Intrinsic Mechanism of CaCl2 Alleviation of H2O2 Inhibition of Pea Primary Root Gravitropism

Normal root growth is essential for the plant uptake of soil nutrients and water. However, exogenous H2O2 inhibits the gravitropic growth of pea primary roots. It has been shown that CaCl2 application can alleviate H2O2 inhibition, but the exact alleviation mechanism is not clear. Therefore, the present study was carried out by combining the transcriptome and metabolome with a view to investigate in depth the mechanism of action of exogenous CaCl2 to alleviate the inhibition of pea primordial root gravitropism by H2O2. The results showed that the addition of CaCl2 (10 mmol·L−1) under H2O2 stress (150 mmol·L−1) significantly increased the H2O2 and starch content, decreased peroxidase (POD) activity, and reduced the accumulation of sugar metabolites and lignin in pea primary roots. Down-regulated genes regulating peroxidase, respiratory burst oxidase, and lignin synthesis up-regulated PGM1, a key gene for starch synthesis, and activated the calcium and phytohormone signaling pathways. In summary, 10 mmol·L−1 CaCl2 could alleviate H2O2 stress by modulating the oxidative stress response, signal transduction, and starch and lignin accumulation within pea primary roots, thereby promoting root gravitropism. This provides new insights into the mechanism by which CaCl2 promotes the gravitropism of pea primary roots under H2O2 treatment.


Introduction
Pea (Pisum sativum L.) is a strategic crop that ensures global food security and is favored for its versatility, nutrient richness, and adaptability [1].The seedling stage of pea plants usually occurs during the low-rainfall season; thus, drought affects the growth of pea seedlings and, consequently, their yield and quality [2].Roots absorb nutrients and water directly into the soil and act as sensors to sense and respond to a variety of external stresses [3].As the only source of water and nutrient uptake for the seedling, the primary root must enter the soil without other support in order to be better adapted to the living environment [4].Therefore, improving root growth capacity is a strategy to increase pea yield.It has been found that positive root geotropism is necessary for plant access to nutrients and water [5].However, exogenous signals can change the plant's root system architecture (RSA) and root growth direction, thus inhibiting plant growth [6].Therefore, it is important to study the pathways that regulate the alteration of RSA and root growth direction.
Reactive oxygen species (ROS) are not only components that induce oxidative damage in the plant body but also act as signaling molecules to regulate plant growth [7].ROS can alter the RSA by regulating lateral root formation in Arabidopsis thaliana [8].In contrast, intracellular ROS homeostasis is regulated by a series of enzymes that include antioxidant enzymes such as NADPH/NADH, superoxide dismutase (SOD) (EC 1.15.1.1),oxidases, and peroxidase (POD) (EC 1.11.1.7)[9,10].It has been found that ROS are involved in the signaling of the plant hormones abscisic acid (ABA) and auxin (IAA), which regulate seed germination, root growth, and differentiation [11].H 2 O 2 mediates NADPH oxidase to enable ABA activation of lateral root development, the synthesis of H 2 O 2 , and cell wall expansion [12][13][14].Interaction between ROS and IAA can regulate root geotropism, lateral and adventitious root formation, and lignification [15].Synergistic interactions occur between IAA and gibberellin (GA), wherein IAA induces the degradation of the DELLA protein, which inhibits GA signaling and is involved in the promotion of GA biosynthesis gene expression in terms of root elongation and root division [16].
In addition, H 2 O 2 is involved in the regulation of calcium signaling pathways.It has been found that the H 2 O 2 produced by tobacco cell inducers may activate H 2 O 2 -sensitive Ca 2+ channels in the plasma membrane, resulting in an increase in cytoplasmic Ca 2+ concentrations [17].Ca 2+ channels can be activated by NADPH oxidase to regulate plant cell expansion and, thus, cell development [18].External signals stimulate the amyloplasts to settle on the endoplasmic reticulum, causing Ca 2+ to be effluxed into the cytoplasm.This may cause a transient increase in cytoplasmic Ca 2+ concentration, which, upon binding to calmodulin, either directly or indirectly activates the downstream signal transduction mechanisms [19].Whereas starch is a key substance in the perception of gravity by the root system [20], changes in Ca 2+ concentration caused by starch deposition regulate the direction of root growth.
It was found that cell wall accumulation limits cell elongation, thereby altering the RSA [21].Cell wall metabolism is a key factor in plant response to environmental stresses and it is mediated by a variety of cell wall-modifying proteins.Soluble arabinogalactan proteins (AGPs) and insoluble extensions (EXTs) are associated with each other in the cell wall, and the EXT/AGP complex acts on the structure of the cell wall to regulate changes in the shape of the cell wall [22,23].Increased H + concentrations activate extensin activity and activate the cell wall, which leads to cell elongation [24].Therefore, it is important to investigate the mechanism of action of exogenous H 2 O 2 and CaCl 2 on the cell walls of the primary roots of pea plants.
From the above, it is clear that H 2 O 2 and Ca 2+ are key signaling molecules that regulate the gravity-oriented nature of plants.It has been shown that exogenous H 2 O 2 inhibits the gravity-oriented force of pea primary roots, while CaCl 2 can alleviate this inhibition to a certain extent, but the specific alleviation mechanism has not been clarified [25].Therefore, it is crucial to study in depth the alleviating effect of CaCl 2 on H 2 O 2 stress.Thus, this study combined transcriptomics and metabolomics to comprehensively analyze the pathways regulating exogenous CaCl 2 to alleviate the inhibition of pea root gravitropism by H 2 O 2 .We also verified the roles of key metabolites and genes by combining the relevant physiological indicators with fluorescence quantification.This study provides a theoretical basis for the gravity-oriented nature of plant roots in adversity.

