AtSYP71 Is Important for Plant Cell Wall Biosynthesis and Stress Adaptation.


 Background: SYP71, the plant-specific Qc-SNARE protein, is reported to regulate vesicle trafficking. SYP71 is localized on the ER, endosome, plasma membrane and cell plate, suggesting its multiple functions. Lotus SYP71 is essential for symbiotic nitrogen fixation in nodules. AtSYP71, GmSYP71 and OsSYP71 are implicated in plant resistance to pathogenesis. To date, SYP71 regulatory role on plant development remain unclear.Results: AtSYP71-knockout mutant atsyp71-4 was lethal at early development stage. Early development of AtSYP71-knockdown mutant atsyp71-2 was delayed, and stress response was also affected. Confocal images revealed that protein secretion was blocked in atsyp71-2. Transcriptomic analysis indicated that metabolism, response to environmental stimuli pathways and apoplast components were influenced in atsyp71-2. Moreover, the contents of lignin, cellulose and flavonoids as well as cell wall structures were also altered.Conclusion: Our findings suggested that AtSYP71 is essential for plant development. AtSYP71 probably regulates plant development, metabolism and environmental adaptation by affecting cell wall homeostasis via mediating secretion of materials and regulators required for cell wall biosynthesis and dynamics.

to rice blast and tolerance to oxidative stress [108]. In wheat, TaSYP71 regulates resistance to biotrophic obligate fungi Pst [112]. Arabidopsis SYP71 can be subverted during TuMV infection to facilitate viral pathogenicity by mediating the fusion of the virus-induced 6K2 vesicles with chloroplasts [111]. However, SYP71 effect on cell wall synthesis is elusive.
Here, we report that AtSYP71 regulated secretion pathway. The secretion of the marker, SecGFP, was severely affected in atsyp71-2 mutant. Knock-down of AtSYP71 disturbed plant early development. Transcriptomic analysis indicated that metabolism, response to environmental stimuli and stresses pathways and cell wall components were in uenced in atsyp71-2 mutant. Moreover, the contents of lignin, cellulose and avonoids as well as cell wall structures were also altered. The response of atsyp71-2 to abiotic stress were abnormal. Our ndings suggested that AtSYP71 affected plant development, metabolism and environmental adaptation via regulation on vesicle tra cking.

