Exogenous proanthocyanidins improve tolerance of Cu-toxicity by amelioration of oxidative damage and re-programming of gene expression in Medicago sativa

Excess copper (Cu) in soil due to industrial and agricultural practices can result in reduced plant growth. Excess Cu resulted in severely retarded root growth with severe discoloration of Alfalfa (Medicago sativa) and Medicago truncatula. Growth in the presence of hydrogen peroxide resulted in similar symptoms that could be partially recovered by the addition of the reductant ascorbic acid revealing damage was likely due to oxidative stress. The addition of proanthocyanidins (PAs) in the presence of Cu prevented much of the damage, including plant growth and restoration of lignin synthesis which was inhibited in the presence of excess Cu. Transcriptome analyses of the impact of excess Cu and the amelioration after PAs treatment revealed that changes were enriched in functions associated with the cell wall and extracellular processes, indicating that inhibition of cell wall synthesis was likely the reason for retarded growth. Excess Cu appeared to induce a strong defense response, along with alterations in the expression of a number of genes encoding transcription factors, notably related to ethylene signaling. The addition of PAs greatly reduced this response, and also induced novel genes that likely help ameliorate the effects of excess Cu. These included induction of genes involved in the last step of ascorbic acid biosynthesis and of enzymes involved in cell wall synthesis. Combined, these results show that excess Cu causes severe oxidative stress damage and inhibition of cell wall synthesis, which can be relieved by the addition of PAs.


Introduction
Copper (Cu) is an essential element that acts as a co-factor for a variety of enzymes involved in various biological processes, including photosynthesis, respiration, reactive oxygen species (ROS) scavenging, cell wall remodeling, and ethylene perception [1,2]. Cu 2+ ions act as a a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 which influence their bioavailability [27]. PAs are natural polyphenolic antioxidants that are widely available in plants, especially in fruits and seeds [28,29], and show excellent protection against free radicals with greater reductant capacity than AsA, tocopherol E, and β-carotene [30]. In Arabidopsis, the germination of PA-deficient mutant seeds was inhibited under oxidative stress conditions, suggesting a loss of PA-related anti-oxidative ability [31]. PA-deficient mutants also exhibit reduced dormancy and faster deterioration during natural aging at room temperature [32], and ROS damage is considered as the key factor inducing seed deterioration. In forage crops, the presence of PAs is an important agronomic trait. PAs bind to proteins in the rumen and reduce the rate of fermentation and methane production, through which more intact proteins to exit the rumen, thus improving nitrogen nutrition [33]. In addition, cucumber seedlings pretreated with exogenous PAs suffered less damage by the abiotic stress, such as high radiation, polyethylene glycol, and cold stress maintained higher content of chlorophyll, abscisic acid, and alternative oxidase protein [34].
Alfalfa (Medicago sativa) is an important perennial leguminous forage species of high nutritional value to livestock. Alfalfa has a good ability to withstand heavy metal toxicity, including that caused by Cd, Ni, Cu, and Hg; most of the information on this response of alfalfa has been confined to studies on antioxidant defense systems [35][36][37][38]. This work was carried out to investigate the changes in seedling growth, physiology and oxidative stress in alfalfa plants in response to Cu toxicity and the associated potentially protective mechanisms of PAs. We also carried out transcriptome analysis in Medicago truncatula to examine the changes that occurred due to Cu toxicity and its amelioration by PAs.

Seed germination test
Seeds of two alfalfa cultivars, WL656 and Southern Hemisphere (SH), were obtained from Hunan Agricultural University, Changsha, China. Seeds were sterilized in 5% (v/v) sodium hypochlorite for 10 min and rinsed 5-6 times with distilled water. Seeds were germinated on sterile filter paper under 16 h light/8 h dark at 25 ± 2˚C, and 60% relative humidity. For pretreatments with PAs and AsA, 5 ml of different concentrations PAs (0, 50, 100, 150, 200, 250 mg L -1 ) and different concentrations of AsA (0, 50, 100, and 150 mg L -1 ) were used, respectively, as germination medium for the first 2 d. For Cu and H 2 O 2 treatments, 5 ml of different concentrations of CuSO 4 •5H 2 O (0, 25, 50, 100, 200 and 400 mg L -1 CuSO 4 •5H 2 O) or different concentrations of H 2 O 2 (0, 90,180, and 450 mM) were added after 2 d of germination of seeds that were pretreated with PA, AsA, or H 2 O. Distilled water was used as the control treatment. Seed germination rate, root length, and root dry weight were measured after 10 d of germination. Each treatment was carried out in triplicates.
The seeds of Medicago truncatula (R108) were polished with sandpaper to promote germination. These seeds were pretreated with 100 mg L -1 PAs or H 2 O for 2 d and then changed to 100 mg L -1 Cu. Seed germination rate and root length were measured after 10 d of germination. Each treatment was carried out in triplicates.

