Physiological and Proteomic Analyses Reveal Effects of Putrescine-Alleviated Aluminum Toxicity in Rice Roots

Zhu Chunquan, Hu Wenjun, Cao Xiaochuang, Zhu Lianfeng, Kong Yali, Jin Qianyu, Shen Guoxin, Wang Weipeng, Zhang Hui, Zhang Junhua

Research Paper

Physiological and Proteomic Analyses Reveal Effects of Putrescine-Alleviated Aluminum Toxicity in Rice Roots

Zhu Chunquan1, #, Hu Wenjun2, #, Cao Xiaochuang1, Zhu Lianfeng1, Kong Yali1, Jin Qianyu1, Shen Guoxin2, Wang Weipeng4, Zhang Hui3, Zhang Junhua1

(State Key Laboratory of Rice Biology, China National Rice Research Institute, Hangzhou 310006, China; State Key Laboratory for Managing Biotic and Chemical Threats to the Quality and Safety of Agro-Products,Zhejiang Academy of Agricultural Sciences, Hangzhou 310021, China; Agricultural Resources and Environment Institute, Jiangsu Academy of Agricultural Sciences, Nanjing 210014, China; College of Environmental and Resource Science, Shanxi University, Taiyuan 030006, China; These authors contributed equally to this work)

The effects of putrescine on improving rice growth under aluminum (Al) toxicity conditions have been previously demonstrated, however, the underlying mechanism remains unclear.In this study, treatment with 50 μmol/L Al significantly decreased rice root growth and whole rice dry weight, inhibited Ca2+uptake, decreased ATP synthesis, and increased Al, H2O2and malondialdehyde (MDA) contents, whereas the application of putrescine mitigated these negative effects. Putrescine increased root growth and total dry weight of rice, reduced total Al content, decreased H2O2and MDA contents by increasing antioxidant enzyme (superoxide dismutase, peroxidase, catalase and glutathione S-transferase) activities, increased Ca2+uptake and energy production. Proteomic analyses using data-independent acquisition successfully identified 7 934 proteins, and 59 representative proteins exhibiting fold-change values higher than 1.5 wererandomly selected. From the results of the proteomic and biochemical analyses, we found that putrescine significantly inhibited ethylene biosynthesis and phosphorus uptake in rice roots, increased pectin methylation, decreased pectin content and apoplastic Al deposition in rice roots. Putrescine also alleviated Al toxicity by repairing damaged DNA and increasing the proteins involved in maintaining plasma membrane integrity and normal cell proliferation. These findings improve our understanding of how putrescine affects the rice response to Al toxicity,which will facilitate further studies on environmental protection, crop safety,innovations in rice performance and real-world production.

aluminum toxicity; antioxidant enzyme;data-independent analysis; putrescine; proteomics; rice

Aluminum (Al) is the most abundant metal in the earth’s crust, and it exists in non-toxic forms under neutral conditions (Schmitt et al, 2016). However, it is easily transformed into the phytotoxic form (especially in the form of Al3+), which is toxic to all living organisms in acidic soils (Rengel, 2004). Due to the rapid and strong interaction between Al and its targets in apoplasts and symplasts, the toxicity of Al for plants is rapid and broad. It includes damage to the structure and function of plasma membranes and cell walls (Illes et al, 2006), and a reduction in the uptake of nutrient elements (Liu and Luan, 2001), leading to respiratory bursts of reactive oxygen species (ROS) (Boscolo et al, 2003), thus inhibiting plant growth (Matsumoto, 2000). It is estimated that half of the potentially arable land worldwide is acidic enough to transform Al into its cationic form and thus limiting crop production (Ryan et al, 2011). Therefore, it is pivotal to improve the Al resistance capacity of crops in acidic soils.

Putrescine is a major component in polyamines and is involved in regulating the plant response to heavy metal stress (Zhu et al, 2019a).For example, the exogenous application of putrescine significantly increases the activities of antioxidant enzymes and reduces peroxidation damage induced by Al toxicity inand(Chen et al, 2008; Mandal et al, 2013). The addition of putrescine also stimulates nitric oxide (NO) production in red kidney bean plants and increases citrate secretion, thereby reducing Al deposition in roots (Wang et al, 2013). Cell walls, especially the pectin and hemicellulose polysaccharide fractions, accumulate the highest proportion of Al agglomerates, and the regulation of cell wall structure is an important means by which plants reduce Al deposition and alleviate Al toxicity (Rengel and Reid, 1997; Yang et al, 2008; Yang et al, 2011). Previous studies have demonstrated that putrescine application significantly inhibits the synthesis of hemicellulose and pectin in wheat and rice, resulting in reduced cell wall Al deposition and improved plant growth under Al toxicity conditions (Yu et al, 2015; Zhu et al, 2019a). However, most of the above studies focused on the physiological mechanism whereby putrescine alleviates Al toxicity.The elucidation of the molecular mechanisms underlying the putrescine-regulated plant response to Al toxicity remains necessary.

Technological advances in ‘omics’ approaches have helped to explore the underlying mechanism by which signaling molecules improve plant stress resistance ability at the protein level via exogenous application, such as the alleviation of salt stress inby NO (Shen et al, 2018) and the alleviation of Al toxicity inby calcium (Ca) (Chen et al, 2019). Decreasing the accumulation of Al in rice in order to improve Al tolerance is important for global crop safety (Foy, 1988; Fageria, 2007). In this study, we combined physiological and proteomic approaches to investigate the protective role of putrescine against Al toxicity in rice. Our objectives were to provide valuable insights into the molecular mechanisms of putrescine-alleviated Al toxicity and to identify key regulatory networks and proteins contributing to Al tolerance in rice. Our results offer a foundation for future genetic improvements to enhance rice productivity and adaptability to Al stress, ultimately reducing the risks of Al to human health.