Root Non-Geostrophic Validation Experiments
In this study, we counted the bending rate (Table S1 and Figure S1 in the Supplementary Materials) and bending degree (Table S2 and Figure S2 in the Supplementary Materials) of pea primordial roots after 24, 36, 54, and 72 h of different treatments.The results showed that the shorter the treatment time, the greater the difference between replicates.Therefore, the main discussion in Section 3 is on the changes seen in the growth of pea primary roots at 72 h and the intrinsic mechanism.
Pea primary roots grew curved under exogenous H 2 O 2 treatment (Figure 1A), and the bending rate and bending degree of primary roots (Figure 1B and Table S3) gradually increased with increasing concentration.The bending rate of primary roots under 150 mmol•L −1 H 2 O 2 treatment was 3.3-fold higher than that of 20 mmol•L −1 H 2 O 2 .The subsequent application of different concentrations of CaCl 2 on top of the 150 mmol•L −1 H 2 O 2 treatment (Figure 1) revealed that CaCl 2 had a mitigating effect on the bending growth of pea primordial roots under the H 2 O 2 treatment (Figure 1A).With increasing CaCl 2 concentration, the bending rate and bending degree of primordial roots had minimum values at the 10 mmol•L−1 CaCl 2 and were significantly lower than for the 150 mmol•L −1 H 2 O 2 treatment (Table S3).The application of 10 mmol•L −1 CaCl 2 to pea primordial roots alone revealed no significant difference in growth status with CK treatment.increased with increasing concentration.The bending rate of primary roots under 150 mmol•L −1 H2O2 treatment was 3.3-fold higher than that of 20 mmol•L −1 H2O2.The subsequent application of different concentrations of CaCl2 on top of the 150 mmol•L −1 H2O2 treatment (Figure 1) revealed that CaCl2 had a mitigating effect on the bending growth of pea primordial roots under the H2O2 treatment (Figure 1A).With increasing CaCl2 concentration, the bending rate and bending degree of primordial roots had minimum values at the 10 mmol•L −1 CaCl2 and were significantly lower than for the 150 mmol•L −1 H2O2 treatment (Table S3).The application of 10 mmol•L −1 CaCl2 to pea primordial roots alone revealed no significant difference in growth status with CK treatment.To further verify the important role of Ca 2+ in mitigating the H2O2 inhibition of root growth toward gravitropism, CaSO4 and KCl, used at the same ionic (Ca 2+ , Cl − ) concentrations as the CaCl2 solution, were applied exogenously (Table S4 and Figure S3).It was found that the pea bending rate (Table S1 and Figure S1) and bending degree (Table S2 and Figure S2) were significantly reduced under the CaSO4 treatment compared with the 150 mmol•L −1 H2O2 treatment, and that these reached a minimum at a Ca 2+ concentration of 10 mmol•L −1 , while KCl treatment had no significant mitigating effect on H2O2 inhibition.
Based on these statistical results, pea primary roots under the 150 mmol•L −1 H2O2 and 150 mmol•L −1 H2O2 + 10 mmol•L −1 CaCl2 treatments were selected for the study of transcriptome and metabolome indexes.

Transcriptome Analysis and Validation of Key DGEs in Pea Primary Roots under H2O2 and CaCl2 Treatment
To investigate the mechanism of action of pea root growth under the CaCl2 mitigation of H2O2 application, transcriptome sequencing was performed on pea primordial roots under different treatments for 72 h.Eukaryotic reference transcriptome (RNA-seq) analyses of nine samples with PCA (Figure S4A) showed high similarity among the biological replicates.FC ≥ 1.5 and FDR < 0.05 were used as screening criteria in the comparison groups of CK vs. CK1, CK vs. T5, and CK1 vs. T5.In these analyses, 2701 differentially expressed genes (1200 up-regulated and 1501 down-regulated), 6857 differentially expressed genes (3242 up-regulated and 3615 down-regulated), and 6683 differentially expressed genes (3139 up-regulated and 3544 down-regulated) were found, respectively To further verify the important role of Ca 2+ in mitigating the H 2 O 2 inhibition of root growth toward gravitropism, CaSO 4 and KCl, used at the same ionic (Ca 2+ , Cl − ) concentrations as the CaCl 2 solution, were applied exogenously (Table S4 and Figure S3).It was found that the pea bending rate (Table S1 and Figure S1) and bending degree (Table S2 and Figure S2) were significantly reduced under the CaSO 4 treatment compared with the 150 mmol•L−1 H 2 O 2 treatment, and that these reached a minimum at a Ca 2+ concentration of 10 mmol•L−1, while KCl treatment had no significant mitigating effect on H 2 O 2 inhibition.
Based on these statistical results, pea primary roots under the 150 mmol•L−1 H 2 O 2 and 150 mmol•L−1 H 2 O 2 + 10 mmol•L−1 CaCl 2 treatments were selected for the study of transcriptome and metabolome indexes.

Transcriptome Analysis and Validation of Key DGEs in Pea Primary Roots under H 2 O 2 and CaCl 2 Treatment
To investigate the mechanism of action of pea root growth under the CaCl 2 mitigation of H 2 O 2 application, transcriptome sequencing was performed on pea primordial roots under different treatments for 72 h.Eukaryotic reference transcriptome (RNA-seq) analyses of nine samples with PCA (Figure S4A) showed high similarity among the biological replicates.FC ≥ 1.5 and FDR < 0.05 were used as screening criteria in the comparison groups of CK vs. CK1, CK vs. T5, and CK1 vs. T5.In these analyses, 2701 differentially expressed genes (1200 up-regulated and 1501 down-regulated), 6857 differentially expressed genes (3242 up-regulated and 3615 down-regulated), and 6683 differentially expressed genes (3139 up-regulated and 3544 down-regulated) were found, respectively (Figure S4B).The Venn diagram of all differential genes (Figure S4C) showed that the total number of differentially expressed genes in the three control groups was 16,848.In addition, there were 456, 630, and 187 specifically expressed differential genes in the CK vs. CK1, CK vs. T5, and CK1 vs. T5 comparison groups, respectively.Nine DEGs were selected based on this transcriptome analysis (Figure 2), including two IAA-related genes (GAT1, IAA26), two ABA-related genes PYL4, the GA-related gene PAT1, the IAA-binding gene ABP19A in the cell wall, one lignin synthesis-related gene CCR1, and three genes related to starch and sucrose metabolism (PGM1, SUS, and BAM3).These RNA-Seq FPKM values of the genes showed a similar trend to the relative expression by qRT-PCR, confirming the authenticity of the transcriptome data.(Figure S4B).The Venn diagram of all differential genes (Figure S4C) showed that the total number of differentially expressed genes in the three control groups was 16,848.In addition, there were 456, 630, and 187 specifically expressed differential genes in the CK vs. CK1, CK vs. T5, and CK1 vs. T5 comparison groups, respectively.Nine DEGs were selected based on this transcriptome analysis (Figure 2), including two IAA-related genes (GAT1, IAA26), two ABA-related genes PYL4, the GA-related gene PAT1, the IAA-binding gene ABP19A in the cell wall, one lignin synthesis-related gene CCR1, and three genes related to starch and sucrose metabolism (PGM1, SUS, and BAM3).These RNA-Seq FPKM values of the genes showed a similar trend to the relative expression by qRT-PCR, confirming the authenticity of the transcriptome data.