Results
AtSYP71 is essential for plant growth and development AtSYP71 gene is composed of nine exons and eight introns (Fig. 1a). To investigate its function, four T-DNA insertion mutants were isolated. In atsyp71-1, T-DNA was inserted into 5'-UTR region. In atsyp71-2 and atsyp71-3, T-DNA cassettes were inserted in different sites in the rst intron. In atsyp71-4, T-DNA was inserted into the seventh intron. RT-qPCR determination indicated that AtSYP71 expression was downregulated by 30% in atsyp71-1, by 70% in atsyp71-2, by 40% in atsyp71-3, and was depleted in atsyp71-4 (Fig. 1b). To con rm AtSYP71 protein accumulation in the mutants, we generated anti-AtSYP71 antibodies and performed the western blot analysis. In atsyp71-1, AtSYP71 protein accumulation decreased slightly. In atsyp71-2 and atsyp71-3, AtSYP71 protein accumulation decreased dramatically. In atsyp71-4, there was no AtSYP71 protein accumulated (Fig. 1c). The root length of seedlings of all the atsyp71 mutants shortened. The greater the AtSYP71 expression decreased, the shorter the root length. In atsyp71-1, the root length of seven-day-old seedlings was slightly shorter than that of wild type, while the root length of atsyp71-2 and atsyp71-3 seedlings were signi cantly shorter than that of wild type (Fig. 1d, 1f). atsyp71-4 seeds could germinate, but the roots were not able to elongate, and the cotyledon margin sometimes was incomplete. In most cases, the true leaves could occur, but were very small, and soon turned yellow and vitri ed and stop developing, and most of the seedlings died when over seven-day old (Fig. 1e). atsyp71-1 plants had no obvious growth and development phenotype, whereas bolting of atsyp71-2 and atsyp71-3 plants was delayed, and the veweek-old plants were much shorter than those of wild type (Fig. 1g). However, there was no signi cant difference in the height of atsyp71 adult (60-day-old) plants (Fig. S1a), suggesting AtSYP71 was essential for morphogenesis and early development. The complemented lines of atsyp71-2 (atsyp71-2 com) and atsyp71-4 (atsyp71-4 com) were generated by introducing pAtSYP71::GFP-AtSYP71 cassette into atsyp71-2 or atsyp71-4 by crossing, respectively. The phenotypes of the complemented lines were restored (Fig. 1c, 1d, 1f, 1g), indicating that AtSYP71 was the causal gene of the phenotypes of atsyp71 mutants. The above results suggested that AtSYP71 plays an essential role in regulating plant growth and development. Since atsyp71-2 and atsyp71-3 had similar phenotypes, we used atsyp71-2 for subsequent analysis.
Secretion was disturbed in atsyp71-2 mutant It has been reported that AtSYP71 is localized on the ER, endosome, plasma membrane and cell plate [79,104,108,[112][113][114], suggesting its multiple functions. AtSYP71 associates with Qa-SNARE KNOLLE, Qb-SNARE NPSN11, and R-SNARE VAMP721 or VAMP722 to form a tetrameric endomembrane type SNARE complex to regulate cytokinesis [114]. But the AtSYP71-partners on the ER are not identi ed yet. Thus, we performed yeast two hybrid (Y2H) analysis and detected the interactions of AtSYP71 with the ERlocalized Qa-SNARE AtSYP81 and Qb-SNARE AtSec20, respectively (Fig. 2a). We reported previously that AtSYP81 and AtSec20 interact with ER-localized MAG2 which forms a tethering complex with MIP1, MIP2 and MIP3 [115]. Thus, we also detected interactions between these proteins and found that AtSYP71 interacted with MIP1, MIP2 and MIP3, respectively; AtSYP81 interacted with MAG2, MIP1, MIP2 and MIP3, respectively; and AtSec20 interacted with MAG2, MIP1 and MIP2, respectively (Fig. 2a). To further con rm the interaction between AtSYP71 and these factors, we performed pull down experiment using myc-AtSYP71-overexpressing (AtSYP71/OE) plants followed by Shotgun LC-MS/MS analysis. As expected, we identi ed MAG2, MIP1 and MIP3 (Fig. 2b). Then, we performed pull down analysis using seven-day-old seedlings of myc-AtSYP71/OE and GFP-AtSYP71/atsyp71-2 lines. Immunoblot analysis with anti-AtSYP81 antibodies detected AtSYP81 in both of the pull down elution (Fig. 2c). These results suggested that AtSYP71 might combine with AtSYP81 and AtSec20 to form a SNARE complex, and coordinate with the MAG2 tethering complex to mediate Golgi-to-ER vesicle transport.
To clarify effects of AtSYP71 down-regulation on vesicle tra cking, we analyzed the secretion using the marker SecGFP [116,117]. Confocal images indicated that in atsyp71-2 root cells, SecGFP displayed reticular-like structures (Fig. 2e, 2f, yellow arrows) which were not observed in wild type (Fig. 2d). This result suggested that AtSYP71 plays essential role on secretion pathway. Then, we detected accumulation of precursors of seed storage proteins (SSPs) which is considered as a marker for ER-tovacuolar transport [115,[118][119][120][121]. As shown in Fig. S1b, there was no detectable SSP precursors accumulated in atsyp71-2 seeds, indicating that the ER-to-vacuole pathway was not affected seriously. It is known that when ER export is severely blocked, numerous newly synthesized proteins are trapped inside the ER and induce ER stress [115,121,122]. We then determined the expression of BiP3 which is considered as an ER stress-speci c marker. The results indicated that BiP3 transcriptional level was not signi cantly altered in atsyp71-2 ( Fig. S1c), suggesting that no severe ER stress was induced, meaning the ER export was not affected seriously. These might because of the complementation from the two AtSYP71 homologues, AtSYP72 and AtSYP73. These results suggested that AtSYP71 probably regulates Golgi-to-ER retrograde pathway timely and spatially.