Detection of H 2 O 2 by 3,3-diaminobenzidine (DAB) staining
Detection of H 2 O 2 by DAB staining was carried out as previously described [39]. Ten-day-old WL656 and SH seedlings were immersed in DAB staining solution (1 mg mL -1 , pH = 5.8), incubated in vacuum for 10 min, and then kept on a shaker for 5 h at 80 rpm in the dark. Samples were then decolorized in 80% ethanol in a boiling water bath. After decoloration, samples were treated with 100% ethanol and then kept at 4˚C for 4 d.

RNA isolation and qRT-PCR
Total RNA was extracted from the plant tissues using a TRIzol kit (TransGen Biotech, Beijing, China) and was reverse transcribed into cDNA using a PrimeScript™ RT Reagent Kit with gDNA Eraser (Takara, Dalian, China) according to the manufacturers' instructions. The qRT-PCR analysis was performed in a 20 μL reaction volume containing 10 μL of SYBR Premix ExTaq™ (Takara, Dalian, China), 2 μL of cDNA, and 0.5 μM of each primer. The primer pairs used for qRT-PCR are listed in S1 Table. ACTIN was used as a reference for normalization. The cycle thresholds were determined using a Roche Light Cycler 480 II sequence detection system (Roche, Shanghai, China).

RNA seq analysis
RNA-seq libraries were performed using the TruSeq Stranded mRNA Library Prep Kit according to the manufacturer's instructions (Illumina) with total RNA isolated as described above, and sequenced on a NextSeq500 system (Illumina) with an average quality score (Q30) of above 95%. Quality control was carried by using FastQC software (https://www. bioinformatics.babraham.ac.uk/projects/fastqc/). A paired two-tailed t-test on the FPKM (per kilobase per million mapped reads) [40] values for each comparison, that is, Cu/control and PAs/control, was conducted. Genes with p-value < 0.05 and |Log 2 FC|�1 were considered significant in each comparison. Fold change was calculated by comparing Cu/control and PA/ control. Log 2 FC was calculated by taking log 2 of the calculated fold change. Genes with FPKM values equal to zero for both replicates in a treatment were not considered in the analysis. Genes that passed the p-value cut off and FPKM threshold in each comparison were used for Venn diagram construction. GO (Gene Ontology) term enrichment analysis was performed on selected overlap groups using the ClueGO plugin for Cytoscape [41]. RNA-seq raw data were deposited at the National Center for Biotechnology Information Sequence Read Archive database under project identifier PRJNA734715.

Histochemical staining of lignin
Histochemical staining of lignin was carried out according to the method described previously [42]. The stem segment of Medicago truncatula samples was sectioned manually using a razor blade. Isolated sections were stained in 5% phloroglucinol staining solution at room temperature for 3 min. Images were acquired using a fluorescence microscope (Olympus BX51, Japan).

Statistical analysis
The data were statistically analyzed using SPSS software (Chicago, USA), and the significance was calculated based on the one-way ANOVA test of three biological replicates. The difference was a significant difference when p � 0.