RESULTS

Mitigative effect of exogenous putrescine on morphological and physiological parameters of rice roots under Al stress

Al treatment (50 μmol/L) significantly increased the Al content in rice roots and shoots, and the addition of 0.1 mmol/L putrescine decreased the Al content (Fig. 1-A and -B). The rice root length and the total dry weight were significantly decreased under the Al treatment compared with the control without Al. However, the application of putrescine significantly mitigated these negative effects (Fig. 1-C to -F).

The results of antioxidant enzyme activities showed that the Al treatment significantly increased the activities of superoxide dismutase (SOD), peroxidase (POD), catalase (CAT) and glutathione S-transferase (GST) in rice roots, and the application of putrescine further increased their activities (Fig. 1-G to -J), indicating that putrescine mobilized the antioxidant system in rice roots to resist Al toxicity. Malondialdehyde (MDA) content and H2O2content both increased under the Al treatment, and the application of putrescine decreased their contents (Fig. 1-K and -L), further confirming that putrescine alleviated the peroxidation damage induced by Al toxicity.

Protein profile of putrescine-alleviated Al toxicity in rice roots

To investigate the underlying mechanism of putrescine- alleviated Al toxicity in rice, 7 934 proteins wereidentified via data-independent acquisitionproteomics(Figs. S1 to S5).Gene Ontology (GO) analysis indicated that in terms of biological processes, most identified proteins belonged to metabolic and cellular processes; for the cellular component, most proteins were localized in the cell and cell parts; and for the molecular function, most proteins were involved in binding and catalytic activity (Fig. 2-A). The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway annotation separated the functions into five groups, including metabolism, genetic information processing, environmental information processing, cellular processes and organismal systems(Fig. 2-B). The EuKaryotic Orthologous Groups (KOG) annotation showed that the top functions were general function prediction only, posttranslational modification, protein turnover and chaperones, and signal transduction mechanisms (Fig. 2-C).

Fig. 1. Effects of putrescine (PUT) on rice response to aluminum (Al) toxicity.

A, Total Al content in rice roots. B, Total Al content in rice shoots. C, Whole rice dry weight. D, Root elongation. E, Relative root elongation. F, Phenotype of rice roots under different treatments. Scale bar, 1 cm. G, Superoxide dismutase (SOD) activity in rice roots. H, Peroxidase (POD) activity in rice roots. I, Catalase (CAT) activity in rice roots. J, Glutathione S-transferase (GST) activity in rice roots. K, Malondialdehyde (MDA) content in rice roots. L, H2O2content in rice roots.

The rice seeds were placed in an incubator for 2 d at 30 oC in darkness until the roots had grown to about 2 cm long, and then treated with Al (50 μmol/L) or PUT (0.1 mmol/L) for another 1 d. After that, the rice roots were collected for measurement. Data are Mean ± SD (= 4). Columns with different lowercase letters are significantly different at< 0.05.

Fifty-nine proteins that changed more than 1.5-fold (< 0.05)are listed in Table S1. The proteins were separated into three sets based on the different comparisons, namely PUT vs CK (PUT/CK), Al vs CK (Al/CK) and Al + PUT vs Al (Al + PUT/Al). Three groups were defined based on the expression patterns (Fig. 3-A). Most of the proteins that belonged to Group 1 were significantly increased in the Al + PUT/Al set and exhibited decreases or no changes in the Al/CK and PUT/CK sets. The proteins in Group 2 were significantly decreased in the PUT/Al or Al/CK set and exhibited no changes in the Al + PUT/Al set. The proteins Group 3 were significantly increased in the Al/CK and PUT/CK sets and showed no changes or decreased in the Al + PUT/Al set (Fig. 3-A).

The number of upregulated proteins that overlapped among the three treatments is shown in a Venn diagram (Fig. 3-B). Among the upregulated proteins, 16 proteins (40.0%) only existed in the Al + PUT/Al set, 14 proteins (35.0%) only existed in the Al/CK set, 6 proteins (15.0%) only existed in the PUT/CK set, and 4 proteins (10.0%) overlapped between the Al/CK and PUT/CK sets. Among the downregulated proteins, 9 (25.7%), 13 (37.1%) and 4 (11.4%) proteins were only present in the Al + PUT/Al, Al/CK and PUT/CK sets, respectively. One protein (2.9%) overlapped in the Al + PUT/Al and Al/CK sets, while eight proteins (22.9%) overlapped in the Al/CK and PUT/CK sets.

To verify the results of proteome in the present study, eight proteins were randomly selected to analyze their transcript expression levels and the results were displayed in the form of Al + PUT/Al, Al/CK and PUT/CK (Fig. 4). The qRT-PCR results presented that five genes showed paralleled expression change patterns in mRNA level and protein level (Fig. 4-A and -B), and there was a significant positive correlation between protein abundance and mRNA expression (Fig. 4-C), confirming the reliability of the proteomic results.

Fig. 2. Protein functional annotation.

A, Gene Ontology analysis. B, Kyoto Encyclopedia of Genes and Genomes pathway annotation. C, EuKaryotic Orthologous Groups function classification of peptide sequences.

The rice seeds were placed in an incubator for 2 d at 30 oC in darkness until the roots had grown to about 2 cm long, and then treated with aluminum (50 μmol/L) or putrescine (0.1 mmol/L) for another 1 d. After that, the rice roots were collected for proteomic analyses.