Transcriptome analysis of atsyp71-2 mutant
In order to explore the regulatory mechanism of AtSYP71 on plant growth and development, we performed transcriptome analysis of wild-type and atsyp71-2 seedlings. The sequencing obtained 34,524 and 33,113 reliable clean reads above 100 bp in wild type and atsyp71-2, respectively. After compared with tophat2 software, 87.68% and 86.59% of clean reads can be matched to the reference genome sequence, respectively. Statistical evaluation of sequencing quality value indicated that base Q30% (i.e. the proportion of sequencing error rate less than 0.1%) was 92.37% and 92.16%, respectively (Fig. 3a), illustrating the high quality of transcriptome sequencing and reliable original data for subsequent analysis. Volcano map showed the overall distribution of the differentially expressed genes (DEGs), and totally 165 DEGs were screened. Red dots represented 69 up-regulated DEGs, blue dots represented 96 down regulated DEGs, and green dots represented genes with no signi cant difference (Fig. 3b).
GO (Gene Ontology) enrichment analysis indicated that the DEGs were mainly concentrated in three aspects: biological process, cellular components and molecular function (Fig. 3c). The biological processes included 26 pathways mainly involved in biotic and abiotic stress, stimuli and defense responses, metabolism and secondary metabolism, and most of DEGs were enriched in stimuli and stress response processes. In cellular components, DEGs were mainly concentrated in cell wall and external encapsulating structure. And in molecular function, DEGs were mainly concentrated in tetrapyrrole binding and heme binding. KEGG (Kyoto Encyclopedia of Genes and Genomes) Enrichment analysis indicated that DEGs were mainly enriched in Biosynthesis of secondary metabolites (20 DEGs), Metabolic pathways (20 DEGs), Glucosinolate Biosynthesis (10 DEGs), 2-Oxocarboxylic acid metabolism (10 DEGs), and Phenylpropanoid biosynthesis (10 DEGs), etc. (Fig. 3d). Above results indicated that down regulation of AtSYP71 resulted in signi cant changes in plant stress response, defense, biosynthesis and metabolism.
The nine PRX genes were in the Extracellular region (GO:0005576) components (Fig. 4A, orange frame). Meanwhile, PRX15, PRX25, PRX49, PRX52 and PRX62 were also in the Cell wall (GO:0005618) components belonging to the External encapsulating structure (GO:0030312) (Fig. 4a, red frames); and PRX25 and PRX62 were in the Plant-type cell wall (GO:0009505) components (Fig. 4a, apricot yellow frame). PRX family proteins which belong to plant speci c Type III peroxidases (C Prxs) are involved in auxin metabolism, lignin and suberin biosynthesis, cross-linking of cell wall components, phytoalexin synthesis, and the metabolism of reactive oxygen species (ROS) and reactive nitrogen species (RNS) [123] and are closely related to cell wall dynamics such as cell wall loosening and strengthening [124].
The alteration of expression of the PRX genes might affect cell wall biosynthesis.
The cell wall components and structures were affected in atsyp71-2 mutant Since expression of many cell wall-and Phenylpropanoid biosynthesis pathway-related genes were altered, we determined the contents of cell wall components and avonoids in atsyp71-2 plants. Our results indicated that the contents of lignin, cellulose and avonoids in atsyp71-2 roots and stems were signi cantly lower than those in wild type (Fig. 5).
To gure out effects of decrease of lignin and cellulose on cell wall structure, we observed the caulome.
The stem cross sections showed that number of xylem and phloem in atsyp71-2 was less than that in wild type, and the size of each xylem and phloem was uneven and the distribution was irregular (Fig. 6a). The statistical results indicated that total area of xylem in atsyp71-2 was signi cantly less than that in wild type (Fig. 6c). Moreover, structure of xylem in atsyp71-2 was incomplete, and alignment of the cells was irregular (Fig. 6b, orange dotted frame). Moreover, in epidermis, cortex, phloem and medullary cells in atsyp71-2, less inclusions mainly composed of cytoplasm and cellulose were accumulated (ochre arrows) compared with that in wild type. Another remarkable difference was that the degree of ligni cation of cell walls of interfascicular ber and pith cells in atsyp71-2 stem decreased signi cantly (red and orange arrows). The statistical results indicated that the thickness of cell wall of interfascicular ber cells was signi cantly thinner than that of wild type (Fig. 6d). These component and structural changes of cell wall in atsyp71-2 might greatly affect the hardness and tenacity of stems and the cell functions, and subsequently affect the plant growth and development.

