in Tris-HCl buffer (pH 8.5, 40 mM) v/v) and PMSF (1 mM), the final concentration is 10 mg/mL, used for the next proteomics analysis. The protein solution was sonicated at 200 W for 15 minutes to promote dissolution, and then centrifuged at 12,000 × g for 15 minutes at 4°C. Discard the pellet and transfer the supernatant to another tube. Then, 10 mM DTT was added to the protein solution to avoid the formation of disulfide bonds. Add 55 mM iodoacetamide (IAM) to the protein solution in the dark to covalently block cysteine. Finally, protein precipitation was obtained by adding 5 volumes of cold acetone for 2 hours at -20 °C. The pellet was dried again, dissolved with 500 μl TEAB (tetraethylammonium bromide, 0.5 M), centrifuged at 12,000 × g for 15 min in a cold room, and the supernatant was collected for proteomics analysis. The Bradford method and Bio-Rad protein assay kit (Bio-Rad, USA) were used to quantify protein concentration.

The protein (100 µg) extracted from the leaves as described above is used for iTRAQ analysis, as shown below. In short, the extracted protein was first digested by Gold Trypsin (Promega, Madison, WI, USA) at a ratio of 30:1 (protein:trypsin) at room temperature for 16 hours, and then dried by vacuum centrifugation. Then follow the recommendations of the 8-fold iTRAQ reagent (AB Sciex Inc., MA, USA) to re-dissolve the dried peptide in TEAB buffer (0.5 M). The proteins extracted from different samples were digested as described above and labeled with iTRAQ reagent with isobaric labeling from 113 to 117 for 2 hours at room temperature. After labeling, the peptide mixture was dried by vacuum centrifugation and re-dissolved in 4 mL of strong cation exchange (SCX) solvent containing 25 mM NaH2PO4, 25% acetonitrile, and 10 mM ammonium formate (pH 2.7). A Shimadzu LC-20AB HPLC pump system (Shimadzu Co., Kyoto, Japan) was used to separate the peptide mixture through an Ultremex SCX column (4.6 × 250 mm). The peptide fraction was eluted with stable 5% buffer B containing 1 M KCl, 25 mM NaH2PO4, and 25% ACN at a flow rate of 1 mL min-1 at pH 2.7 for 7 minutes, followed by a linear gradient of 5-60 within 20 minutes Add% Buffer B, add 60–100% Buffer B within 2 minutes. Finally, 100% buffer B was used for elution. The absorbance at 214 nm was selected to monitor the elution peak, and a total of 20 fractions were collected. Each SCX fraction was desalted and re-dissolved in buffer C (5% acetonitrile and 0.1% formic acid), and centrifuged at 20,000 × g for 10 minutes to collect the supernatant. Finally, take 5 μL of the supernatant and use Shimadzu LC-20 AD nanoHPLC (Shimadzu Co. Kyoto, Japan) to perform HPLC-mass spectrometry analysis with a C18 column (inner diameter 200 μm). The peptides are eluted with 5% buffer D ( 95% ACN, 0.1% formic acid) for 5 minutes, then add 3-35% buffer D within 35 minutes, add 60-80% buffer D within 2 minutes, and finally wash with 80% buffer D at a flow rate of 250 Nanoliters/minute. Then the spray voltage of 2.5 kV, 30 psi N gas, 15 psi atomizer gas and heater temperature were 150 °C. The Orbitrap analyzer acquires a full-scan mass spectrum in information-related acquisition (IDA) mode with a mass range of 100-2400 m/z and a detection resolution of more than 30,000 (FWHM). The 30 strongest precursor ion peaks with a threshold exceeding 120 cps and a charge state of 2 to 5 were selected for collision-induced fragmentation. Use dynamic exclusion within 40 seconds to prevent repeated selection of peptides.

The original LC-MS/MS data files are converted into general Mascot files (mgf) by Proteome Discoverer 1.2 software, and searched using the Chinese Lactobacillus proteome database. Trypsin is used as a proteolytic enzyme, allowing one missed cut. The peptide mass and fragment mass tolerance values ​​are 10 ppm and 0.1 Da, respectively. Search parameters include iTRAQ 8 heavy quantification, oxidation of methionine residues, and pyroglutamate formation of N-terminal glutamine residues as variable residues and carbamoylmethyl formation of cysteine ​​residues as Fixed modification. Only peptides with a significant score exceeding 20, 99% confidence interval and false discovery rate (FDR) <1.5% were used for quantitative analysis, and the cutoff value was higher than 1.5 times or lower than 0.6 times and p value <0.05 was identified as Significantly differentially expressed proteins.

In order to understand the mechanism of the AlCl3 stress response of mixed Liriodendron chinense, we first aimed to optimize our experimental conditions. The photosynthesis efficiency of plants is a good way to assess the physiological health of plants to cope with various stresses. Therefore, by examining the dose-dependent response of the Fv/Fm ratio to increasing AlCl3 concentration, we determined how AlCl3 toxicity affects leaf photosynthesis. The Fv/Fm ratio is an effective indicator of leaf photosynthetic capacity. We found that treatment of hybrid Liriodendron with AlCl3 concentrations ranging from 5 to 50 μM gradually reduced the Fv/Fm ratio: once the AlCl3 concentration reached 100 μM, the Fv/Fm ratio dropped rapidly (Supplementary Figure 1). Based on our dose-response curve, we chose to use a 30 μM AlCl3 concentration for further experiments in this study.