Metabolomic Analysis of Pea Primary Roots under H2O2 and CaCl2 Treatments
To further elucidate the mechanism of root growth under the effect of H2O2 alleviated by CaCl2, we performed qualitative and quantitative metabolomic analyses on nine samples, and a total of 647 metabolites were detected.In the CK vs. CK1, CK vs. T5, and CK1 vs. T5 comparison groups, 303 differential metabolites (176 up-regulated and 127 downregulated), 257 differential metabolites (81 up-regulated and 176 down-regulated), and

Metabolomic Analysis of Pea Primary Roots under H 2 O 2 and CaCl 2 Treatments
To further elucidate the mechanism of root growth under the effect of H 2 O 2 alleviated by CaCl 2 , we performed qualitative and quantitative metabolomic analyses on nine samples, and a total of 647 metabolites were detected.In the CK vs. CK1, CK vs. T5, and CK1 vs. T5 comparison groups, 303 differential metabolites (176 up-regulated and 127 down-regulated), 257 differential metabolites (81 up-regulated and 176 downregulated), and 280 differential metabolites (124 up-regulated and 156 down-regulated) were found, respectively (Figure S4D).Correlation analysis showed that the correlation coefficients (R 2 ) of the biological replicates of the samples were all greater than 0.9 (Figure S4E), and the PCA showed that the samples from different treatments were better separated (Figure S4F), suggesting that the metabolomic data were highly credible.
The results of the GO enrichment analysis indicated that the alteration of pea primary root growth by exogenous CaCl 2 and H 2 O 2 may be achieved through oxidative stress, the alteration of cell wall components, and the activation of phytohormone signaling.

CaCl 2 and H 2 O 2 Affect Oxidative Stress within Pea Primary Roots
The "hydrogen peroxide catabolic process (GO:004274)" and "response to oxidative stress (GO:0006979)", which were significantly enriched in the GO biological processes, were analyzed for KEGG enrichment to further refine the DEGs-enriched pathways.It was found that the above DEGs were heavily enriched in the "phenylpropanoid biosynthesis" (ko00940) pathway (Figure S6A,B).A total of 24 DEGs were expressed in the "phenylpropanoid biosynthesis" pathway in the CK vs. CK1 and CK1 vs. T5 comparative groups, which were mainly the key genes regulating peroxidase.The heat-map visualization of DEGs co-expressed in the CK vs. CK1 and CK1 vs. T5 comparison groups (Figure 3A) revealed that 9 were up-regulated and 15 were down-regulated in the CK group, 15 were up-regulated and 9 were down-regulated in the CK1 group, and all of them were downregulated in the T5 group, compared with the 3 treatment groups.By determining the POD (Figure 3C) and SOD (Figure 3D) activities of pea primary roots at 36-, 54-, and 72-h time intervals, it was found that the trends of the enzyme activities were basically the same at the different time intervals.Among them, the SOD and POD activities of the CK1 group were significantly higher than those of the CK group after 72 h.Compared with the CK1 group, the SOD activity of the T5 group was elevated, while the POD activity was significantly lower.
Oxidative stress in the root system alters the endogenous ROS content.DEG enrichment showed (Figure 3B) that the respiratory burst oxidase gene (RBOH) was activated by CaCl 2 and H 2 O 2 treatment, and the endogenous H 2 O 2 content of the root system was significantly reduced by exogenous H 2 O 2 treatment (Figure 3E).In contrast, the endogenous H 2 O 2 content after the application of exogenous CaCl 2 on the basis of H 2 O 2 stress increased significantly compared with CK1, but was lower than that of the CK treatment, and the trends of the H 2 O 2 content were basically the same at different time intervals.After the DAB staining of pea primary roots cultured for 72 h in the CK, CK1, and T5 treatment groups, the results showed (Figure 3F) that the primary roots were more lightly colored than the CK group under the action of exogenous H 2 O 2 , whereas the CaCl 2 alleviation treatment resulted in deeper primary root coloration than the H 2 O 2 treatment.This further indicated that CaCl 2 might alleviate the inhibition of exogenous H 2 O 2 on pea primary root growth by regulating endogenous H 2 O 2 .What is compared here is the significance between different treatments at all times.(F) H2O2 staining of primary roots, wherein pea primary roots cultured for 72 h were stained in DAB staining solution for 2 h.Photographs were taken to observe the staining results.(G) Primary root starch staining, wherein pea primary roots cultured for 72 h were stained in Lugol's iodine solution for 10 min, and photographs were taken to observe the staining results.
Oxidative stress in the root system alters the endogenous ROS content.DEG enrichment showed (Figure 3B) that the respiratory burst oxidase gene (RBOH) was activated by CaCl2 and H2O2 treatment, and the endogenous H2O2 content of the root system was significantly reduced by exogenous H2O2 treatment (Figure 3E).In contrast, the endogenous H2O2 content after the application of exogenous CaCl2 on the basis of H2O2 stress increased significantly compared with CK1, but was lower than that of the CK treatment, and the trends of the H2O2 content were basically the same at different time intervals.After the DAB staining of pea primary roots cultured for 72 h in the CK, CK1, and T5 treatment groups, the results showed (Figure 3F) that the primary roots were more lightly colored than the CK group under the action of exogenous H2O2, whereas the CaCl2 alleviation treatment resulted in deeper primary root coloration than the H2O2 treatment.This further indicated that CaCl2 might alleviate the inhibition of exogenous H2O2 on pea primary root growth by regulating endogenous H2O2.What is compared here is the significance between different treatments at all times.(F) H 2 O 2 staining of primary roots, wherein pea primary roots cultured for 72 h were stained in DAB staining solution for 2 h.Photographs were taken to observe the staining results.(G) Primary root starch staining, wherein pea primary roots cultured for 72 h were stained in Lugol's iodine solution for 10 min, and photographs were taken to observe the staining results.