Stress Response of atsyp71-2 was altered
Since most of DEGs in transcriptome of atsyp71-2 mutant were related to environmental stimuli and stress responses, we rstly detected the activities of peroxidases. The activities of superoxide catalase (CAT) and peroxidase (POD) were signi cantly increased (Fig. 7a, 7b), and the activities of superoxide dismutase (SOD) and ascorbate peroxidase (APX) decreased signi cantly in atsyp71-2 (Fig. 7c, 7d). Then, we performed salt, drought and H 2 O 2 treatments. Under 120 mM NaCl treatment, root length of atsyp71-2 only reduced by 5% compared with 34% and 33% decline of that of wild type and AtSYP71/OE, respectively ( Fig. 7e-7g). Under 75 µm H 2 O 2 treatment, root length of atsyp71-2 only reduced by 9.1% compared with 40.4% and 45.8% decline of that of wild-type and AtSYP71/OE, respectively ( Fig. 7h-7j). Under 150 mM Mannitol treatment, root length of atsyp71-2 increased by 8.4% compared with 35.3% and 35.5% decline of that of wild type and AtSYP71/OE, respectively, respectively ( Fig. 7h-7j). These results indicated that response to stress in atsyp71-2 altered signi cantly.

Discussion
AtSYP71 is essential for plant growth and development via regulating secretion of cell wall-related proteins and chemical compounds That AtSYP71 is a Qc-SNARE with multi-localization on the ER, endosome, PM and cell plate, which are right on the secretion and cycling pathways [79,104,[108][109][110][111][112][113][114], suggests its diverse functions in plant growth and development. AtSYP71 is proposed to be involved in multiple membrane fusion steps during the secretion process in Arabidopsis [109]. It is also clari ed that AtSYP71 binds with Qa-KNOLLE, Qb-SNARE NPSN11 and R-SNAREs VAMP721 and VAMP722 to form a SNARE complex to regulate cytokinesis [114]. De cient of AtSYP71 de nitelyaffected AtSYP71-dependent vesicle tra cking and localized organelle functions. The knock-out mutant atsyp71-4 could germinate, but the roots could not elongate, the leaves were small and the seedlings died soon (Fig. 1C). These phenotypes resembled syp71 amiR -mutant and npsn11 syp71 amiR -double mutant which were cytokinesis defective [114]. This might because that depletion of AtSYP71 seriously disrupted delivery of materials necessary for cell plate formation and the plasma membrane integrity and lead to failure of morphogenesis and subsequent lethality at early development stage. Even the knock-down mutants, atsyp71-2 and atsyp71-3 also displayed obvious defects on plant growth and development. On the other hand, Lotus LjSYP71 plays an important role in symbiotic nitrogen xation, but the Ljsyp71 mutant grows similarly to wild-type plant when supplied with combined nitrogen. OsSYP71 and TaSYP71 are involved in plant resistance to pathogens, but the mutants didn't show obvious phenotypes under normal growth conditions [108,114].
These suggested that LjSYP71, OsSYP71 and TaSYP71 are not essential for plant growth and development. This probably because that SYP71 orthologues in different species gain functional division during evolutionary process.
Laccases and peroxidases (i.g. PRXs) required for lignin biosynthesis are glycosylproteins [13,22,53]. The ER and the Golgi apparatus are responsible for protein glycosylation. Thus, post transcriptional modi cation of laccases and peroxidases as well as other enzymes were probably disrupted in atsyp71 mutants. Secretion, cycling and homeostasis of PM-localized proteins and their motility in the PM depend on the PM components [32][33][34]. For the above reasons, it is easy to conjecture that secretion, homeostasis of PM and PM-localized proteins were perturbed and subsequently affected expression of many cell wall-related proteins in atsyp71-2 mutant. For example, AT4G01700-encoding protein belongs to Chitinase family which is related to cellulose biosynthesis and cell wall remodeling [128]. The EXPA proteins are responsible for controlling cell wall extension and developmental processes, including cell dissociation and separation [129][130][131][132]. LTP1 and LTP2 belong to a family of lipid transfer proteins, and LTP2 plays a role in maintaining the integrity of the cuticle-cell wall interface [133]. LTPG1, a PM-localized lipid transfer protein, regulates cuticular lipid composition [134,135]. BBE enzymes catalyze the biosynthesis of the secondary metabolites, isoquinoline alka loids [136]. BBE19/OGOX1, an oligogalacturonide (OG) oxidase, is involved in plant immunity. OGs, a major component of pectin, are a well-known class of damage-associated molecular patterns (DAMPs) that activate immunity and protect plants against microbes [137]. XTH22 is a cell wall-modifying enzyme and is rapidly upregulated in response to environmental stimuli. XTH20, a putative xyloglucan endotransglycosylase/hydrolase expressed primarily in the main and lateral roots, is involved in cell proliferation in incised in orescence stems [138][139][140]. PP2-B13, a phloem protein 2-like protein, belongs to F-box-like domain superfamily.