The growth phenotype of hybrid Liriodendron in response to aluminum toxicity. The 1-week-old seedlings were treated with 30 μM AlCl3 for the specified time and photographs were taken. The experiment was carried out in triplicate, the results were similar, and a set of photos was shown. B AlCl3 inhibits leaf growth related parameters, including Fv/Fm, MDA, stomatal conduction and transpiration. One-week-old seedlings were treated with 30 μM AlCl3 for the specified time, and these growth-related parameters were measured. The experiment is carried out in triplicate, and the plotted value is the mean ± standard deviation of three biological replicates

Then, we further characterized the vigor/photosynthesis ability of hybrid Liriodendron leaf to 30 μM AlCl3. We found that after 7 days of treatment, the leaves of hybrid Liriodendron exposed to AlCl3 turned yellow, while the Fv/Fm ratio gradually decreased during this period, from ~0.803 to ~0.427. In the control plants, the Fv/Fm ratio remained stable at ~0.79 (Figure 1A, B). Lipid peroxidation is commonly used to assess the intensity of oxidative stress. Lipid peroxidation produces a variety of reactive aldehydes, such as malondialdehyde (MDA); therefore, the level of MDA is used to assess environmental oxidative stress. AlCl3 treatment also increased the level of MDA, reflecting the oxidative damage of membrane lipids. In addition, we found that leaf transpiration rate and stomatal conductance levels, which reflect additional parameters of photosynthetic activity, also decreased after AlCl3 treatment (Figure 1B). Therefore, these data indicate that AlCl3 has a negative impact on leaf vigor and photosynthetic capacity of Liriodendron hybrids.

Next, we aimed to identify genes related to aluminum stress response in hybrid Liriodendron chinense. To this end, we conducted a proteomic study using the iTRAQ proteomics method, which allowed us to analyze the differential protein abundance in hybrid Liriodendron leaf treated with 30 μM AlCl3 for 1, 3, 5, and 7 days, using Untreated plants are used as control. Three biological replicates are repeated for each sample used for iTRAQ analysis, and protein prediction and quantification are performed through Mascot software and public plant protein database and our own L. chinense transcriptome. Different abundance proteins are divided into three groups: group 1 (AlCl3 treatment for 1 day/untreated control), group 2 (AlCl3 treatment for 3 days/untreated control) and group 3 (AlCl3 treatment for 5 days/untreated control) ). Proteins with an abundance change of> 1.5 times are considered to have significantly increased expression; conversely, a protein with an abundance change of <0.6 times is considered to have significantly reduced expression. In total, we identified 198 proteins that showed significant changes after AlCl3 treatment (Figure 2, Supplementary Tables 1 and 2). The 198 identified proteins are divided into 8 groups according to their biological functions; most of the proteins belong to the largest group of material and energy metabolism, followed by the group of plant hormone signals and antioxidant proteins. Among the 198 differentially regulated proteins, 39 proteins with increased expression and 50 proteins with decreased expression were up-regulated or down-regulated at all time points, respectively.

The functional classification of proteins showing differential accumulation under AlCl3 stress. B Venn diagram analysis of differentially accumulated proteins after 30 μM AlCl3 stress; 1 day/control (1D/CK) means the number of differential proteins between AlCl3 treatment and control conditions for 1 day; 3 days/control (3D/CK) means AlCl3 treatment The number of different proteins after 3 days and the control condition; 5 days/control (5D/CK) means the number of difference proteins after 5 days AlCl3 treatment and the control condition; 7 days/control (7D/CK) means the difference protein number after AlCl3 treatment for 7 days and the control The number of different proteins compared under the conditions. C Heat map clustering of leaf protein abundance curves under AlCl3 stress. The 1-week-old hybrid Liriodendron seedlings of somatic embryos were treated with AlCl3 stress for 1, 3, and 7 days. Seedlings without AlCl 3 stress were used as controls. Compare the difference in protein abundance between treatment and control samples through iTRAQ. Different colors represent the difference in protein abundance ratio between treatment and control, as shown in the bar graph at the bottom of the figure.

We also performed a hierarchical cluster analysis to determine the differentially expressed proteins during aluminum stress. We noticed the accumulation level of antioxidant proteins, such as glutathione S-transferase (Lchi06330), monodehydrogenase reductase (Lchi27219), polyphenol oxidase (Lchi16057), peroxidase 17 (Lchi20649), Peroxidase 72 (Lchi10278Lchi1112alzyme) and), were up-regulated. Several proteins related to protein synthesis or stability, such as proteasome α type 1 B subunit (Lchi26499), plant UBX domain protein 4 (Lchi21723), aspartyl protease family protein (Lchi27243) and COP9 signal body Complex subunit 7 (Lchi31560) and proteins related to epigenetic regulation, such as histone H2A (Lchi09914), thioredoxin M type (Lchi12001), mRNA preprocessing protein 40 A (Lchi23330), DEAD-box ATP Dependent RNA helicase 53 (Lchi32037) and putative DNA repair protein RAD23-3 (Lchi24553) also accumulated differentially after aluminum stress. In addition, there are a series of transcription factors, such as zinc finger CCCH domain protein (Lchi14090, Lchi14538), WRKY protein (Lchi09560, Lchi03796, Lchi13688), bZIP transcription factor (Lchi10492, Lchi02202, Lchi09267 and other transcription factors), Lchi16241), Later it is also subject to different adjustments.

According to reports, organic acids can act as aluminum chelating agents when transported outside the cell, helping to avoid aluminum toxicity4. Consistent with this, we have found several proteins encoding malate transporter homologues, namely MATE1 (Lchi06125) and MATE2 (Lchi26133), which are up-regulated by aluminum stress, indicating that they may be involved in enhancing the effect of hybrid Liriodendron on aluminum stress. Tolerance.