Energy production and related enzyme activity

Al treatment significantly inhibited the production of ATP in rice roots, however, the application of putrescine mitigated this negative performance under the Al toxicity conditions (Fig. 5-A). In addition, the protein content of ATPase, which is involved in ATP synthesis, was measured by western blot, and the results showed that Al toxicity significantly decreased the ATPase content, while the application of putrescine increased its abundance (Fig. 6).

Ca uptake and salicylic acid (SA) production

Ca content in rice roots decreased significantly under the Al toxicity conditions and increased following putrescine application (Fig. 5-B). The accumulation of SA significantly increased under the Al toxicity conditions, and the application of putrescine decreased the SA content. However, a single application of putrescine increased the SA content without Al treatment (Fig. 5-C).

Ethylene production and related gene expression

The application of putrescine under the Al toxicity conditions significantly decreased ethylene emissions from rice roots compared to the single Al treatment (Fig. 5-D). The activity of 1-aminocyclopropane-1- carboxylic acid (ACC) oxidase (ACO), which is responsible for ethylene production in plants, was also measured. ACO displayed the same tendency as the ethylene emissions (Fig. 7-A), i.e., Al induced ACO activity and putrescine inhibited ACO under the Al toxicity conditions.

Fig. 3. Hierarchical clustering analysis (A) and Venn diagram (B) for selected differentially expressed proteins of rice roots in Al/CK, Al + PUT/Al and PUT/CK sets.

Upregulated and downregulated proteins are indicated in red and green, respectively, in A. The intensity of the colors indicates the abundance of the protein, as shown in the bar. The rice seeds were placed in an incubator for 2 d at 30 oC in darkness until the roots had grown to about 2 cm long, and then treated with Al (50 μmol/L) or PUT (0.1 mmol/L) for another 1 d. After that, the rice roots were collected for proteomic analyses. Three independent biological replicates were tested. Al, Aluminum; PUT, Putrescine.

Fig. 4. Comparison between protein and mRNA expression changes of eight randomly selected proteins.

A, Comparison of expression changes at protein level. The data of protein fold change are from the results of data-independent acquisition proteomics. B, Comparison of expression changes at mRNA level. Total RNA was extracted from rice root after 24 h of treatment with the aluminum (Al) concentration in the solution of 50 μmol/L and putrescine (PUT) concentration of 0.1 mmol/L. The relative expression of genes was defined CK as ‘1’. Thegene was used as a reference gene. C, Correlation between proteins and genes.

O1, Os11t0702100-01, class III chitinase homologue; O2, Os11t0151700-01, purple acid phosphatase; O3, Os06t0594600-01, BAHD acyltransferase; O4, Os07t0194500-01, 2OG-Fe(II) oxygenase domain containing protein; O5, Os10t0361000-01, lipoxygenase; O6, Os05t0176100-05, cellulose synthase BoCesA1; O7, Os06t0175500-01, epsin-like; O8, Os07t0639000-01, class III peroxidase 46. The results of selected genes’s relevant expression are calculated in the form of Al + PUT/Al, Al/CK and PUT/CK. Data are Mean ± SD (= 4). Columns with different lowercase letters are significantly different at< 0.05.

Fig. 5. Effects of putrescine (PUT) on rice response to aluminum (Al) toxicity.

A, ATP content in rice roots. B, Ca content in rice roots. C, Salicylic acid content in rice roots. D, Ethylene emission rate in rice roots. E, Pectin content in rice roots. F, Pectin content on root surface. Thepectin content on root surface was indicated by red color. Scale bar is 1 mm.G, Degree of pectin demethylesterification in rice roots. H, Apoplast Al concentration in rice roots.

The rice seeds were placed in an incubator for 2 d at 30 oC in darkness until the roots had grown to about 2 cm long, and then treated with Al (50 μmol/L) or PUT (0.1 mmol/L) for another 1 d. After that, the rice roots were collected for measurement. Data are Mean ± SD (= 4). Columns with different lowercase letters are significantly different at< 0.05.

The expression offamily geneswas also measured for the Al + PUT/Al, Al/CK and PUT/CK sets.The results showed that the expression patterns ofandexhibited the same tendency as ACO protein abundance (Fig. 7-B and Table S1),exhibiting decreased in the Al + PUT/Al set, increased in the Al/CK set and no changes in the PUT/CK set.

Fig. 6.Effects of putrescine (PUT) on relative abundance of lipoxygenase and ATP synthase proteins.

The rice seeds were placed in an incubator for 2 d at 30 oC in darkness until the roots had grown to about 2 cm long, and then treated with aluminum (Al, 50 μmol/L) or PUT (0.1 mmol/L) for another 1 d. After that, the rice roots were collected for western blot. The values are compared with CK and indicate the band intensities. There were four replicates for each experiment, and only one experiment was displayed.

Al toxicity significantly induced the activity, protein abundance and gene expression of lipoxygenase, while the application of putrescine under the Al toxicity significantly decreased the activity, protein abundance and gene expression of lipoxygenase (Figs. 4-B, 6 and 7-C). This was further confirmed by the production of ethylene under different treatments (Fig. 5-D).

Pectin synthesis and apoplastic Al concentration

Pectin is negatively charged and is involved in the deposition of Al in the plant cell walls (Zhu et al, 2018a). Our results indicated that the Al treatment increased the pectin content in rice roots, whereas the application of putrescine under the Al toxicity conditions decreased the pectin content (Fig. 5-E and -F). The Al treatment significantly increased the pectin demethylesterification degree in rice roots compared with the control. However, the application of putrescine under the Al toxicity conditions decreased the pectin demethylesterification degree compared with the single Al treatment (Fig. 5-G). In addition, the apoplastic Al concentration increased under the Al toxicity conditions and decreased after putrescine application (Fig. 5-H).