PAs improved the tolerance of alfalfa seedling exposed to excess Cu
The effects of PA pretreatment on high Cu toxicity on alfalfa were assessed by using WL656 and SH cultivars. Two days old seedlings were treated with medium containing 25 to 400 mg L -1 CuSO 4 •5H 2 O (defined as Cu25 to Cu400). Cu toxicity symptoms appeared on roots grown with Cu25 to Cu100 medium ( Fig 1A). Root Growth was severely inhibited in Cu200 and Cu400. Leaves underwent browning or senescence when Cu concentration was over 100 mg L -1 . Therefore, 100Cu was chosen for Cu treatment but did not cause lethality (Fig 1A and S1  Fig). Cu toxicity was alleviated when seeds were pretreated with different concentration of PAs (from 50 to 250 mg L -1 , defined as PA50 to PA250) for both cultivars WL656 and SH (Fig 1B), and PA100 showed optimum effects against Cu toxicity. Root growth was inhibited when PA concentration was greater than 200 mg L -1 (Fig 1B). The combination of Cu100 and PA100 were chosen for further experiments. WL656 and SH had pale roots with abundant root hair

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The role of proanthocyanidins in protection against copper toxicity ( Fig 1C). After exposure to excess Cu, the phenotype of root exhibited growth retardation, the roots changed to brown or green and were shorter without any visible root hair. PAs pretreatment partially mitigated Cu toxicity in the roots, although the root status did not recover to that of the control level. Roots remained whitish or light brown with observable root hair formation ( Fig 1C). While Cu treatment significantly (P < 0.05) decreased root length and root dry weight by~60% and~80%, respectively, PA treatment significantly (P < 0.05) improved root length and root dry weight (Fig 1D and 1E).

PAs improved the performance of alfalfa seedling under oxidative stress
The effect of excess Cu stress and PAs treatments on ROS generation in alfalfa plants was assessed by in vivo histochemical staining with DAB. There was an increase in the formation of dark brown lesions on leaves and roots, especially the root tips, after exposure to Cu stress (Fig 2A and S2 Fig). When plants were pretreated with PAs, the dark brown lesions reduced in leaves and roots, and the color of leaves almost recovered to that observed in the control (Fig 2A and S2 Fig).
Seedling development was inhibited after exposure to an increasing concentration of H 2 O 2 from 90 mM to 450 mM. The phenotype of plants treated with 450 mM H 2 O 2 was very similar to those treated with Cu100, with severely inhibited root development ( Fig 2B). Root length decreased progressively with increasing H 2 O 2 stress. Under minor H 2 O 2 stress (90 mM H 2 O 2 ), the root length of plants recovered to that of the control after PAs pretreatment. However, the root length of plants under 450 mM H 2 O 2 treatment recovered less than 20% compared to that of the control. PAs treatment significantly (P < 0.05) improved root length for both WL656 and SH treated with different concentrations of H 2 O 2 (Fig 2C and 2D). Analysis of DAB staining revealed that dark brown lesions increased with increasing H 2 O 2 concentrations. PAs reduced the formation of dark brown lesions, especially under high H 2 O 2 stress ( Fig 2E). Thus, PAs may protect alfalfa seedlings from Cu toxicity by eliminating ROS such as H 2 O 2 produced due to excess Cu.

PAs alleviated oxidative stress induced by excess Cu by its antioxidant capacity
AsA is an excellent antioxidant that protects plants from oxidative damages via its ROS scavenging capability [37]. Different concentrations of AsA from 50 to 150 mg L -1 were used to treat both WL656 and SH before treating with excess Cu. AsA improved the performance of alfalfa seedling at each concentration with observable white and elongated roots and reduced DAB staining (Fig 3A and S2 Fig). Likewise, pretreatment with both 100 and 150 mg L -1 PAs showed a similar protective effect as did AsA against Cu stress. AsA treatment significantly (P < 0.05) increased root length under Cu stress, and the effect was more significant (P < 0.05) when treated with 150 mg L -1 AsA than with 50 or 100 mg L -1 AsA. PAs treatment at the same concentration showed significantly increased root length than that after AsA treatment (Fig 3B and 3C). Therefore, PAs may act as a strong reductant to improve the tolerance of alfalfa seedlings to high Cu stress.