In Arabidopsis, STOP1 belongs to the nuclear zinc finger protein family; it can directly activate the expression of AtALMT1 or MATE, and is involved in reducing the toxicity of H and Al3 rhizomes14. Our data also showed that zinc finger proteins homologous to STOP1 (Lchi25591) were up-regulated in response to aluminum stress, indicating that there may be a common mechanism between hybrid tulip tree and other plants. These results indicate that our proteomics data indeed accurately reflect the transformation response of hybrid Liriodendron tulipifera to aluminum stress.

In addition to the previous proteins involved in the aluminum stress response, we also detected changes in the expression levels of GAD and succinate semialdehyde dehydrogenase (SSADH) homologs, both of which are enzymes involved in the plant GABA biosynthetic pathway . GABA can be used as a signal molecule to participate in different physiological processes, including growth, development and defense responses. Our iTRAQ results showed that the abundance of two GAD homologs (Lchi33118 and Lchi05759) and one SSADH homolog (Lchi21261) was up-regulated (Supplementary Table 1) after 1 or 3 days of aluminum stress treatment, indicating GABA signaling The role of tulip tree in the hybridization stress response.

One-week-old hybrid Liriodendron seedlings of somatic embryos were subjected to 30 μM AlCl3 stress for a specified period of time, and then GABA content (A) and GAD activity (B) were measured. The experiment was performed in triplicate. Data represents the mean ± SD of three biological replicates

To test this hypothesis, we determined the GABA levels in the leaves of hybrid Liriodendron chinense after exposure to aluminum stress. 30 μM AlCl3 treatment induced a rapid increase in GABA content in hybrid Liriodendron chinense leaves, which reached 10.35 μmol g-1 FW after 24 hours of treatment, and maintained a high level during the next 36 hours of treatment (Figure 2). 3A). Based on these data, we also found that GAD enzyme activity (responsible for GABA biosynthesis) also increased after aluminum stress. The Al-induced GAD activity reached a peak after 48 hours of Al stress and remained high until AlCl3 treatment for 60 hours (Figure 3B). These data support our proteomics data, indicating that aluminum stress induces an increase in GAD protein abundance, and that GABA has a potential role in the aluminum stress response of hybrid Liriodendron chinense.

Our above iTRAQ results show that aluminum stress increases the expression of proteins encoding antioxidant enzymes, including catalase (Lchi14208), peroxidase (Lchi17145, Lchi20649, Lchi10272) and MDHAR (Lchi27219), which may be removing ROS. Play a role in aluminum, such as H2O2 and O2−, which may accumulate due to aluminum stress. To determine whether the aluminum stress response actually leads to ROS production, we measured aluminum-induced ROS accumulation, focusing on H2O2 and O2-. We found that applying aluminum stress for 3 days induces rapid accumulation of H2O2 and O2-, and pretreatment with exogenous GABA can alleviate aluminum-induced ROS generation (Figure 4A). These results indicate that GABA has the effect of scavenging ROS in hybrid Liriodendron, which is also consistent with previous studies in plants.

One-week-old hybrid Liriodendron seedlings of somatic embryos are subjected to AlCl3 stress or AlCl3 stress, and additional aminooxyacetic acid (AOA), vigabatrin (Vir), L-allylglycine (L-allyl) are added Or GABA for 3 days. Then, ROS content, including H2O2 and O2- (A), and antioxidant enzyme activities, including MDHAR, DHAR, APX, and GR activity (B) were measured. Control: Take the sample without aluminum stress as the control; 3 d: 30 μM AlCl3 stress for 3 days; AOA: 30 μM AlCl3 stress, add 1 mM aminooxyacetic acid for 3 days; Vir: 30 μM AlCl3 stress, add 100 μM ammonia Acrylic acid for 3 days; L-allyl: 30 μM AlCl3 stress, 1 mM L-allylglycine added for 3 days; GABA: 30 μM AlCl3 stress with 10 mM GABA added for 3 days. The experiment was performed in triplicate, and the data represents the mean ± SD of three biological replicates. Different letters indicate statistically significant differences determined by Tukey's multiple comparison test (p <0.05)

To confirm these results, we used chemical inhibitors specifically designed to change the intracellular GABA concentration. Aminoacetic acid (AOA) and vigabatrin are recognized GABA transaminase inhibitors, which can inhibit the conversion of GABA to SSDHA, leading to an increase in endogenous GABA content. L-allylglycine is a GADase inhibitor that can inhibit the production of GABA. Pretreatment with AOA or vigabatrin can counteract the accumulation of ROS induced by AlCl3 (Figure 4A) and enhance the activity of antioxidant enzymes. This is consistent with the increase in GABA levels, while L-allylglycine treatment increased AlCl3-induced ROS damage and decreased antioxidant enzyme activity (Figure 4B). Similar to AOA or vigabatrin, additional GABA attenuated the increase in AlCl3-induced antioxidant enzyme activity (Figure 4B), indicating that GABA signaling has a putative effect in protecting hybrid Liriodendron tulipifera from AlCl3-induced oxidative damage.

Proline promotes plant environmental stress response by changing the level of cell penetration. Proline 5-carboxylate synthase (P5CS) and proline 5-carboxylate reductase (P5CR) are two essential enzymes that catalyze the biosynthesis of proline in plants. We found that the protein levels of P5CS (Lchi29824) and P5CR (Lchi04198) were significantly up-regulated in hybrid Liriodendron chinense in response to aluminum stress, indicating that they may play a role in aluminum stress tolerance. To investigate this hypothesis, we monitored the proline levels of hybrid Liriodendron tulipifera subjected to aluminum stress. Starting from 3 days after treatment, aluminum stress induced a significant increase in proline levels, and then the levels slowly decreased again (Figure 5). Additional GABA treatment increased aluminum-induced proline accumulation, which was maintained after 3 to 5 days of aluminum stress. In addition, treatment with inhibitors AOA or vigabatrin or direct addition of GABA strongly increased AlCl3-induced proline biosynthesis, while additional L-allylglycine treatment reduced AlCl3-induced proline biosynthesis, indicating The new role of GABA in proline biosynthesis during aluminum stress (Figure 5).