Fig. 7. Effects of putrescine (PUT) on ethylene emission and phosphorus (P) uptake in rice roots.

A, 1-aminocyclopropane-1-carboxylic acid oxidase (ACO) activity. B, Comparison ofexpression changes in the form of Al + PUT/Al, Al/CK and PUT/CK. Total RNA was extracted from rice roots after 24 h of treatment. C, Lipoxygenase activity. D, Cell sap P content. E, Apoplast P content. F, Relative expression ofunder aluminum (Al) conditions. Total RNA was extracted from rice roots after 24 h of treatment. Relative expression ofunder Al treatment was defined as ‘1’, andthegene was used as a reference gene.

Al concentration in the solution is 50 μmol/L and PUT concentration is 0.1 mmol/L. Data are Mean ± SD (= 4). Columns with different lowercase letters (A?C) or stars (D?F) are significantly different at< 0.05.

Phosphorus (P) uptake and related gene expression

Putrescine application not only decreased the cell sap P content, but also decreased the apoplastic P content (Fig. 7-D and -E) under the Al toxicity conditions. In addition, the P uptake gene expression patterns showed that, with the exception ofand, which both increased, and, which exhibited no change, the other eight relatedall decreased after putrescine was applied under the Al toxicity conditions (Fig. 7-F), indicating that putrescine inhibited P uptake under Al toxicity.

DISCUSSION

Mechanism of rice response to Al toxicity

In this study, the Al treatment significantly increased the total Al content in rice roots, inhibited rice root elongation and decreased rice seedling dry weight (Fig. 1-A to -F). These results were in accordance with the growing evidence that Al stress severely affects the material metabolism of plants and inhibits plant growth (Matsumoto, 2000; Zhang et al, 2019).Carbon metabolism plays an important role in the production of ATP and in the accumulation of plant dry matter (Hu et al, 2014). In the study, proteins similar to hexokinase (Os05t0187100-02), vacuolar ATP synthase subunit C (Os05t0593100-01), plastidic ATP/ADP- transporter (Os02t0208100-01), ATP/ADP carrier protein(Os05t0302700-01) and Ca2+-ATPase (Os03t0281600-01), which are related to the energy production pathway, were identified and decreased under Al toxicity conditions (Fig. 3-A and Table S1). Among them, hexokinase catalyzes the first step of the glycolytic pathway, which converts hexoses to hexose 6-phosphates (Jang et al, 1997). ATP synthase is associated with the generation of mitochondrial cristae morphology and is responsible for the synthesis of ATP(Paumard, 2002). ATP/ADP-transporter is responsible for the uptake and transport of ATP in organisms (Mayinger and Meyer, 1993). The decrease in ATP content and ATPase (ATP synthase) protein abundance under the Al toxicity conditions (Figs. 5-A and 6) further confirmed that Al toxicity inhibits energy production and transportation to affect rice growth. Furthermore, the low energy production in rice roots under the Al toxicity conditions increased the abundance of oligouridylate binding protein (Os11t0620100-03) (Fig. 3-A), which is thought to play a role in isolating poorly-translated mRNAs under low energy stress (Sorenson and Bailey-Serres, 2014).

Calcium (Ca2+) functions as a messenger to regulate plant responses to biotic and abiotic stress (Elizabeth et al, 2005), and Al toxicity significantly affects Ca2+uptake in plants (Hossain et al, 2014). In our study, thetotal Ca content in rice roots (Fig. 5-B), the abundances of Ca-dependent protein kinases (Os03t0788500-01 and Os11t0171500-01), similar to calmodulin-binding receptor-like kinase (Os09t0123300-01) and calcineurinB-like protein 2 (Os12t0597000-01) were all decreased under the Al toxicity conditions (Fig. 3-A and Table S1), suggesting that Al interferes with the Ca2+signal in rice to inhibit rice growth.

Al induces ethylene emissions and increases ethylene- aggravated Al toxicity in plants (Sun et al, 2007, 2010). In the present study, ACO (Os05t0149400-01) and lipoxygenase (Os10t0361000-01), which are related to ethylene biosynthesis, were significantly increased under the Al toxicity conditions (Fig. 3-A and Table S1). Among them, ACO is responsible for catalyzing ACC to produce ethylene (Yu et al, 2016). Lipoxygenase is responsible for the oxidation of fatty acids and is involved in the ACC-mediated formation of ethylene (Siedow, 1991). In addition, the ethylene emission rate, ACO activity, lipoxygenase activity, lipoxygenase abundance and associated gene expression were all induced by Al toxicity compared with the control (Figs. 4-B, 5-D, 6 and 7), confirming that Al induced ethylene emissions and then aggravated the growth of rice roots.

The increase in MDA and H2O2contents in the Al/CK set (Fig. 1-K and -L) confirmed the finding that Al toxicity induces oxidative stress in plants (Wu et al, 2017; Riaz et al, 2018). Increasing antioxidant system enzyme activity to reduce peroxidation damage is a common strategy for alleviating Al toxicity in plants (Manevich et al, 2002; Wu et al, 2017; Riaz et al, 2018). In this study, five antioxidant proteins, namely haem peroxidase (Os07t0104500-01), class III peroxidase46 (Os07t0639000-01), similar to 1-cys peroxiredoxin(Os07t0638400-01) and GST proteins (Os10t0528300-01 and Os09t0367700-01), were significantly induced by Al toxicity (Fig. 3-A and Table S1). Among them, GST is responsible for catalyzing the addition of-glutathione to electrophilic compounds and protecting plants from oxidant reactive species (Frova, 2003). Class III peroxidases catalyze the redox reaction of H2O2with a variety of organic and inorganic hydrogen donors in plants (Welinder et al, 1992). In addition, the activities of SOD, POD, CAT and GST under the Al conditions were all significantly increased compared with the control (Fig. 1-G to -J), further confirming that rice stimulated antioxidant system enzymes to counter the peroxide damage induced by Al toxicity.