Effect of CaCl 2 and H 2 O 2 on the Contents of Starch and Soluble Sugar
The transcriptome and metabolome analyses revealed that some DEGs and DAMs were enriched in the "starch and sucrose metabolic pathway".Further analysis of this metabolic pathway showed that the expression of 11 DEGs (VCINV, ISA2, PGM1, ISA2, LECRKS7, LECRKS5, LECRKS4, LECRK71, BAM3, and BMY1) was significantly downregulated by exogenous H 2 O 2 (Figure 4A).After alleviation by CaCl 2 , the expression of three DEGs (ISA2, LECRKS7, and BMY1) was significantly down-regulated and BAM3 was significantly up-regulated.This suggests that CaCl 2 and H 2 O 2 may induce starch synthesis and metabolism in the primary roots of pea plants by regulating the expression of genes related to the "starch and sucrose metabolic pathway".LECRKS7, LECRKS5, LECRKS4, LECRK71, BAM3, and BMY1) was significantly down-regulated by exogenous H2O2 (Figure 4A).After alleviation by CaCl2, the expression of three DEGs (ISA2, LECRKS7, and BMY1) was significantly down-regulated and BAM3 was significantly up-regulated.This suggests that CaCl2 and H2O2 may induce starch synthesis and metabolism in the primary roots of pea plants by regulating the expression of genes related to the "starch and sucrose metabolic pathway".Five key soluble sugars were detected in the metabolome (Figure 4B), and the results showed that "D-Melibiose, Sucrose, Turanose, D-(+)-Maltose Monohydrate, and Maltose" abundance was up-regulated in response to H2O2 and up-regulated after CaCl2 alleviation Five key soluble sugars were detected in the metabolome (Figure 4B), and the results showed that "D-Melibiose, Sucrose, Turanose, D-(+)-Maltose Monohydrate, and Maltose" abundance was up-regulated in response to H 2 O 2 and up-regulated after CaCl 2 alleviation compared to the CK1 group.By determining the contents of starch (Figure 4C) and soluble sugar (Figure 4D) in the primary roots at different time intervals, it was found that the differences in the starch and soluble sugar contents at 36 h and 54 h were smaller.In contrast, the soluble sugar content in pea primary roots significantly increased by 1.87-fold, while starch content significantly decreased under the effect of H 2 O 2 at 72 h.The soluble sugar and starch contents in the T5 group almost recovered to the level of the CK group.It suggests that it is possible for CaCl 2 administration to alleviate the inhibition of root gravitropism by H 2 O 2 by regulating the content of starch.
To investigate the effects of exogenous H 2 O 2 and CaCl 2 treatments on the amount and distribution of starch accumulation, we stained pea primary roots with Lugol's iodine solution.The results showed (Figure 3G) that the starch in the CK group was mainly distributed in the root tip and the coloring was darker; in the CK1 group, the starch was uniformly distributed throughout the primordial roots and the coloring became lighter than that of the CK group; in the T5 group, the starch was distributed throughout the primordial roots and the coloring became darker than that of the CK1 group.Overall observation, followed by the freehand sectioning of transverse sections of primordial roots (Figure S7), revealed that the starch granules were significantly enlarged after H 2 O 2 treatment, and the size of the starch granules was restored to the CK level after CaCl 2 alleviation.
gravitropism by H2O2 by regulating the content of starch.
To investigate the effects of exogenous H2O2 and CaCl2 treatments on the amount and distribution of starch accumulation, we stained pea primary roots with Lugol's iodine solution.The results showed (Figure 3G) that the starch in the CK group was mainly distributed in the root tip and the coloring was darker; in the CK1 group, the starch was uniformly distributed throughout the primordial roots and the coloring became lighter than that of the CK group; in the T5 group, the starch was distributed throughout the primordial roots and the coloring became darker than that of the CK1 group.Overall observation, followed by the freehand sectioning of transverse sections of primordial roots (Figure S7), revealed that the starch granules were significantly enlarged after H2O2 treatment, and the size of the starch granules was restored to the CK level after CaCl2 alleviation.

Effects of CaCl 2 and H 2 O 2 on Phytohormone Signal Transduction in Pea Primary Roots
Transcriptome analysis showed that a large number of DEGs were enriched in "Plant hormone signal transduction (ko04075)", and the key genes regulating phytohormone signaling under different treatments will be analyzed in the following.

Effects of CaCl2 and H2O2 on Phytohormone Signal Transduction in Pea Primary Roots
Transcriptome analysis showed that a large number of DEGs were enriched in "Plant hormone signal transduction (ko04075)", and the key genes regulating phytohormone signaling under different treatments will be analyzed in the following.
A total of 31 DEGs were detected in the GA signaling pathway, and 6 DEGs were co-expressed in the CK vs. CK1 and CK1 vs. T5 comparison groups (Table S6).Among them, the scarecrow-like transcription factor (PAT1), scarecrow-like protein (SCL14/33), and transcription factor phytochrome-interacting factor-like 15 (PIL15) were down-regulated by 2.330, 1.417, 1.546, and 1.476-fold, respectively, in the CK vs. CK1 comparison group, and were up-regulated by 4.103, 1.591, 1.616, and 2.082-fold, respectively, in the CK1 vs. T5 comparison group (Figure 6C).This shows that pea primordial roots with applied CaCl 2 under exogenous H 2 O 2 treatment significantly regulated the expression of key genes in phytohormone signaling.
A total of 31 DEGs were detected in the GA signaling pathway, and 6 DEGs were coexpressed in the CK vs. CK1 and CK1 vs. T5 comparison groups (Table S6).Among them, the scarecrow-like transcription factor (PAT1), scarecrow-like protein (SCL14/33), and transcription factor phytochrome-interacting factor-like 15 (PIL15) were down-regulated by 2.330, 1.417, 1.546, and 1.476-fold, respectively, in the CK vs. CK1 comparison group, and were up-regulated by 4.103, 1.591, 1.616, and 2.082-fold, respectively, in the CK1 vs. T5 comparison group (Figure 6C).This shows that pea primordial roots with applied CaCl2 under exogenous H2O2 treatment significantly regulated the expression of key genes in phytohormone signaling.

Discussion
The growth morphology of pea primary roots was altered under the action of exogenous H 2 O 2 , while the application of CaCl 2 could alleviate the phenomenon [25].In order to investigate the intrinsic mechanism, this study was carried out by applying different concentrations of H 2 O 2 and CaCl 2 , counting the relevant growth indexes (germination potential, bending rate, and bending degree), and determining the relevant physiological indexes (POD, SOD, lignin content, etc.).Through transcriptome sequencing and metabolome assays, we analyzed the key pathways of DEG and DAM enrichment in pea primary roots under different treatments, with a view to finding the key genes and metabolite interactions of H 2 O 2 and CaCl 2 that regulate root growth and development.

CaCl 2 and H 2 O 2 Treatments Affect Pea Primary Root Growth toward Gravitropism
The growth and development of the primary root is essential for the early growth of peas [26].In this study, the germination potential of pea seeds increased in the presence of high concentrations of H 2 O 2 (Table S7 and Figure S8), suggesting that H 2 O 2 could promote the germination rate of pea seeds, thereby verifying the findings of Barba-Espin et al. [27].Jiang et al. [28] found that H 2 O 2 caused the non-directional growth of the primary roots of Lathyrus quinquenervius and wavy growth of the primary roots of Arabidopsis, which may be caused by the uneven distribution of calcium ions and IAA [29].In our study, we found that the geotropism-related growth of pea primordial roots was inhibited under H 2 O 2 treatment, while the bending rate and bending degree of pea primordial roots increased as the concentration of exogenous H 2 O 2 increased (Figure 1).This inhibition was relieved by the application of CaCl 2 , which is consistent with the findings of Li et al. [25].To further investigate whether the alleviation was caused by Ca 2+ or Cl − , this study demonstrated that Ca 2+ was a key factor in alleviating the inhibition of pea gravitropism by H 2 O 2 by replacing the counterion to apply CaSO 4 and KCl with the same particle concentration (Ca 2+ , Cl − ) as CaCl 2 .However, the exact manner of its mitigation is currently unknown.Root tips usually have more gravity-sensing signals; however, it has been found that plants can still sense gravity after the root crown has been removed [30,31], suggesting that the root tip is not the only gravity-sensing site [32].The gravity-oriented nature of the roots was weakened after the removal of the medial columella cells from the roots of maize seedlings, but the tendency of gravity-oriented growth was maintained [33].Therefore, the whole primary root was selected for analysis in this study, in order to screen the key factors of exogenous H 2 O 2 and the CaCl 2 regulation of root growth more comprehensively.