One-week-old hybrid Liriodendron seedlings of somatic embryos were subjected to AlCl3 stress (3 days) or AlCl3 stress, and added AOA, vigabatrin (Vir), L-allylglycine or GABA for a specified time, and then proline was measured content. The experiment was performed in triplicate, and the data represents the mean ± SD of three biological replicates. Different letters indicate statistically significant differences determined by Tukey's multiple comparison test (p <0.05)

Aluminum treatment may trigger ALMT activity, leading to the extracellular transport of malic acid or citrate and the chelation of aluminum ions. Previous studies have found that ALMT is responsible for the secretion of malic acid, while MATE exports citrate in most plants, such as Arabidopsis, rice, and wheat35. We found that aluminum treatment did increase the accumulation of homologs of MATE1 (Lchi06125, named LchMATE1) and MATE2 (Lchi26133, named LchMATE2) in hybrid tulip tree (Supplementary Table 1 and Supplementary Figure 2), indicating that lemon in hybrid tulip tree The possibility of acid salt exudation may function aluminum stress tolerance. We next tried to confirm the up-regulation of LchMATE by measuring the extracellular citrate content of hybrid tulip tree in response to aluminum stress. AlCl3 treatment caused a strong increase in citrate, and this response could be further enhanced by additional GABA treatment (Figure 6A, B). The pretreatment of enzyme inhibitors AOA and vigabatrin similarly increased the exudation of citrate, while the treatment of L-allylglycine reduced the exudation of citrate, indicating that the GABA signal additionally controls the organic acid-mediated exudation. The aluminum chelation is a stress response. We also studied the changes of malic acid in hybrid Liriodendron chinense after AlCl3 or GABA treatment. Although AlCl3 treatment significantly increased the malate content, additional GABA treatment did not further increase the malate content (Supplementary Figure 3), indicating that malate metabolism is not the main way for GABA to resist AlCl3 stress.

The one-week-old hybrid Liriodendron seedlings of somatic embryos are subjected to AlCl3 stress or AlCl3 stress, and AOA, vigabatrin (Vir), L-allyl glycine (L-allyl) or GABA are added, followed by citric acid exudation and LchMATE1 /2 transcription was measured. The experiment was performed in triplicate, and the data represents the mean ± SD of three biological replicates. Different letters indicate statistically significant differences determined by Tukey's multiple comparison test (p <0.05). A The effect of GABA or different inhibitors on citrate exudation after 3 days of treatment. One-week-old hybrid Liriodendron seedlings of somatic embryos were added with different inhibitors or GABA under 30 μM AlCl3 stress or 30 μM AlCl3 stress, respectively, for 3 days, and the amount of citrate exudation was measured. B AlCl3 stress induces the transcription of LchMATE1/2. One-week-old hybrid Liriodendron seedlings of somatic embryos were subjected to 30 μM AlCl3 stress for a specified period of time, and the transcription level of LchMATE1/2 was measured by RT-qPCR analysis. The effect of C GABA concentration on the increase in transcription of LchMATE1/2 induced by Al. The 1-week-old somatic embryos hybridized with Liriodendron tulipifera seedlings were treated with 30 μM AlCl3 or 30 μM AlCl3 supplemented with different concentrations of GABA for 24 h, and the transcription level of LchMATE1/2 was measured by RT-qPCR analysis. Control: sample without Al stress; Al: 30 μM AlCl3 treatment; Al 5: 30 μM AlCl3 and additional 5 mM GABA; Al 10: 30 μM AlCl3 and additional 10 mM GABA; Al 50: 30 μM AlCl3 and additional 50 mM GABA; Al 100: 30 μM AlCl3 and additional 100 mM GABA. D. The effect of different inhibitors on the transcription increase of LchMATE1/2 induced by aluminum. The one-week-old hybrid Liriodendron seedlings of somatic embryos were subjected to 30 μM AlCl3 stress or 30 μM AlCl3 and added the above different inhibitors for 24 hours, and the transcription level of LchMATE1/2 was measured by RT-qPCR analysis

Next, we tried to determine whether the regulation of AlCl3 stress by LchMATE1 or LchMATE2 only occurs at the translation level, or whether it also involves transcriptional regulation. Through RT-qPCR experiments, we found that AlCl3 induces a dose-dependent transcription response in LchMATE1 or LchMATE2, and GABA synergistically further increases the transcription level of LchMATE1/2 induced by AlCl3 (Figure 6C). Consistent with these results, AOA and vigabatrin treatment also synergistically increased AlCl3-induced citrate exudation, while L-allylglycine treatment had the opposite effect (Figure 6D). There are two isomers of GABA in plants, namely α-aminobutyric acid (AABA) and β-aminobutyric acid (BABA). It has been shown that BABA can enhance the resistance of plants to diseases and abiotic stresses. Many studies have shown that there is a difference between BABA and GABA36,37. As an isomer of GABA, AABA has not been well studied. Hybrid Liriodendron was further processed with AlCl3, GABA, and AABA to determine whether the regulation of LchMATE1 or LchMATE2 was performed by this GABA analog. RT-qPCR data showed that the transcription of LchMATE1 or LchMATE2 could hardly be regulated by AABA in hybrid Liriodendron chinense (Supplementary Figure 4).