The application of SA alleviates Al toxicity in rice (Zhu et al, 2020). The probenazole protein (PBZ, Os12t0555500-01) functions upstream of SA and induces the accumulation of SA in rice (Takayoshi et al, 2007). In this study, the abundance of PBZ was increased under the Al toxicity conditions (Table S1), and together with the increased SA content in rice roots under the Al toxicity conditions (Fig. 5-C), suggesting that rice might increase SA content to alleviate Al toxicity.

Putrescine regulates cell structure and composition to alleviate Al toxicity in rice

Previous studies have demonstrated that Al and putrescine both regulate cell wall biosynthesis in plants. For example, Al toxicity significantly increasesthe cell wall polysaccharide content in rice (Yang et al, 2008), and the application of putrescine decreases the cellulose content in pollen tubes of tea () (Etinba-Gen et al, 2019) and decreases the hemicellulose content in rice (Zhu et al, 2018a, 2020). In this study, the pectin content was significantly increased under the Al toxicity conditions (Fig. 5-E and -F) and was accompanied by an increase in hydroquinone glucosyltransferase (Os02t0242900-00) and UDP- glycosyltransferases (Os01t0597800-01, Os05t0215300-01 and Os01t0638000-01) (Fig. 3-A and Table S1), both of which are involved in cell wall polysaccharide synthesis (Yokoyama et al, 1990; Ridley et al, 2001). Although it had no effect on rice root growth under no Al conditions (Fig. 1-D to -F), the exogenous application of putrescine regulated the proteins involved in cell wall synthesis in the present study. For example, the abundance of xyloglucan endotransglucosylase/hydrolases (Os06t0696400-01), which regulates cell expansion (Rose et al, 2003), increases after the application of putrescine. Xylan-acetyltransferase (Os11t0107000-01), which is responsible for the acetylation of xylans (Zhong et al, 2018), xyloglucan 6-xylosytransferase (Os02t0529600-01), which is responsible for the biosynthesis of cellulose-xyloglucan (Vuttipongchaikij et al, 2012), and cellulose synthase BoCesA1 (Os05t0176100-05), were all decreased (Fig. 3-A and Table S1). The above results confirmed that both Al and putrescine affect rice cell wall material synthesis and composition.

Removing Al from cell walls by modifying the cell wall structure and pectin content significantly increases rice root growth under Al toxicity conditions (Zhu et al, 2018a, 2019a, 2019b). Pectin is the main polysaccharide possessing negative charges in the cell wall, and its methylation degree is regulated by pectin methylesterase and pectin methylesterase inhibitor (Horst et al, 2010). In this study, the abundance of a pectinesterase inhibitor domain-containing protein (Os06t0711800-01) significantly increased after the application of putrescine under the Al toxicity conditions (Fig. 3-A and Table S1). These patterns were accompanied by a lower pectin demethylesterification degree and pectin content (Fig. 5-E to -G), indicating that putrescine regulates pectin content and structure to reduce apoplastic Al content (Fig. 5-H).

Ethylene is associated with cell wall polysaccharide synthesis in plants, and our previous studies found that the addition of putrescine significantly inhibits ethylene emissions in rice to reduce Al content in rice cell walls (Zhu et al, 2019a). After applying putrescine under the Al toxicity conditions, the contents of ACO and lipoxygenase (Os05t0149400-01 and Os10t0361000-01),ethylene emissions and the ACO activity all significantly decreased (Table S1; Figs. 3-A, 5-D, 6 and 7). These patterns were accompanied by lower pectin content (Fig. 5-E and -F), which further confirmed that putrescine inhibits ethylene and then reduces Al deposition in cell walls. Further study of the expression ofgenes found that the expression patterns ofand(Fig. 7-B) in the three comparison groups were the same as ACO, further confirming the credibility of the proteomic research and verifying that putrescine inhibits ethylene emissions via the ACO pathway under Al toxicity conditions.

The limitation of P under Al toxicity conditions significantly increases Al tolerance by decreasing phospholipids and pectins (Maejima et al, 2014). In our study, the metallophosphoesterase domain- containing protein (Os03t0725300-01), which exists in the P-uptake protein purple acid phosphatase (Matange et al, 2015), and the purple acid phosphatase geneboth decreased following the application of putrescine under the Al toxicity conditions (Table S1; Fig. 3-A and Fig. 4-B). In addition, the application of putrescine under the Al toxicity conditions significantly decreased the P concentration in rice root cell sap and apoplast, and was accompanied by the decreased expression levels of the P transporters///////(Fig. 7-D to -F). This further confirmed that putrescine alleviates the Al toxicity by decreasing P uptake and thereby decreasing the pectin content as well as the absorption of Al in cell walls.