PAs improved the growth rate of Medicago truncatula (R108) seedling under Cu stress
Alfalfa is autotetraploid and the genomes of different cultivars vary. Although the alfalfa genome has been sequenced, the genomic information is still emerging [43,44]. To explore the underlying mechanism of PAs-mediated protection in alfalfa, we choose Medicago truncatula

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The role of proanthocyanidins in protection against copper toxicity R108 for further studies. PAs showed similar protection in R108 seedling development compared to two alfalfa cultivars, WH656 and SH. Seedling growth and root length in these cultivars were severely inhibited after exposure to different concentrations of Cu compared to those in the control (Fig 4A). Seedlings pretreated with 100 mg L -1 PAs could alleviate Cu toxicity with elongated root length (Fig 4B).

Differentially expressed genes in Medicago truncatula R108 under high Cu stress
RNAseq analysis was carried out to compare the effect of Cu treatment with or without PAs pretreatment with that in the control. A paired two-tailed t-test was conducted on the FPKM values for different comparisons. Gene expressions changed two-fold and p-value < 0.05 was considered significant in each comparison. A total of 1,791 differentially expressed genes  Table). Treatment with excess Cu induced more DEGs compared to PAs pretreatment, with 663 up-regulated, and 528 down-regulated DEGs. Plants pretreated with PAs (+ PAs + Cu) under Cu stress showed 293 up-regulated and 461 down-regulated DEGs (Fig 5B). Excess Cu induced a greater than a two-fold increase in the expression of over 398 genes, and in the majority of the cases this was reduced after PAs pretreatment, indicating the ability of PAs to prevent the changes in gene expression. Many of the induced genes were related to pathogen defense so that excess Cu could trigger a defense response, in agreement with previous reports in Arabidopsis of activation of ethylene and salicylate defense pathways due to excess Cu [45].
Enrichment analysis of DEGs for cellular components showed that genes related to the extracellular region, an intrinsic component of plasma, plasma membrane, and cell wall were more enriched in response to excess Cu treatment. However, more enriched DEGs were related to the extracellular region, external encapsulating structure, and cell wall when plants were pretreated with PAs (Fig 5C and 5D). Molecular function enrichment analysis showed that genes related to antioxidant activity were enriched in response to Cu treatment (S3 Fig). The transcript expression levels of genes involved in antioxidant activity were tested in both alfalfa cultivars WL656 and SH (S4 Fig). The relative transcript levels of manganese/iron superoxide dismutase (MsG0380016734), copper/zinc superoxide dismutase (MsG0680031329), Catalase (MsG0380017935) and Glutathione reductase (MsG0680031828) were all up-regulated under Cu treatment, this was reduced after PAs treatment, indicating that PAs likely decreased the oxidative stress, consistent with the above-mentioned physiology. The growth stage and ROS level indicated that PAs pre-treatment could ameliorate Cu toxicity. A high accumulation of ROS was reported to signal initiation of cell wall formation [46]. Therefore, we investigated whether this had any effect on genes associated with the cell wall. RNA-seq data revealed changes in the expression of a variety of genes and most of them were associated with cell wall or extracellular processes (Fig 5C). It was apparent that although the addition of PAs could rescue growth in the presence of Cu at a toxic level, it did not restore the status of seedlings to the normal growth path (Fig 6, S3 Table). Examination of the genes encoding proteins associated with cell wall synthesis revealed that the pattern of PAs

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The role of proanthocyanidins in protection against copper toxicity pretreatment was not similar to that in wild type or the control. Thus, 26 genes that control cell wall components were down-regulated after Cu treatment, still remained low after PAs pretreatment (Fig 6); in fact, five more genes, including peroxidase family protein, glycoside hydrolase family protein 17, class I chitinase, pectinesterase inhibitor, and myosin II heavy chain family protein were down-regulated with PAs and Cu treatment compared to Cu alone (Fig 6). The expression of two genes associated with cell wall increased after PAs and Cu treatment-cellulose synthase-like protein and GDP-L-galactose phosphorylase, the latter is a key enzyme that catalyzes the last step of ascorbic synthesis in plants [47,48]. Thus, PAs treatment

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The role of proanthocyanidins in protection against copper toxicity appeared to up-regulate the synthesis of a crucial anti-oxidant, AsA, which, as shown above, can ameliorate the toxic effects of Cu toxicity (Fig 3). Also, endo-1,4-beta-glucanase (MTR_3g0822440), which plays a key role in cell wall synthesis and growth, exhibited notable up-regulation. The increase here after PAs and Cu treatment, compared to both untreated and