It was found that aluminum stress in hybrid Liriodendron chinense leads to an increase in GABA synthesis, which mediates downstream reactions such as ROS reduction and organic acid exudation. We investigated whether GABA can indeed protect hybrid Liriodendron from AlCl3 induced damage. We found that compared with the levels in control plants, exogenous GABA treatment or AOA and vigabatrin treatment did increase the photosynthetic Fv/Fm ratio, increase the chlorophyll level, and reduce the MDA level of hybrid Liriodendron chinense after AlCl3 exposure (Figure 7A), B). Consistent with this, L-allylglycine treatment had opposite effects on these parameters (Figure 7A, B).

One-week-old hybrid Liriodendron seedlings of somatic embryos were subjected to AlCl3 stress or AlCl3 stress with additional inhibitors as described above for 3 days. The leaf Fv/Fm, MDA content (A), total chlorophyll content (B) and relative root growth (C) were measured. The experiment was performed in triplicate, and the data represents the mean ± SD of three biological replicates. Different letters indicate statistically significant differences determined by Tukey's multiple comparison test (p <0.05)

Another parameter that reflects the tolerance of plant seedlings to AlCl3 stress is the average relative net root growth (RNRG), which compares the root length before and after AlCl3 treatment23. We again found that compared with the control plants, GABA, AOA or vigabatrin treatments increased the tolerance of roots to AlCl3 stress, while L-allylglycine decreased the tolerance (Figure 7C). These data indicate that GABA signaling increases tolerance to AlCl3 by protecting hybrid Liriodendron from the damaging effects of AlCl3 toxicity.

The leaf vigor/photosynthesis ability of hybrid Liriodendron chinense was further characterized by responding to 30 μM AlCl3 and 30 μM AlCl3, supplemented with 10 mM GABA. We found that after 7 days of treatment, the leaf vigor/photosynthetic capacity of hybrid tulip tree treated with AlCl3 GABA was better than that of the AlCl3 treatment group (Supplementary Figure 3). Although the Fv/Fm ratio of leaves treated with AlCl3 GABA decreased, it was still higher than that of leaves treated with AlCl3. AlCl3 GABA treatment also reduced the MDA content, indicating that GABA reduced the oxidative damage of membrane lipids. In addition, we found that compared with AlCl3 treatment, leaf transpiration rate and stomatal conductance level (an additional parameter reflecting photosynthetic activity) also increased after AlCl3 GABA treatment. Therefore, these data indicate that GABA AlCl3 actively helps the leaves of hybrid Liriodendron to resist aluminum stress.

So far, our results indicate that GABA enhances the tolerance of hybrid Liriodendron to aluminum stress, which is consistent with previous studies that showed that wheat varieties that are tolerant to aluminum contain high levels of GABA23,24. In the plant kingdom, GABA has a wide range of regulatory functions in growth, development, abiotic stress response and defense38,39. In addition, GABA can play a role in different kingdoms because plants, animals and fungi respond to GABA38,39,40. These results also indicate that GABA signaling, as a response to aluminum stress, is a more widely conserved pathway in higher plants. To investigate this, we turned to the model system Arabidopsis. The Arabidopsis GABA-T-deficient pop2-1 mutant showed higher endogenous GABA content, while the Arabidopsis gad1/2 double mutant contained low levels of GABA22. Therefore, we placed wild-type Col, pop2, and gad1/2 double mutant seedlings under AlCl3 stress, and then measured their physiological responses. We first confirmed that in Arabidopsis, compared with unstressed seedlings, Al stress also reduced the Fv/Fm ratio and total chlorophyll content, and increased the MDA content (Figure 8A, B). Consistent with our hypothesis, the pop2-1 mutant with high endogenous GABA content exhibits reduced aluminum toxicity because it shows higher Fv/Fm values ​​and total chlorophyll content, lower MDA levels, and increased RNRG (Figure 8C). In contrast, the gad1/2 mutant with lower GABA levels showed lower Fv/Fm values ​​and total chlorophyll content, higher MDA levels, and lower RNRG. These findings indicate the key role of GABA in enhancing the tolerance of plants to aluminum stress; in addition, this function may be conserved in a wide range of plant species.

The role of GABA in AlCl3 stress signaling is conserved in Arabidopsis. The experiment was performed in triplicate, and the data represents the mean ± SD of three biological replicates. Different letters indicate statistically significant differences determined by Tukey's multiple comparison test (p <0.05). A, B The effect of AlCl3 on leaf photosynthesis-related Fv/Fm, MDA content and total chlorophyll content in wild-type Col and gad1/2 and pop2 mutants. One-week-old seedlings were treated with 30 μM AlCl3 for 3 days, and the Fv/Fm ratio, MDA content (A), and total chlorophyll content (B) were measured. The effect of C AlCl3, GABA and GABA biosynthesis inhibitor treatment on relative root elongation. One week old seedlings were treated with 30 μM AlCl3 or 30 μM AlCl3 and the different additional chemicals mentioned above for one week, and the relative root elongation was measured

Most woody plants grow on acid soils and show tolerance to aluminum stress, but the mechanism of this tolerance is still unclear. To solve this problem, we used hybrid tulip tree, a Chinese tulip tree that has been sequenced and developed as a woody plant model system in our laboratory because of its ecological and economic value for wood cultivation25. We chose to use proteomics methods to identify genetic pathways involved in aluminum stress resistance. We have found that multiple pathways are responsive to aluminum toxicity, and have detected changes in the biosynthetic pathways of auxin, BR and GA. We also detected changes in transcription factors, such as WRKY and alkaline HLH, which are related to aluminum stress tolerance in Arabidopsis; for example, Arabidopsis WRKY46 plays a role in aluminum tolerance16. Proteins involved in protein degradation and methylation status also regulate aluminum resistance in Arabidopsis 41,42. Based on these findings, we suggest that the hybrid tulip tree adopt a variety of strategies to control its aluminum stress response.