Activation of Oxidative Stress in Primary Roots by CaCl 2 and H 2 O 2 Treatments
ROS play an important role in shaping the RSA by regulating root growth and lateral root formation [34].Transcriptome analysis showed that the applied H 2 O 2 induced oxidative stress in the primary roots, and the enriched pathway was the "phenylpropane metabolic pathway".The DEGs in this pathway mainly regulate peroxidase.The study by Wan et al. [35] showed that exogenous H 2 O 2 treatment could enhance the cold resistance of oilseed rape seedlings by inducing the accumulation of antioxidant substances and activating the activity of antioxidant enzymes.In this study, we found that 11 peroxidase genes (including GSVIVT00023967001, PER12, PNC2, PRX112, POD, PER52, Psat5g250040, PER55, PNC1, PER10, and PER25) were up-regulated in response to H 2 O 2 , whereas all of them were down-regulated under CaCl 2 treatment.Barley root growth under high Cd stress was inhibited, while cationic POD isozymes accumulated Cd and were concentration-dependent [36].In this study, the POD and SOD activities in primary roots were significantly elevated by H 2 O 2 , while POD activity was reduced by CaCl 2 alleviation.SOD is at the core of antioxidant enzymes, and its elevated activity scavenges free radicals and enhances membrane permeability [37].It was found that exogenous Ca 2+ could improve the antioxidant capacity of black algae to enhance its resistance to Cd [38].
ROS have a dual role in the plant body, one as stressors that trigger oxidative stress and the other as signaling molecules that are involved in plant development [39].It was found that respiratory burst oxidase D (RBOHD) induces the production of cytoplasmic ectodomain ROS [40].In this study, exogenous H 2 O 2 stress led to a decrease in endogenous H 2 O 2 content (Figure 3E), whereas the content of endogenous H 2 O 2 in the root system increased after CaCl 2 application.This suggests that Ca 2+ may be regulating the endogenous H 2 O 2 to alleviate the non-gravitropic nature of primary roots due to exogenous H 2 O 2 stress, which is similar to the findings of Liu et al. [41].It was found that endogenous H 2 O 2 synthesis was reduced by the exogenous H 2 O 2 inhibition of sallow bean [28] root vigor, while the endogenous H 2 O 2 content of pea primordial roots [41] was significantly reduced by the activation of antioxidant enzyme systems.We further confirmed that exogenous H 2 O 2 inhibited endogenous H 2 O 2 accumulation using DAB staining (Figure 3F), and hypothesized that this was possibly because exogenous H 2 O 2 accelerated the clearance of endogenous H 2 O 2 by POD; the specific mechanism of action needs to be further explored.Transcriptome analysis showed that the expression of RBOH-related genes (RBOHA, RBOHB, RBOHC, RBOHE, and RBOHH) was activated upon the application of H 2 O 2 .This suggests that RBOH may be a key gene in the exogenous H 2 O 2 regulation of endogenous H 2 O 2 content changes.It was found that the expression of AtRBOHC regulates root development in Arabidopsis [42], and it can be hypothesized that the RBOH gene in this study may be related to pea root development, which needs further verification.

Effect of CaCl 2 and H 2 O 2 on Starch Metabolism in Primary Roots
According to the starch-equilibrium body hypothesis, starch-filled amyloplasts are asymmetrically distributed in the root system during gravity perception, thus inducing asymmetric growth signal transmissions [43,44].In this study, exogenous H 2 O 2 decreased the starch content and increased the soluble sugar content in primary roots.Exogenous H 2 O 2 also decreased the distribution of starch in the root tip, which finding is similar to the findings of Zhou et al. [29].It suggests that H 2 O 2 may attenuate root gravitropism by converting starch to sugar in the root tip.The application of CaCl 2 could also alleviate the acceleration of starch metabolism induced by H 2 O 2 , thus restoring the gravity-oriented nature of roots to some extent.Interestingly, the starch granules were significantly enlarged under the H 2 O 2 treatment compared to the CK treatment, which may also be a key factor leading to the change in gravitropism, the exact mechanism of which remains to be further verified.
Starch consists of straight-chain starch and branched-chain starch, in which straightchain starch is synthesized by granule-bound starch synthase (gss1) activity [45,46].It was found that the reduced expression of the PGM1 gene, a key starch-synthesizing gene in the root tip of Arabidopsis, resulted in diminished root geotropism [47].In contrast, in pgm1 mutants, the deposition of amyloid-free plastids is blocked, leading to a slowing of the gravitropic response in roots and shoots [48].In this study, the transcriptome and fluorescence quantification results showed that H 2 O 2 down-regulated the expression of PGM1 (Figures 2 and 4A), a key gene for starch synthesis, and its expression was upregulated after CaCl 2 alleviation.It has been found that Ca 2+ stabilizes α-amylase activity modulating gravitational sensitivity; therefore, Ca 2+ and amylase are the controlling factors in stabilizing starch content in cells [49][50][51].Combined with the results of starch content measurements, we can speculate that PGM1 is a key gene in the H 2 O 2 -regulated changes seen in starch content in pea primary roots.