PP2 is component of the phloem protein bodies found in sieve elements. PP2 proteins can directly bind with the chitin cell wall and play important roles in defense against pathogens, photoassimilate transport and wound healing [141][142][143]. BGLU25 belongs to beta-glucosidase family which is involved in cellulose degradation [72,144].
Beyond that, biosynthesis or/and secretion of some chemicals may also be affected in atsyp71 mutants.
Hemicelluloses are synthesized at the Golgi apparatus are then delivered to the cell wall by transport vesicles [10,23,24]. Release of hemicelluloses is controlled by the process of vesicle fusion to the PM which is regulated by SNARE complex including Qc-SNARE AtSYP71. Thus, secretion of hemicelluloses probably was disrupted.
The contents of monolignols and avonoids synthesized by the Phenylpropanoid biosynthesis pathway decreased in atsyp71-2 mutant (Fig. 5). A β-D-glucosidase, BGLU25, functioning on Phenylpropanoid biosynthesis pathway, was also changed (Fig. 4). These suggested that Phenylpropanoid biosynthesis pathway was affected. Monolignols, the materials for lignin biosynthesis, are synthesized in the cytoplasm or in close to the ER [52,53] and then are secreted by PM-localized transporters such as ABC transporters [54][55][56][57]. and the transport of avonoids and monolignols is unclear, it cannot be ruled out that the functions of ABCG1 and other transporters changed and affected the transport of avonoids and monolignols. Another aspect that cannot be ignored is that the biosynthesis of both avonoids and monolignols is related to the ER. The effect of de cient of AtSYP71 on the ER will certainly in uence their biosynthesis. Further study is needed to clarify mechanism underlying AtSYP71 regulation on relating organelle functions, secretion of proteins and chemical compounds as well as homeostasis of PM-localized proteins.
AtSYP71 is closely related to plant stress responses Abiotic stresses affect plant growth and development [145][146][147] (Fig. 7A-D). In addition to PRXs, expression of many redox-related enzymes also altered. For example, Endothelial amine oxidase AOC3, Proline oxidase family FAD-linked oxidoreductase POX1, FAD linked oxidase AT4G20830 and AT5G44400, Multicopper oxidase LAC1 and Copper amine oxidase AT1G31710 (supplemental Table S1). There may be several in uencing factors. One is due to defects on vesicle transport. Since the secretion pathway was blocked in atsyp71-2, e ciency of the enzyme delivery to destinations might decrease. The second reason could be defects on posttranscriptional modi cation. For example, expression of UDP-glucosyltransferase, UGT74E2 and AT2G18560 changed (supplemental Table S1). The changes in enzyme activities affected ROS homeostasis and resulted in defects on cellular functions and cell wall structures which leading to weakened plant adaptabilities to environmental stress.
The non-enzymatic components contain ascorbic acids, α-tocopherol, avonoids, phenolic compounds glutathione, carotenoids and lipids which mitigate oxidative damage by their antioxidant activities through utilization of H 2 O 2 [151,152].  Table S1), which could affect lax biosynthesis. Considering of defense function of avonoids, the decrease of avonoid contents could be one of the reasons for the change of stress response of atsyp71-2 mutant.
Besides the enzymatic and non-enzymatic processes, sugars are also antioxidant based on redox balance. Sugars affect gene expression through sugar-speci c signaling regulators such as hexokinase (HXK), Snf1-related kinase 1, and INV, which regulate the expression of stress-related genes such SODs, heat shock proteins (HSP) and glutathione-S-transferases (GST) [154,181] Table S1). These alterations might severely affect plant adaptation to environmental stimuli. Moreover, in addition to WRKY and MYB family transcription factors, some other stress response TFs also altered, such as AP2/ERF domain superfamily members, ERF107, ERF018, TINY, AIL6 and RAP2-3; Zinc nger domain-containing BT1, AT5G49665, ZFP3 and RHA4A.
In conclusion, AtSYP71 modulated plant metabolic pathways, development and environmental adaptabilities via regulatory on vesicle tra cking.