Other possible pathways by which putrescine alleviates Al toxicity

Maintaining the integrity of plasma membranes, normal cell proliferation and repairing damaged DNA are essential for plant resistance to environmental stress (Hu et al, 2015). In this study, nine relevant proteins, including acyl-CoA-binding protein (ACBP) (Os03t0243600-01), similar to atypical receptor-like kinase MARK (Os03t0712400-02), B3 domain transcriptional repressor (Os07t0563300-01), ribosome biogenesis protein Nop16 domain containing protein (Os05t0367100-01), similar to E3 ubiquitin protein ligase UPL2 (Os12t0428600-01), similar to GTP- binding proteins (Os01t0667600-01, Os01t0179700-01 and Os02t0653800-01) and similar to ethylene- responsive small GTP-binding protein (Os07t0239400-01), were increased following the putrescine application under the Al toxicity conditions (Fig. 3-A and Table S1). Among them, ACBP plays an important role in intermembrane acyl-CoA transport and also donates acyl-CoA for β-oxidation and glycerolipid synthesis (Rasmussen et al, 1994). Cell surface receptor-like kinases (RLKs) are the receptors of small ligands and are important for plant tissue morphogenesis (Ingram and Waites, 2006), such as the regulation of cell proliferation in(Vaddepalli et al, 2011). The B3 domain superfamily proteins are important transcription factors and are involved in regulating seed germination (Suzuki and McCarty, 2008). Ribosome-biogenesis protein is involved in seedling growth (Gachomo et al, 2014) and DNA repair (Gaudet et al, 2011). Ubiquitin ligases (E3) are refined post-translational modification proteins that are involved in improving root growth and increasing environmental stress resistance in plants (Shu and Yang, 2017). The G proteins perceive extracellular stimuli and transmit signals to ion channels, enzymes and other effector proteins to affect numerous cellular behaviors, such as oxidative stress (Joo et al, 2005) and cell proliferation (Ullah et al, 2001).

In addition, the NB-ARC domain protein (Os11t0673600-00) is involved in recognizing pathogens and activating subsequent innate immune responses in plants (van Ooijen et al, 2008); the Ser/Thr protein kinases (Os01t0323000-01), which are involved in regulating intracellular metabolic processes and interfering with the signaling pathways of the infected host cell (Wehenkel et al, 2008), the heat-shock proteins/ chaperones (Os05t0587300-01), which are associated with re-establishing normal protein conformation and helping plants resist Al stress (Wang et al, 2004), were all significantly increased after the application of putrescine under the Al toxicity conditions (Fig. 3-A and Table S1). The increased abundance of the above proteins in the Al + PUT/Al set indicated the multidimensional role of putrescine in the alleviation of Al toxicity in rice.

Fig. 8. Schematic models for rice response to aluminum (Al) toxicity and putrescine (PUT) alleviate Al toxicity.

A, Response mechanism of rice roots to Al toxicity. The presence of Al inhibited the synthesis of ATP, disturbed the Ca2+signal, aggravated the oxidative stress, induced ethylene emissions and increased the pectin content in rice. The rice plants increased peroxidase activity and the accumulation of salicylic acid to resist Al toxicity.

B, A hypothetical model displaying the pathway of PUT-alleviated Al toxicity in rice through an increased pectin methylation degree and decreased pectin content to remove cell wall Al content. Application of PUT under the Al toxicity conditions decreased ethylene emissions by decreasing the lipoxygenase, 1-aminocyclopropane-1-carboxylic acid (ACC) oxidase and aminotransferase protein contents. It inhibited pectin synthesis and increased the pectin methylation level in rice root cell walls. In addition, application of PUT decreased metallophosphoesterase, purple acid phosphatase and UDP-glucosyltransferase to inhibit pectin synthesis in rice roots. All the above processes improved the cell wall Al exclusion and ultimately alleviated Al toxicity in rice.

In conclusion, Al toxicity significantly reduced energy synthesis, disturbed Ca2+signaling, aggravated oxidative stress, induced ethylene emission and increased the polysaccharide content in cell walls (Fig. 8-A). To cope with Al toxicity, antioxidant enzyme activity and the signaling molecule SA increased in rice roots (Figs. 1-G to -J and 5-C). Putrescine alleviated Al toxicity by regulating a set of proteins involved in material metabolism,energy pathway, stress response, cell structure, signal transduction, and transcription and translation pathways. One possible pathway by which putrescine alleviated Al toxicity in rice is shown in Fig. 8-B. In this potential pathway, the addition of putrescine significantly decreased lipoxygenase proteins and thereby decreased ACO abundance, resulting in a decrease in aminotransferase abundance to inhibit ethylene emissions. The decreasedethylene emissions increased the pectinesterase inhibitor and pectin methylesterase content, thereby increasing the degree of pectin methylation. The addition of putrescine also inhibited pectin synthesis by decreasing the purple acid phosphatase, metallophosphoesterase domain-containing protein and UDP-glucuronosyl/ UDP-glucosyltransferase contents. The increase in pectin methylation and decrease in pectin content together removed the cell wall Al content, thereby improving root growth under the Al toxicity conditions.

METHODS

Experimental design and rice culture conditions

Nipponbare rice seeds were soaked in deionized water for 1 d, transferred into 0.5 mmol/L CaCl2and then placed in an incubator for another 2 d at 30 oC in darkness until the roots had grown to about 2 cm long. Four treatments were tested: control (CK), putrescine addition (PUT), aluminum addition (Al), and putrescine with aluminum addition (Al + PUT). Each treatment included 40 seedlings in 2 L black plastic pots. The root elongation under Al toxicity conditions reflects the Al tolerance in plants (Matsumoto, 2000). Therefore, in the present study, the root growth time and the Al concentration were selected based on the combination that inhibited 50% of the root elongation after 24 h of treatment (Fig. S6), and the PUT concentration was selected based on our previous study (Zhu et al, 2019a). The concentration of PUT was 0.1 mmol/L and the Al concentration was 50 μmol/L. The solution pH was adjusted to 4.5. After 24 h of treatment, the rice roots were removed with a blade for further study.

Total Al and Ca contents, and whole rice dry weight measurements

Fresh roots were collected and dried in an oven until a constant weight. The dry roots (0.1 g) were digested in 2 mL of HNO3: HClO4(4:1) at 120 oC. The digestion step was stopped after the solution became clear, and the solution was then diluted to 50 mL with ultrapure water. Al and Ca concentrations were measured via an inductively-coupled plasma mass spectrometry (Shen et al, 2002). Ten whole rice seedlings were dried in an oven at 75 oC until a constant weight. The weights were then recorded and the single rice seedling weight was calculated.