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The role of proanthocyanidins in protection against copper toxicity only Cu-treated samples suggests that an additional enzyme is activated to drive growth [49]. Furthermore, the induction of glycoside hydrolase family 17 protein and chitinase with Cutreatment reduced significantly in the presence of PAs, and the breakdown of the cell wall was prevented [50]. Finally, the large increase in the expression of a gene encoding myosin after PA and Cu treatment plays a role in the exocytosis of cellulose synthase complexes [51]. Relative transcript expression levels of eight orthologous genes encoding proteins associated with cell wall synthesis were tested in alfalfa cultivars WL656 and SH, including cellulose synthaselike protein D3, GDP-L-galactose phosphorylase, Chitinase (Class Ib) / Hevein, Chitinase (Class I) / Hevein, cellulose synthase E1-like protein, glycoside hydrolase family 17 protein, cytochrome P450 family 71 protein, endo-1,4-beta-glucanase. Transcripts in alfalfa showed similar changes as transcripts in Medicago truncatula, confirming that cell wall synthesis was disturbed after Cu treatment, and PA alleviated Cu toxicity in cell wall synthesis (S5 Fig). In terms of transcription factors, several ethylene AP2 type transcription factors changed in abundance with excess Cu, including Medtr1g098460 and Medtr7g055857 by greater than five-fold that was ameliorated by PAs (S4 Table). There are conflicting reports about ethylene induction in Cu toxicity [52,53], but it appears that at least some factors related to ethylene signalling are altered, but this may be due to alterations of either ABA or auxin, that have also been reported to be involved in Cu toxicity. A TCP (Teosinte branched1/Cincinnata/proliferating)-like transcription factor was also induced by more than five-fold and was reduced after PAs treatment. TCP transcription factors can reduce meristem activity and resulting growth [54]. Almost three times the amount of transcription factors were altered after PAs and Cu treatment compared to that after Cu treatment alone. A variety of other transcription factors were also induced by Cu, including a negative regulator of transcription (Medtr4g070340) and a TAZ domain transcription factor (Medtr5g015680), that interacts with a variety of other transcription factors.

Discussion
Cu toxicity in the soil can result in a reduction of plant germination and growth, and also pose a threat to human health as excess Cu levels may accumulate in food. This study reveals that excess Cu resulted in severe oxidative stress leading to retarded growth and development, similar to that was observed after H 2 O 2 was added, and these symptoms could be partially relieved upon the addition of AsA and PAs. Oxidative stress occurred in seedlings exposed to excess Cu and it was expressed as considerably greater staining of H 2 O 2 than in control plants. H 2 O 2 is an important redox molecule as it is remarkably stable (half-life > 1 ms) in plant cells [57]. H 2 O 2 can be transported by cell membrane aquaporins and react with DNA and proteins by oxidation of cysteine and methionine residues, causing long-distance oxidative damage [57,58]. Enhanced levels of H 2 O 2 were also observed in wheat (Triticum aestivum L.), rice (Oryza sativa L.), and Arabidopsis thaliana under Cu stress [21,59,60]. In this study, alfalfa seedlings treated with a high concentration of H 2 O 2 showed a similar phenotype as that after excess Cu treatment, indicating that Cu toxicity in plants was induced partially by H 2 O 2