Effects of CaCl 2 and H 2 O 2 on Calcium Signaling in Primary Roots
Instantaneous changes in Ca 2+ are early events in the plant's response to a variety of environmental signals [52].It was found that cold stress induces Ca 2+ signaling in plant cells, involving the activation of Ca 2+ channels and Ca 2+ pumps [53,54].Water stress causes hypoxia in plant roots, and by knocking down CAX (Ca 2+ /H + exchanger) and ACA (Ca 2+ -ATPase), it was found that the harmful effects of water stress on roots were alleviated by ACA knockdown [55].In plant cells, CaM, calmodulin neurophosphatase b-like proteins (CBLs), CMLs, and CDPKs (CPKs) can bind to free calcium in the cytoplasm, triggering a conformational change of the proteins that can lead to downstream physiological and biochemical responses [56][57][58].Ca 2+ is involved in plant root geotropism.An earlier study found that gravity leads to the asymmetric distribution of Ca 2+ gradients within pea and maize roots [59], and that the application of Ca 2+ chelating agents resulted in the retardation of root geotropism [60].Gravity-stimulated Ca 2+ is involved in regulating differential changes in extracellular pH in the elongation zones of Arabidopsis roots on both the ground-oriented and far-ground sides in response to auxin, resulting in a change in root orientation [61].In addition, primary Arabidopsis roots were less gravity-oriented in the presence of exogenous H 2 O 2 .The expression of MCA1, which encodes a Ca 2+ -permeable mechanosensitive channel, was significantly increased and Ca 2+ levels were higher in cells on the inner side of bent roots than in those on the outer side [29].Through transcriptome analysis, we predicted the subcellular localization of key genes regulating calcium signaling and found that ACA12/13, CPK1/17, KIC, CAM3, CML25, and CAMTA5 were activated by H 2 O 2 and CaCl 2 .It was further demonstrated that root geotropism was regulated by Ca 2+ .

Effect of CaCl 2 and H 2 O 2 on the Cell Wall of Primary Roots
Under water stress, the genes regulating those enzymes related to maize isoflavone biosynthesis are up-regulated and lignin is increased in the elongation zone [72].The phenylpropane pathway is one of the sources of the lignin found in plant cells.POD is the last enzyme in the lignin synthesis pathway and high POD activity increases lignification [73].Large accumulations of lignin under drought stress limit cell-wall extension in soybean roots [74].The EXPA and XET proteins play important roles in cell wall expansion [75].In our study, the genes regulating EXPA and XET showed 20 DEGs upregulated and 11 DEGs down-regulated in the CK treatment group, 8 DEGs up-regulated and 23 DEGs down-regulated in the CK1 treatment group, and 9 DEGs up-regulated and 22 DEGs down-regulated in the T5 treatment group.This indicates that the curved growth of pea primary roots under H 2 O 2 treatment may be related to the activities of EXPA and XET proteins.Cell wall extension depends on the deposition of cell wall components and the modification of cell wall structure to balance rigidity and extensibility.It was shown that the cytoskeletal network, the deposition of cell wall components, Ca 2+ homeostasis, ROS, ectoplasmic pH changes, and cell-wall-modifying proteins regulate cell wall extension [76][77][78].To better adapt to the external environment, the plant body must establish the correct cell shape and size [79].Therefore, it can be hypothesized that exogenous H 2 O 2 may promote lignin accumulation by increasing POD activity [80], thus altering the cell wall extensibility of pea primary roots and affecting the normal growth of root cells.After the application of CaCl 2 , there were no significant changes in the genes regulating the expression of EXPA and XET proteins compared to the CK1 group, while the lignin content was reduced compared to the CK group.

Plant Materials and Treatment
The Longwan 1 pea was used as the experimental material.Pea seeds of uniform size and full grains were selected; they were first rinsed with running water for 30 min, then sterilized with 75% alcohol for 30 s, and finally rinsed with sterile water 4-5 times.The seeds were placed in petri dishes with two layers of filter paper, then 20 seeds were placed in each petri dish.Finally, 20 mL of culture solution was added and the seeds were incubated in the incubator (Zhejiang Topu Yunnong Technology Co., Ltd.; Zhejiang, China) at a constant temperature of 25 • C for 72 h in the dark.
The culture solution concentration was screened with reference to the method used by Li et al. [25].H 2 O 2 concentration was screened with six concentration gradients of 0, 20, 80, 150, 200, and 300 mmol•L −1 H 2 O 2 (Sinopharm Group Chemical reagent Co., Ltd.; Shanghai, China).Each petri dish was considered as one replicate, and three replicates were set up.At the four time points of 24, 36, 54, and 72 h, the germination potential, bending rate, and bending degree of peas in different experimental groups were counted; 150 mmol•L −1 was determined to be the optimal inhibitory concentration.
For CaCl 2 concentration screening, a control group with CK (deionized water), CK1 (150 mmol•L −1 H 2 O 2 ), CK2 (10 mmol•L −1 CaCl 2 (Tianjin Guangfu Technology Development Co., Ltd.; Tianjin, China)), and a mitigation group (H 2 O 2 + different concentrations of CaCl 2 ) (Table 1) were set up to carry out the experiment.The specific treatment concentrations of CaCl 2 are given in Table 1.Each petri dish was considered as one replicate, and three replicates were set up.At the four time points of 24, 36, 54, and 72 h, the germination potential, bending rate, and bending degree of peas in the different experimental groups were measured.T5 (150 mmol•L −1 H 2 O 2 + 10 mmol•L −1 CaCl 2 ) was finally determined as the optimal mitigation concentration.The primary roots of the CK, CK1, and T5 groups treated for 36, 54, and 72 h were taken and stored at −80 • C for subsequent index measurements.Note: Deionized water was used for the configuration of all culture solutions in the experiment, excluding ionic forms other than water molecules.

Root Germination Potential, Bending Rate and Bending Degree Statistics
The germination potential, bending rate, and bending degree of pea seeds under different treatments were measured and 3 replications for each were performed (1 replicate per petri dish, 20 seeds per dish).
The statistical criterion for germination potential is as follows [80]: the length of the radicle of the seed is equal to the length of the seed.
Germination potential (%) = (number of seeds germinating normally at 72 h/total number of seeds per dish).
The statistical criterion for bending primary roots is as follows [41]: primary roots were considered to be bent if the angle between the tip growth angle and the direction of gravity was greater than 180 • , i.e., bent, and the bending angle was measured using ImageJ 1.53q software (https://imagej.net/,accessed on 17 July 2023) and Java 1.8.0_322 (64-bit) [81].