More specifically, we found that several metabolic enzymes involved in the synthesis of signal molecule GABA, including GAD (Lchi33118, Lchi05759 and succinate semialdehyde dehydrogenase (Lchi21261)), have increased expression levels under aluminum stress. Accumulated evidence indicates that GABA has physiological functions in plant growth, development and defense responses19,20,21. Consistent with this finding, we found that aluminum stress did induce an increase in GAD1 activity, and the GABA produced by GAD1 also increased after aluminum stress. Inhibition of GAD1 enzyme significantly reduced aluminum-induced GABA production by using the specific inhibitor L-allylglycine activity, supporting the hypothesis that aluminum-induced GABA biosynthesis depends on GAD activity. In plants, GABA is dynamically degraded by GABA-Tase VOA and Vir. We also processed hybrid VOA or Vir Liriodendron seedlings, and found that this treatment further enhanced Al-induced GABA biosynthetic enzyme activity and impaired GABA degradation by inhibiting GABA-T. The important role of GABA shunt in preventing ROS generation and cell death during ultraviolet or heat stress has been reported. Here, our iTRAQ data shows that aluminum stress up-regulates the protein levels of several antioxidant enzymes, including CAT and SOD. After exposure to aluminum stress, rapid accumulation of ROS (mainly H2O2 and O2−) and an increase in antioxidant enzyme activity were observed. In addition, we found that supplementing GABA or enhancing endogenous GABA levels through AOA or Vir can enhance aluminum-induced antioxidant enzyme activity and reduce ROS accumulation. At the same time, inhibiting GAD1 enzyme activity by using L-allylglycine can damage GABA levels and reduce GABA levels. Antioxidant enzyme activity leads to an increase in the accumulation of ROS. A previous study showed that GABA shunt is involved in ROS metabolism during ultraviolet or salt stress. Our findings here are consistent with this finding and prove that GABA effectively increases antioxidant enzyme activity to scavenge ROS during aluminum stress. Our iTRAQ results also showed that aluminum stress induced protein accumulation in P5CS, which is a key enzyme for proline biosynthesis. Proline also accumulates in large quantities in plants under environmental stress. Environmental stress induces the production of proline to balance cell osmotic pressure 33, 34, 43, and proline has been shown to have multiple antioxidant functions44. Here, we found that aluminum stress induces proline biosynthesis. Additional GABA or GABA-Tase inhibitor treatment further increased aluminum-induced proline biosynthesis, while inhibition of GABA biosynthesis by L-allylglycine also reduced aluminum-induced proline biosynthesis. These data indicate that GABA also controls aluminum-induced proline biosynthesis, which can be used for multiple antioxidant reactions under aluminum stress. Unlike drought stress, when proline accumulates at high levels, it is considered not only an osmotic agent, but also a signaling molecule that can protect against oxidative damage.

Secreted organic acids, including malate and citrate, chelated aluminum plays a crucial role in the tolerance of plants to aluminum stress4. ALMT promotes the efflux of malic acid, while MATE promotes the efflux of citric acid10. Our iTRAQ results indicate that aluminum stress triggers the upregulation of MATE1 and MATE2. RT-qPCR results also showed that aluminum stress increased the transcription level of MATE1/2, indicating that MATE-mediated citrate may play a role in aluminum tolerance. Consistent with this finding, we found that aluminum stress did lead to an increase in citrate in the roots of hybrid Liriodendron chinense, and the citrate efflux capacity also increased after aluminum stress. These results indicate that citrate efflux chelation of aluminum enhances the tolerance of hybrid Liriodendron to aluminum stress. A previous study showed that GABA regulates ALMT for malic acid efflux. Here, we also found that additional GABA, AOA or Vir treatments increased the transcription of MATE1/2 and citrate efflux, which may explain why GABA signaling enhanced the tolerance of hybrid Liriodendron to aluminum stress. In contrast, inhibiting GABA biosynthesis by using L-allylglycine impairs aluminum-induced MATE1/2 transcription and reduces citric acid efflux. These data further support the critical role of GABA signaling in promoting aluminum tolerance in citrate efflux. In addition, we found that GABA signal also enhanced the aluminum-induced malic acid efflux. In wheat roots, aluminum-activated malate transport also promotes GABA transport; therefore, whether or how GABA signaling controls malate or citric acid outflow through ALMT or MATE1/2 needs further research.

In summary, our proteomics method uses quantitative iTRAQ technology to analyze the differential expression of proteins in hybrid Liriodendron chinense leaves subjected to aluminum stress, and finds that hybrid Liriodendron chinense may adopt a variety of strategies to enhance its tolerance to aluminum stress: Changes in metabolism, sugar and proline biosynthesis, differential transcription factor expression, autophagy, and ubiquitin-dependent protein degradation into anion transport. Through further research, we revealed the new role of the non-classical amino acid GABA, which not only controls the hybrid Liriodendron but also controls many aspects of the aluminum stress response in Arabidopsis. Therefore, our results help to further explain how plants resist aluminum toxicity. These findings should contribute to the widespread application of genetic engineering in hybrid tulip tree and other plants to improve their aluminum tolerance.

Kochian, LV, Pineros, MA & Hoekenga, OA. Physiology, genetics and molecular biology of aluminum resistance and toxicity in plants. Plant soil 274, 175–195 (2005).

Horst, WJ, Wang, Y. & Eticha, D. The role of root apoplasts in aluminum-induced inhibition of root elongation and aluminum resistance in plants: a review. install. robot. 106, 185–197 (2010).