Conclusion
The AtSYP71-knockout mutant atsyp71-4 was lethal at early development stage, suggesting that AtSYP71 is essential for plant development. Disturbance of secretion of SecGFP, the secretion marker, suggested that AtSYP71 is required for secretion pathway. Thus, we propose that depletion of AtSYP71 might block the delivery of materials and enzymes required for cell wall biosynthesis and lead to failure of morphogenesis. Transcriptome analysis of the AtSYP71-knockdown mutant, atsyp71-2, revealed the disruptions of metabolism, stress response and extracellular components. In addition, contents of lignin, cellulose and avonoids also altered signi cantly. These results suggested that AtSYP71 regulates cell wall formation and secondary metabolism. The defects on cell wall structures subsequently affected plant response to abiotic stress. Our ndings suggested that AtSYP71 regulates plant development, metabolism and environmental adaptation probably by affecting cell wall homeostasis via mediating secretion of materials and regulators required for cell wall biosynthesis and dynamics.

Abiotic Stress Treatments
For the NaCl, osmotic and oxidative stress treatments, the seeds were sown on 1/2MS medium with 120 mM NaCl, 150 mM mannitol, or 75 mM H 2 O 2 and grown vertically. The root length was measures using ImageJ software.

Antibody Preparation
To prepare antibodies against AtSYP71, a polypeptide (Cys-LPARIEAIPDGTAGGPKSTSAWTPSSTTSRPDIKFDSDGRFDDDYFQESN) was synthesized and conjugated to a Carrier protein KLH linked by a N-terminal Cys residue. The peptide-KLH conjugates were injected into two rabbits to generate antibodies. The antibodies were subjected to ProteinA/G puri cation from the serum and ELISA detection. Polypeptide synthesis and antibody preparation were commissioned to GL Biochem (Shanghai) Ltd.

Confocal Microscopy
Fluorescent images were obtained using a point scanning confocal microscope (Leica TCS SP8).

Yeast Two-Hybrid Assay
For the yeast two-hybrid assay, the fragments of AtSYP71 (cytosolic region), MIP2 (Sec39 domain) and MIP3 were ampli ed using cDNA obtained from seedlings with speci c primers and ligated into pEASY-Blunt vector (TransGen, #CB101-01), respectively. After Sanger sequencing con rmation, the fragments were transferred into pGADT7 or pGBKT7 vectors, respectively. AtSYP81, AtSEC20 and MAG2 constructs were generated in our previous study [115,121].
The paired constructs were introduced into Saccharomyces cerevisiae strain AH109 (Clontech) and selected on SD/-Leu/-Trp medium. The interactions were detected on SD/-Leu/-Trp/-His/-Ade medium.

RNA Extraction and RT-qPCR
Total RNA was extracted as described previously [122] using 9-day-old seedlings grown on 1/2MS medium horizontally. Quantitative reverse transcription-polymerase chain reaction (RT-qPCR) was performed according to the manufacturer's instructions. The speci c primers are listed in Supplementary estimate the in uence of non-uniquely mapped reads on gene expression we also mapped reads using the same software and parameters as indicated above, but allowing multiple mapping (up to 10 hits). For each gene, total gene reads (TGR) was determined as the sum of all reads mapped to this gene. To avoid bias due to different library sizes, TGR values were normalized by a size factor as described in Anders and Huber [185].

Identi cation of DEGs
DEGs were identi ed using the R package 'DESeq' [185]. was used as a standard for calculation of cellulose content. Three repeats per sample.

Determination of total avonoids
The avonoid content was determined by colorimetric assay [188]. A 250 μL of standard solution of rutin at different concentrations or appropriately diluted samples were added to 10 ml volumetric ask containing 1 ml of distillate water, respectively, then, 75 μl of NaNO 2 (5%) was added and mixed thoroughly. After 6 min of incubation, 75 μl of AlCl 3 (10%) was added, fully mixed and incubated for 6 min, then 500 μl of NaOH (1N) was added. Immediately, the solution was diluted by adding 2.5 ml of methanol and mixed thoroughly. Absorbance of the mixture was determined by spectrophotometer at 506 nm wavelength versus the prepared blank. Total avonoid compounds in plant were indicated as mg rutin equivalents (CE mg/ml). Three repeats per sample.

Analysis of enzyme activities
For determination of activities of ROS-scavenging enzyme, 0.2 g of roots from nine-day-old seedlings

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Supplementary Files
This is a list of supplementary les associated with this preprint. Click to download. TableS1.xlsx TableS2.xlsx atsyp71sypplement gs.pdf