H 2 O 2 , Starch, and Lignin Staining
The diaminobenzidine (DAB) method was used to stain the primary roots of pea plants to detect H 2 O 2 changes [82,83].Primary roots from CK, CK1, and T5 specimens treated for 72 h were taken and placed in 1 mg•mL −1 DAB (Hefei Bomei Biotechnology Co., Ltd.; Hefei, China) staining solution, then treated in the dark at a constant temperature of 25 • C for 2 h.The DAB solution was poured out, and the primordial roots were rinsed with distilled water 4-5 times to remove the staining solution on the surface of the material, and the staining was observed; the darker the yellow color, the greater the H 2 O 2 content.
The pea primary roots were stained using Lugo's iodine solution [84] (iodine 4.5-5.5% (Tianjin Guangfu Technology Development Co., Ltd.; Tianjin, China) and potassium iodide 9.5-10.5% (Tianjin Guangfu Technology Development Co., Ltd.; Tianjin, China)) method to detect starch changes.Primary roots from the groups with CK, CK1, and T5 treatment for 72 h were placed in 9-millimeter disposable Petri dishes, to which 20 mL of staining solution was added (the concentration of staining solution could be adjusted according to the starch content of the material), and the staining was photographed and observed after 10 min.
Pea root tips were stained using the phloroglucinol method [85] to detect changes in lignin at 72 h under the CK, CK1, and T5 treatments.Pea root tips of 1 cm were placed in 2 mL centrifuge tubes and fixed for more than 24 h by adding 1.8 mL of FAA 70% fixative.Paraffin sections (apical transverse sections) were completed by Wuhan Xavier Biotechnology Co. (Wuhan, China).A total of 1% phloroglucinol (Yuanye Biotechnology technology company, Shanghai, China) solution (95% ethanol) was added dropwise to the sections and stained for 2 min.Then, 25% HCl was added dropwise for 2 min to develop the color.The samples with completed color development were quickly placed under a microscope (Leica Microsystems, Wetzlar, Germany) to observe the lignin distribution throughout the cross-section of the root tip and photographed.

Transcriptome Sequencing
Three biological replicates of pea primary roots from each of the CK, CK1, and T5 treatment groups were made.RNA sequencing analysis was performed by Biomarker Technologies Co., Ltd.(Beijing, China).Total RNA from the pea primary roots was extracted using the RNA Prep Pure Plant Kit (Tiangen, Beijing, China) according to the instructions provided by the manufacturer, and RNA sequencing analysis was performed using the Hieff NGS Ultima Dual-mode mRNA Library Prep Kit for Illumina (Yeasen Biotechnology (Shanghai) Co., Ltd., Shanghai, China).The libraries were finally sequenced on the Illumina NovaSeq platform.The raw data were further processed using the bioinformatics analysis platform BMKCloud (www.biocloud.net,accessed on 5 October 2023).Reads containing adapters were removed from the raw data, and these clean reads were mapped to the reference genome of Pisum_sativum_v1a (GCA_900700895.2,NCBI).
Genes with a DESeq2 corrected p-value < 0.05 and a fold change of ≥1.5 [86] were designated as differentially expressed.Differentially expressed genes (DEGs) were functionally annotated using the GO [87] and KEGG [88] databases.

Quantitative Real-Time PCR
To verify the reliability of the transcriptome sequencing data, nine DEGs were selected from the transcriptome data for qRT-PCR.Three replications were made for each treatment.The qRT-PCR primer sequence information is given in Table S8.qRT-PCR was performed after RNA reverse transcription using a LightCycler ® 96 Real time-PCR machine (Roche, Mannheim, Germany).The reaction system used a total of 20 µL: 10 µL of 2 × Talent qPCR premix, 6.8 µL of RNase-Free ddH 2 O, 2 µL of 75 ng/µL cDNA, and the forward and reverse primers of 10 µmol•L −1 were 0.6 µL each.The qRT-PCR reaction program was used as referenced by Lu et al. [86].The relative expression of nine genes was calculated by the 2 −∆∆Ct method, using β-tubulin [89] as the housekeeping gene.

LC-MS/MS Analysis
The same samples were used for the metabolome as for the transcriptome.Metabolome assays was performed with the UPLC-ESI-MS/MS system (UPLC, Waters Acquity I-Class PLUS; MS, Applied Biosystems QTRAP 6500+) (Waters, Milford, MA, America).The total peak area was normalized to the original peak area information for subsequent analysis.The screening criteria were: FC > 1, p-value < 0.05, and VIP > 1 [86].Spearman correlation analysis and principal component analysis (PCA) were used to determine the reproducibility of within-group and quality-control samples.

Conclusions
In this study, we comprehensively revealed the key pathways through transcriptomics and metabolomics that function to alleviate the inhibition of pea primary root gravitropism by H 2 O 2 with exogenous CaCl 2 (Figure 8).The transcriptome results indicated that CaCl 2 may alleviate H 2 O 2 stress by regulating the pea primary root oxidative stress response, starch and sucrose metabolism, calcium signaling and phytohormone signaling, and cell wall composition under a gravity field.Among them, PGM1, which regulates starch synthesis, is a key gene for gravity perception in pea primary roots.The metabolome and physiological and biochemical results showed that CaCl 2 might alleviate the inhibition of H 2 O 2 on the gravitropic movement of pea primary roots by regulating POD and SOD activities, increasing the content of endogenous H 2 O 2 and starch, and decreasing the accumulation of lignin to alleviate the inhibition of H 2 O 2 on the gravitropic movement of pea primary roots.This study not only contributes to the study of the pathways of pea root growth under adversity stress but also provides theoretical references for the study of root vectorial movement.

Conclusions
In this study, we comprehensively revealed the key pathways through transcriptomics and metabolomics that function to alleviate the inhibition of pea primary root gravitropism by H2O2 with exogenous CaCl2 (Figure 8).The transcriptome results indicated that CaCl2 may alleviate H2O2 stress by regulating the pea primary root oxidative stress response, starch and sucrose metabolism, calcium signaling and phytohormone signaling, and cell wall composition under a gravity field.Among them, PGM1, which regulates starch synthesis, is a key gene for gravity perception in pea primary roots.The metabolome and physiological and biochemical results showed that CaCl2 might alleviate the inhibition of H2O2 on the gravitropic movement of pea primary roots by regulating POD and SOD activities, increasing the content of endogenous H2O2 and starch, and decreasing the accumulation of lignin to alleviate the inhibition of H2O2 on the gravitropic movement of pea primary roots.This study not only contributes to the study of the pathways of pea root growth under adversity stress but also provides theoretical references for the study of root vectorial movement.

Figure 2 .
Figure 2. qRT-PCR analysis of DEGs in the primary roots under H2O2 and CaCl2 treatments.We selected nine DEGs regulating key metabolic pathways for qRT-PCR validation.The qRT-PCR values were compared with gene FPKM values to validate the reliability of the transcriptomic data.Different lower-case letters are the results of significance analyses of Duncan's multiple range test, indicating statistically significant differences (p < 0.05).What is compared here is the significance of the same indicator between different treatments.Genes included GAT2.1, IAA26, PYL4, PGM1, PAT1, ABP19A, CCR1, SUS2, and BAM3.CK: water only, CK1: 150 mmol•L −1 H2O2, and T5: mmol•L −1 H2O2 + 10 mmol•L −1 CaCl2; the same apply in the figures below.