CAS PubMed PubMed Central Article Google Scholar 

Thad, H. et al. Toxicity and tolerance of aluminum in plants: crop plants to adapt to acidic soils. Biometal 29, 187–210 (2016).

CAS PubMed article Google Scholar 

Kochian, LV, Pineros, MA, Liu, J. & Magalhaes, JV Plant adaptation to acidic soils: the molecular basis of crop aluminum resistance. Anu. Pastor plant biology. 66, 571–598 (2015).

CAS PubMed article Google Scholar 

Uexküll, HRV & Mutert, E. The global scope, development and economic impact of acid soils. Plant Soil 171, 1-15 (1995).

Matsumoto, H. Cell biology of aluminum toxicity and tolerance in higher plants. internationality. Priest cell. 200, 1–46 (2000).

CAS PubMed article Google Scholar 

Ahonen-Jonnarth, U., Goransson, A. & Finlay, RD Growth and nutrient absorption of ectomycorrhizal Pinus sylvestris seedlings in natural substrates treated with high concentrations of aluminum. Tree Physiology. 23, 157–167 (2003).

CAS PubMed article Google Scholar 

Grisel, N. et al. The transcriptome response of the roots of white poplar (Populus tremula) to aluminum stress. Bmc Plant Biology. 10, 185 https://doi.org/10.1186/1471-2229-10-185 (2010).

Sasaki, T. etc. Wheat gene encoding aluminum-activated malate transporter. Plant J.: Cellular Molecules. biology. 37, 645–653 (2004).

Hoekenga, OA, etc. AtALMT1 encodes a malate transporter and is identified as one of several genes essential for aluminum tolerance in Arabidopsis. Process National Academy of Sciences. science. United States 103, 9738–9743 (2006).

CAS PubMed article Google Scholar 

Ligaba, A., Katsuhara, M., Ryan, PR, Shibasaka, M. & Matsumoto, H. The BnALMT1 and BnALMT2 genes in rapeseed encode aluminum-activated malate transporters, which can enhance the aluminum resistance of plant cells. Plant Physiology. 142, 1294–1303 (2006).

CAS PubMed PubMed Central Article Google Scholar 

Magalhaes, JV, etc. A gene in the multi-drug and toxic compound extrusion (MATE) family confers aluminum tolerance in sorghum. Nat. Gene. 39, 1156–1161 (2007).

CAS PubMed article Google Scholar 

Upadhyay, N. etc. The multitasking capabilities of MATE transporters in plants. J. Experience. Botany, https://doi.org/10.1093/jxb/erz246 (2019).

Iuchi, S. etc. The zinc finger protein STOP1 is essential for proton tolerance in Arabidopsis and regulates key genes for aluminum tolerance. Process National Academy of Sciences. science. United States 104, 9900–9905 (2007).

PubMed article CAS PubMed Central Google Scholar 

Tsutsui, T., Yamaji, N. & Feng Ma, J. Identification of cis-acting elements of ART1, which is a C2H2 zinc finger transcription factor for aluminum tolerance in rice. Plant Physiology. 156, 925–931 (2011).

CAS PubMed PubMed Central Article Google Scholar 

Ding, ZJ, Yan, JY, Xu, XY, Li, GX & Zheng, SJ WRKY46 acts as a transcription repressor of ALMT1 and regulates the secretion of malate induced by aluminum in Arabidopsis. Plant J. 76, 825–835 (2013).

CAS PubMed article PubMed Central Google Scholar 

Wang, JP, Raman, H., Zhang, GP, Mendham, N. & Zhou, MX aluminum tolerance in barley (Hordeum vulgare L.): physiological mechanisms, genetics, and screening methods. J. Zhejiang University Science. B 7, 769–787 (2006).

CAS PubMed PubMed Central Article Google Scholar 

Taylor, GJ, etc. Direct measurement of the absorption and distribution of aluminum in Chara corallina single cells. Plant Physiology. 123, 987–996 (2000).

CAS PubMed PubMed Central Article Google Scholar 

Shelp, BJ, Bown, AW & McLean, MD Metabolism and function of γ-aminobutyric acid. Trends in plant science. 4, 446–452 (1999).

CAS PubMed article PubMed Central Google Scholar 

Kinnersley, AM & Turano, FJ Gamma Gamma Aminobutyric Acid (GABA) and Plant Responses to Stress. Crit. Pastor Plant Science. 19, 479–509 (2000).

Bouche, N. & Fromm, H. GABA in plants: just a metabolite? Trends in plant science. 9, 110–115 (2004).

CAS PubMed article Google Scholar 

Renault, H. et al. The Arabidopsis pop2-1 mutant revealed that GABA transaminase is involved in salt stress tolerance. Bmc Plant Biology. 10, 20 (2010).

PubMed PubMed Central article CAS Google Scholar 

Ramesh, SA etc. GABA signaling regulates plant growth by directly regulating the activity of plant-specific anion transporters. Nat. Community. 6, 7879 (2015).

CAS PubMed PubMed Central Article Google Scholar 

Ramesh, SA etc. Al-activated malate transporter can promote GABA transport ([OPEN]). Plant Cell 30, 1147–1164 (2018).

CAS PubMed PubMed Central Article Google Scholar 

Chen, J. et al. The genome of Liriodendron tulipifera reveals angiosperm phylogeny and species pair differentiation. Nat. Plant 5, 18-25 (2019).

CAS PubMed article Google Scholar 

Huo, A. et al. Establishment of a transient gene expression system in protoplasts of hybrid mesophyll cells of Liriodendron tulipifera. PLoS ONE 12, e0172475 (2017).

PubMed PubMed Central article CAS Google Scholar 

White, XG, etc. Interpretation of the protective effect of nitric oxide on salt stress at the physiological and proteome level of maize. J. Proteome research. 10, 4349–4364 (2011).