Cell sap and apoplastic Al and P concentration measurements

Rice root apices (1 cm, 10 segments for one replicate) were placed on the surface of 0.45 μm filters in ultra-free MC tubes (1.5 mL, Millipore, Billerica, MA, USA). The tubes were stored at -80 oC for 1 d and then heated to 25 oC to break the cells. The cell sap was collected via centrifugation (Xia et al, 2010). The residues after centrifugation were used to extract apoplastic solution with 1 mL of 2 mol/L HCl. Inductively coupled plasma (ICP) was used to assay the P concentration, and ICP-mass spectrometry (ICP-MS) was used to measure the Al concentration.

Measurement of enzyme activity

For the ROS enzyme measurements, the crude enzyme solution was extracted from 0.1 g fresh roots using 50 mmol/L phosphate buffer solution (pH 7.8). SOD activity was measured according to Beauchamp and Fridovich (1971), and the inhibition of 50% of nitroblue tetrazolium photochemical reduction was defined as one unit of SOD activity. POD activity was assayed according to the changes in tetraguaiacol after crude enzyme solution was added to the reaction mixture (Chen et al, 2013). CAT activity was assayed based on the rate of decrease of H2O2in the reaction system mixture (Dhindsa et al, 1981). GST activity was measured according to the production of 1-chloro-2,4-dinitrobenzene and glutathione at 340 nm absorbance (Farinati et al, 2009).

For the measurement of lipoxygenase activity, the enzyme solution was extracted in 0.1 mol/L phosphate buffer solution (pH 7.5) from 0.5 g fresh rice roots, and its activity was assayed based on the changes at 234 nm absorbance before and after 5 min of reaction after being mixed with 0.1 mL of enzyme solution, 2.5 mL of 100 mmol/L phosphate buffer solution (pH 6.0), and 0.4 mL of 100 mmol/L phosphate solution (pH 8.0) containing 0.25% Tween-20 and 8.6 mmol/L of linoleic acid (Yang et al, 2012).

Measurement of ATP content

We extracted and measured ATP in rice roots according to the method described by K?pnick et al (2018). Fresh rice root homogenate was created by adding 1 mL of cold 0.83 mol/L perchloric acid (Sigma Aldrich, St. Louis, MO, USA) to 0.1 g rice roots on ice. The supernatant was collected after being centrifuged and adjusted to a neutral pH by the addition of 3 mol/L KOH. The BacTiter-Glo Microbial Cell Viability Assay (Promega, Madison, USA) was used to determine the ATP content in a white opaque-walled 96-well microplate (Greiner Bio-One, Frickenhausen, Germany).

Measurement of MDA and H2O2 contents

Fresh rice root homogenate was created by adding 10% trichloroacetic acid to 0.1 g rice roots on ice. MDA was extracted at 95 oC after mixing 0.5% trichloroacetic acid and 10% thiobarbituric acid mixture with the supernatant at a 1:1 ratio. MDA content was measured at 440, 532 and 600 nm (Wang et al, 2017). H2O2was extracted by 3-amino-1,2,4- triazole and measured at 410 nm after being mixed with 0.1% TiCl4dissolved in 20% H2SO4(Yang et al, 2007).

Measurements of pectin content and its demethylesterification degree

Cell wall materials were extracted according to Zhu et al (2018b). The pectin was extracted three times from the cell wallusing 1 mL of 100 oC distilled water. Pectin content was assayed based on the uronic acid content. Pectin demethylesterification degree was assayed according to Zhu et al (2020). Pectin deposition in root apices (0–1 cm) was stained with 0.02% ruthenium red solution and observed by a light stereomicroscope (M205 FA, Leica, Germany) (Ballance et al, 2012).

Measurement of ethylene emission and ACO activity

Two grams of fresh roots were collected and incubated in 20 mL glass vials sealed with a silica gel plug at 30 oC for 2 h in dark. Ethylene was measured by a gas chromatograph (GC-2010 Plus, Shimadzu, Japan) after injecting 10 mL of incubated gas, and its emission rate was calculated based on the fresh weight of rice roots and the incubation time (Yu et al, 2016). ACO activity was measured based on the ethylene emissions (Yu et al, 2016).

qRT-PCR

Total fresh rice root (0.1 g) homogenate was created using liquid nitrogen and TRIzol reagent (Invitrogen, Germany) for total RNA extraction. One microgram of RNA was reverse transcribed into cDNA. The reaction mixture for qRT-PCR was as follows: 1 μL of 10-fold-diluted cDNA, 5 μL of SYBR Premix ExTaq (TaKaRa, Japan), 0.4 μL of forward primer, 0.4 μL of revise primer and 3.2 μL of RNA-free water. The sequences of selected genes and reference gene primers are listed in Table S2 (Xia et al, 2013). The relative expression levels of selected genes were calculated according to Livak and Schmittgen (2001) andwas used as a reference gene.

Protein extraction and digestion

Fresh rice root (approximately 1 g) homogenate was created with liquid nitrogen and 2 mL lysis buffer containing 2% sodium dodecyl sulfate (SDS), 1× Protease Inhibitor Cocktail (Roche Ltd. Basel, Switzerland), and 8 mol/L urea, and the protein was extracted according to Zhu et al (2016). The quality of the extracted protein was measured by SDS-polyacrylamide gel electrophoresis (SDS-PAGE), and the banding was clear with no dragging or fuzzy phenomena observed (Fig. S7). The protein concentration was assayed using a BCA Protein Assay Kit (Chen et al, 2017) and digested according to Zhu et al (2016).