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The role of proanthocyanidins in protection against copper toxicity oxidative damage. PAs pretreatment increased alfalfa seed germination, enhanced healthy root development, and root hair formation, demonstrating the ameliorative role of PAs in reducing Cu toxicity. On the other hand, PAs pretreatments showed similar protective effects on alfalfa plants as those by AsA under Cu stress. AsA, the most abundant non-enzymatic antioxidant in plants, is oxidized to monodehydroascorbate by the reaction catalyzed by ascorbate peroxidase, and thus detoxifies oxidative damage caused by H 2 O 2 [61,62]. Increasing concentration of PAs and AsA alleviated Cu toxicity on alfalfa plants by increasing root length, and PAs showed better performance than AsA at the same concentrations, indicating that PAs is a powerful antioxidant. DAB staining also confirmed that PAs reduced the accumulation of H 2 O 2 in the root and leaf. The results indicate that Cu stress possibly induces an increase in H 2 O 2 level and that PAs pretreatment could provide protection to plants by detoxification of H 2 O 2 damage.
While it is clear that oxidative stress was the primary source of damage in excess Cu, lignin biosynthesis was noticeably reduced in the presence of excess Cu in this study. Lignin is found in plant secondary cell walls and the biosynthesis and polymerization of lignin require H 2 O 2 , its synthesis is often induced in the presence of pathogens and excess heavy metals [63]. Conversely, lignin was much reduced but was restored by the addition of PAs in our study. The inhibition of lignin biosynthesis here may be due to a number of reasons, primarily cell death by autophagy resulting from the excess Cu levels in this study, as has been reported previously for excess heavy metals, then respiration and photosynthetic disruption to produce energy in the form of ATP and the precursors for lignin biosynthesis, as lignin is the second most abundant biopolymer, and oxidative damage to both protein and lipids would result in the inability to transport the precursors to the cell surface. The addition of a reductant, either AsA or PAs, resulted in partial restoration of many parameters by relieving this oxidative stress and the restoration of lignin biosynthesis. In fact, this appeared to be an overall trend in the treatment with Cu alone, because gene ontology analysis showed that a variety of up-regulated DEGs were enriched in extracellular activities. Moreover, many of these up-regulated genes were greatly reduced by pretreatment with PAs, to the extent that the alfalfa could germinate and survive. Thus, pretreating seeds with reductants or compounds may be an effective strategy to prevent the massive oxidative damage and ensure the establishment of plants in Cu-rich soils that may be toxic to their development. Once established, the plant can carry out a variety of processes, involving lignin biosynthesis to tolerate high levels of Cu.
Besides Cu, a number of other heavy metals can also accumulate in the soil including aluminum, Cd, and zinc. Additionally, a pathogen attached to young seedlings can greatly inhibit the establishment. A common response to all these root stresses appears to be involved in the secondary wall and lignin synthesis combined with increased activity of anti-oxidant stress systems [63]. Thus, Cd tolerance in alfalfa reveals the importance of glutathione-based anti-oxidant systems [64]. Overexpression of glutaredoxin in Arabidopsis reduced the accumulation of heavy metals and increased the level of antioxidant molecules and various physiological parameters [65]. In our study here, the induction of AsA biosynthesis was a notable response to excess Cu, and the tolerance could be enhanced by the addition of PAs. Thus, a combination of coating the seeds with strong anti-oxidants such as PAs prior to sowing and selection of anti-oxidant optimized germplasm will be helpful in improving tolerance to excess Cu and other oxidative stress. Although our assays indicated that exogenous addition of PAs could improve alfalfa tolerance to excess Cu, in accordance with previous reports [31], we suspected that the content of endogenous PAs on the seed coat may also affect the germination of alfalfa. More assays need to be carried out in the future to support our speculations, such as selecting different cultivars of alfalfa and analyze the relationship of PAs content in seed coat and the data of germination and other phenotypes in the excess Cu stress.
AsA biosynthesis occurs in the mitochondria. In this study, the addition of AsA and the induction of the last step of AsA biosynthesis were found to be key aspects. Mitochondria, particularly mitochondrial citrate synthase, aggregation of mitochondria, and mitochondrial programmed cell death are linked to metal toxicity [66][67][68]. Overexpression of the mitochondrial alternative oxidase alleviates mitochondrial-induced cell death due to aluminum toxicity [69]. Given the central role of mitochondria in REDOX biology, programmed cell death, and stress signalling, optimizing mitochondrial biogenesis and activity would be another strategy to improve the tolerance of plants to heavy metal.

Conclusion
Excess Cu results in strong oxidative stress that cannot be overcome by plant defense alone. The addition of PAs partially but not totally enables basic plant defense including lignin biosynthesis and anti-oxidant systems, to help plants to overcome this stress, and allow plant germination and seedling growth. Multiple strategies, chemical and genetic, can be deployed to overcome heavy metal tolerance.