CAS PubMed article Google Scholar 

Hu, XY, Neil, SJ, Cai, WM & Tang, ZC Oligogalacturonic acid induces the expression of defense genes to increase the cytosolic calcium and hydrogen peroxide in Arabidopsis. Cell Resources 14, 234–240 (2004).

CAS PubMed article Google Scholar 

Elstner, EF & Heupel, A. Inhibition of the formation of nitrite from hydroxylammonium chloride: a simple determination of superoxide dismutase. anus. Biochemical. 70, 616–620 (1976).

CAS PubMed article Google Scholar 

Zhang, GJ & Bown, AW Rapid determination of γ-aminobutyric acid. Phytochemistry 44, 1007–1009 (1997).

Cheng, TL, etc. Quantitative proteomics analysis showed that S-nitrosoglutathione reductase (GSNOR) and nitric oxide signals enhanced poplar defenses against cold stress. Plant 242, 1361–1390 (2015).

CAS PubMed article Google Scholar 

Bouche, N., Fait, A., Bouchez, D., Moller, SG & Fromm, H. The mitochondrial succinate semialdehyde dehydrogenase shunted by γ-aminobutyric acid needs to limit the level of reactive oxygen intermediates in plants. Process National Academy of Sciences. science. United States 100, 6843-6848 (2003).

CAS PubMed article PubMed Central Google Scholar 

Szabados, L. & Savoure, A. Proline: a multifunctional amino acid. Trends in plant science. 15, 89-97 (2010).

CAS PubMed article PubMed Central Google Scholar 

Verslues, PE & Sharma, S. Proline metabolism and its effects on plant-environment interactions. Arabidopsis Volume 8, e0140 (2010).

PubMed PubMed Central article Google Scholar 

Liu, J., Magalhaes, JV, Shaff, J. & Kochian, LV Aluminum-activated citrate and malate transporters from the MATE and ALMT families function independently to confer aluminum tolerance in Arabidopsis. Plant J.: Cellular Molecules. biology. 57, 389–399 (2009).

Zimmerli, L., Jakab, G., Métraux, J.-P. & Mauch-Mani, B. Enhancement of β-aminobutyric acid on Arabidopsis pathogen-specific defense mechanisms​​. Process National Academy of Sciences. science. 97, 12920 (2000).

CAS PubMed article Google Scholar 

Cohen, Y. The story of BABA-induced resistance. Plant parasites 29, 375 (2001).

Seifikalhor, M., Aliniaeifard, S., Hassani, B., Niknam, V. & Lastochkina, O. Different roles of γ-aminobutyric acid in dynamic plant cell responses. Plant Cell Report 38, 847–867 (2019).

CAS PubMed article PubMed Central Google Scholar 

Ramesh, SA, Tyerman, SD, Gilliham, M. & Xu, B. Gamma-aminobutyric acid (GABA) signaling in plants. cell. Mole. life sciences. 74, 1577–1603 (2017).

CAS PubMed article PubMed Central Google Scholar 

Shelp, BJ, Bown, AW & Faure, D. Extracellular γ-aminobutyric acid mediates communication between plants and other organisms. Plant Physiology. 142, 1350 (2006).

CAS PubMed PubMed Central Article Google Scholar 

Geng, X. etc. LEUNIG_HOMOLOG transcriptional co-repressor mediates aluminum sensitivity through the methyl esterification of root cell wall pectin regulated by pectin methyl esterase 46 in Arabidopsis. Plant J.: Cellular Molecules. biology. 90, 491–504 (2017).

Zhang, Y. et al. The F-box protein RAE1 regulates the stability of the aluminum-resistant transcription factor STOP1 in Arabidopsis. Process National Academy of Sciences. science. United States 116, 319–327 (2019).

CAS PubMed article Google Scholar 

Khan, N., Bano, A., Rahman, MA, Rathinasabapathi, B. & Babar, MA Non-targeted metabolic analysis based on UPLC-HRMS revealed the changes in the metabolome of chickpea (Cicer arietinum) after long-term drought stress. Plant, cell environment. 42, 115–132 (2019).

Bose, J., Rodrigo-Moreno, A. and Shabala, S. ROS homeostasis of halophytes in the context of salt stress tolerance. J. Experience. robot. 65, 1241–1257 (2014).

CAS PubMed article Google Scholar 

This work was supported by the National Natural Science Foundation of China (32071784 and 31770715), the Natural Science Foundation of Jiangsu Province (BK20181176), the Qinglan Project of Jiangsu Province, and the Key Discipline Construction Project of Jiangsu Province.

The contributions of these authors are the same: Pengkai Wang, Yini Dong

Key Laboratory of Forest Genetics and Biotechnology of Ministry of Education, Nanjing Forestry University, Collaborative Innovation Center for Sustainable Forestry in South China, Nanjing, 210037

Pengkai Wang, Yini Dong, Liming Zhu, Zhaodong Hao, Lingfeng Hu, Jisen Shi, and Jinhui Chen

Suzhou Agricultural Vocational and Technical College, Suzhou, 215008

Shanghai Key Laboratory of Bioenergy Crops, School of Life Sciences, Shanghai University, Shanghai 200444

College of Forestry, Nanjing Forestry University, Nanjing 210037

College of Biology and Environment, Nanjing Forestry University, Nanjing 210037

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Wang, P., Dong, Y., Zhu, L. etc. The role of γ-aminobutyric acid in the tolerance of woody plant Liriodendron × tulip to aluminum stress. Horticulture Research 8, 80 (2021). https://doi.org/10.1038/s41438-021-00517-y

DOI: https://doi.org/10.1038/s41438-021-00517-y

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