Western blot

The total protein was extracted according to the above method, and the Bradford method was used to determine the protein content (Bradford, 1976). The SDS-PAGE method was used to separate proteins (20 μg for each sample), which were transferred into a 0.45-μm polyvinylidene di?uoride (PVDF) membrane immediately (Laemmli, 1970). The protein blot was probed with a primary antibody of lipoxygenase (AS06 128, Agrisera, Sweden), ATP synthase (AS08 370, Agrisera, Sweden) and monoclonal anti-actin (plant) antibody produced in mice (A0480, Sigma, USA) at dilutions of 1:1000, 1:10000 and 1:1000, respectively, after resting on PVDF membrane for 2 h at 25 oC. We then probed the secondary antibody (HRP-labeled Goat Anti-Rabbit IgG (H+L) and HRP-labeled Goat Anti-Rat IgG (H+L), 1:1000 dilution, Beyotime, China) for 2 h at 25 oC. Enhanced chemiluminescence (Pierce, Waltham, WA, USA) was used to obtain blot images, and the Quantity One software (Bio-Rad, Hercules, CA, USA) was used to analyze band intensities.

High pH reverse-phase separation

The digested protein was re-dissolved in 0.02 mol/L ammonium formate (pH 10.0). An Ultimate 3000 System (Thermo Fisher Scientific, MA, USA), which was equipped with a reverse- phase column (XBridge C18 column, 4.6 mm × 250.0 mm, 5 μm; Waters Corporation, MA, USA), was used to fractionate the digested protein mixture for 40 min in 0.02 mol/L of ammonium formate (containing 80% acetonitrile, pH 10.0) with a column flow rate of 1 mL/min at 30 oC. The vacuum-dried fractions were collected for further study.

Data-independent analysis (DIA): nano-high-performance liquid chromatography (HPLC)-MS/MS analysis

The online nanospray liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis was conducted on an Orbitrap Fusion Lumos equipped with an EASY-nLC 1200 system (Thermo Fisher Scientific, MA, USA). Three microliters of peptides dissolved in 0.1% formic acid solution sample were injected into an analytical column (Acclaim PepMap RSLC C18, Thermo Fisher Scientific, MA, USA; 75 μm × 25 cm, particle size of 3 μm and pore size of 75 μm) using 0.1% formic acid solution. The column flow rate was 200 nL/min, the temperature was 40 oC, the electrospray voltage was 2 kV, and each run was 120 min. The mass spectrometer was run under the DIA mode, and the parameters for the MS were set according to Zhao et al (2020). The differentially expressed proteins (DEPs) with more than 1.5-fold change and< 0.05 (Student’s-test) were considered statistically significant.

Spectronaut X (Biognosys AG, Switzerland) was used to analyze the DIA raw data according to Zhao et al (2020). A 1% false discovery rate-corrected-value was applied as a cutoff for precursors and proteins. The major group quantities were calculated from the top-3 filtered peptides. The proteins that changed more than 1.5-fold and obtained avalue < 0.05 (Student’s-test) were defined as significantly different.

Protein functional annotation and enrichment analysis

The UniProt (http://www.uniprot.org) and NCBI protein databases (http://www.ncbi.nlm.nih.gov) were used to search the protein functions. The functions of DEPs were determined using GO annotation (https://www.ebi.ac.uk/GOA/), and the KEGG pathway analysis (https://www.kegg.jp/kegg/pathway.html) was used to analyze protein and enrichment functions. The UniProt-GOA database (http://www.ebi.ac.uk/GOA/) and the InterProScan software were used for GO annotations. Cluster version 3.0 was used to create three hierarchical clusters of selected proteins: PUT/CK, Al/CK and Al + PUT/Al. Tree-view version 1.1.3 was used to plot the results.

Statistical analysis

All experiments were performed with four independent biologicalreplicates, except the proteomic experiment with three independent biological replicates. One-way analysis of variance (ANOVA) was used to analyze the data, and a post hoc Tukey’s test was used to compare the mean values at< 0.05 (SPSS 13.0). R (version 3.0.0; R Development Core Team) was used to perform multivariate analyses using the ‘vegan’ package (Ma et al, 2015).

ACKNOWLEDGEMENTS

This study was supported by the Zhejiang Provincial Natural Science Foundation of China (Grant No. LQ19C020007), the National Natural Science Foundation of China (Grant Nos. 31901452, 31771733, 32001104 and 31872857), the Key Research and Development Program of Zhejiang Province, China (Grant No. 2021C02002), the Open Project of State Key Laboratory of Rice Biology of China (Grant No. 20190402), and the Basic Research Foundation of National Commonweal Research Institute of China (Grant No. 2017RG004-2).

SUPPLEMENTAL DATA

The following materials are available in the online version of this article at http://www.sciencedirect.com/journal/rice-science; http://www.ricescience.org.

Fig. S1. Numbers of different peptide molecular weights from the database.

Fig. S2. Numbers of different peptide lengths from the database.

Fig. S3. Values of identity statistics, Pearson’s correlation coefficient, principal component analysis and partial least squares discriminant analysis of samples from four treatments.

Fig. S4. Distribution of peptide numbers identified.

Fig. S5. Box diagram of all samples after normalization.

Fig. S6. Relative root elongation under different Al concentrations and treatment times.

Fig. S7. Protein quality examined by SDS-PAGE.

Table S1. Differentially expressed proteins in rice roots under different treatments.

Table S2. List of primers used.

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Zhang Junhua (zhangjunhua@caas.cn); Zhang Hui (9833672@qq.com)

12 October 2020;

1 March 2021

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