Figure 2 .
Figure 2. qRT-PCR analysis of DEGs in the primary roots under H 2 O 2 and CaCl 2 treatments.We selected nine DEGs regulating key metabolic pathways for qRT-PCR validation.The qRT-PCR values were compared with gene FPKM values to validate the reliability of the transcriptomic data.Different lower-case letters are the results of significance analyses of Duncan's multiple range test, indicating statistically significant differences (p < 0.05).What is compared here is the significance of the same indicator between different treatments.Genes included GAT2.1, IAA26, PYL4, PGM1, PAT1, ABP19A, CCR1, SUS2, and BAM3.CK: water only, CK1: 150 mmol•L −1 H 2 O 2 , and T5: mmol•L −1 H 2 O 2 + 10 mmol•L −1 CaCl 2 ; the same apply in the figures below.

Figure 3 .
Figure 3. Oxidative stress regulates pea RSA growth.(A) Heat map of peroxidase gene expression in the "phenylpropane metabolic pathway".(B) Heat map of respiratory burst oxidase (RBOH) gene expression.(C) POD activity.(D) SOD activity.(E) Endogenous H2O2 content.Different lower-case letters are the results of significance analyses of Duncan's multiple range test, indicating statistically significant differences (p < 0.05).What is compared here is the significance between different treatments at all times.(F) H2O2 staining of primary roots, wherein pea primary roots cultured for 72 h were stained in DAB staining solution for 2 h.Photographs were taken to observe the staining results.(G) Primary root starch staining, wherein pea primary roots cultured for 72 h were stained in Lugol's iodine solution for 10 min, and photographs were taken to observe the staining results.

Figure 3 .
Figure 3. Oxidative stress regulates pea RSA growth.(A) Heat map of peroxidase gene expression in the "phenylpropane metabolic pathway".(B) Heat map of respiratory burst oxidase (RBOH) gene expression.(C) POD activity.(D) SOD activity.(E) Endogenous H 2 O 2 content.Different lower-case letters are the results of significance analyses of Duncan's multiple range test, indicating statistically significant differences (p < 0.05).What is compared here is the significance between different treatments at all times.(F) H 2 O 2 staining of primary roots, wherein pea primary roots cultured for 72 h were stained in DAB staining solution for 2 h.Photographs were taken to observe the staining results.(G) Primary root starch staining, wherein pea primary roots cultured for 72 h were stained in Lugol's iodine solution for 10 min, and photographs were taken to observe the staining results.

Figure 4 .
Figure 4. Starch metabolism regulates pea RSA growth.(A) The transcriptome and metabolome were combined to analyze the "starch and sucrose metabolic pathway".(B) Heat map of the relative sugar contents as detected by the metabolome.(C) Starch content.(D) Soluble sugar content.Different lower-case letters are the results of significance analyses of Duncan's multiple range test, indicating statistically significant differences (p < 0.05).What is compared here is the significance between different treatments at all times.Red and blue boxes indicate up-and down-regulated genes, respectively.The main stem of the graph represents the key metabolites obtained based on the KEGG database (DAMs detected by the metabolome have an orange background).The normalized mean expression of each gene is represented by a colored cell, based on a color scale.

Figure 4 .
Figure 4. Starch metabolism regulates pea RSA growth.(A) The transcriptome and metabolome were combined to analyze the "starch and sucrose metabolic pathway".(B) Heat map of the relative sugar contents as detected by the metabolome.(C) Starch content.(D) Soluble sugar content.Different lower-case letters are the results of significance analyses of Duncan's multiple range test, indicating statistically significant differences (p < 0.05).What is compared here is the significance between different treatments at all times.Red and blue boxes indicate up-and down-regulated genes, respectively.The main stem of the graph represents the key metabolites obtained based on the KEGG database (DAMs detected by the metabolome have an orange background).The normalized mean expression of each gene is represented by a colored cell, based on a color scale.

Figure 5 .
Figure 5. Calcium signaling is involved in pea RSA growth.Red and blue boxes indicate up-and down-regulated genes, respectively.Black arrows: the putative regulatory relationships of key genes for calcium signaling (calcium in the cytoplasm in the presence of exogenous H 2 O 2 may bind to calmodulin proteins such as CaM, CML25, and CPK1/17, thereby activating calcium signaling).The normalized mean expression of each gene is represented by a color-scale-based colored cell.The ICONS on organelles and cell membranes represent calcium transporters.The purple hexagon (ACA12/13) and orange rectangle (CAMTA5) are calcium channel proteins, and the blue icon is calcium ion binding protein (KIC).

Figure 6 .
Figure 6.Key genes regulating IAA, ABA, and GA signaling.(A) IAA signaling.(B) ABA signaling.(C) GA signaling.Red and blue boxes indicate up-and down-regulated genes, respectively.The trunk of the graph represents key genes obtained based on the KEGG database.The normalized

Figure 6 .
Figure 6.Key genes regulating IAA, ABA, and GA signaling.(A) IAA signaling.(B) ABA signaling.(C) GA signaling.Red and blue boxes indicate up-and down-regulated genes, respectively.The trunk of the graph represents key genes obtained based on the KEGG database.The normalized mean expression of each gene is represented by a color cell based on the color scale.All blue backgrounds in the figure represent key protein regulatory pathways of signaling pathways.

Figure 7 .
Figure 7. Cell wall involvement in pea RSA growth.(A) Venn diagram of the DEGs associated with the "plant cell wall synthesis" pathway.(B) Heat map of the expression of the key genes for lignin synthesis.(C) Heat map of the DEGs regulating the cell wall relaxation factors.(D) Lignin content.Different lower-case letters are the results of significance analyses of Duncan's multiple range test, indicating statistically significant differences (p < 0.05).What is compared here is the significance between different treatments at all times.(E) Resorcinol lignin staining.Sections of 72-h pea primary roots were taken and stained with resorcinol, then quickly placed under a light microscope (×100) to observe the degree of lignin accumulation.The darker the color, the greater the lignin accumulation.The black arrows point to layers of cells where lignin accumulates more.

Figure 8 .
Figure 8. Model of the mechanism by which CaCl2 mitigates the H2O2 inhibition of pea primordial roots toward gravitropism.By summarizing previous research along with the results of the present study, it can be hypothesized that CaCl2 may alleviate the mechanism of H2O2 inhibition of pea

Figure 8 .
Figure 8. Model of the mechanism by which CaCl 2 mitigates the H 2 O 2 inhibition of pea primordial roots toward gravitropism.By summarizing previous research along with the results of the present study, it can be hypothesized that CaCl 2 may alleviate the mechanism of H 2 O 2 inhibition of pea primary root growth toward gravitropism.Under the gravitational field, exogenous H 2 O 2 induced oxidative stress responses in pea primary roots, which mainly consisted of elevated regulatory POD and SOD activities and reduced endogenous H 2 O 2 content.Meanwhile, the reduced H 2 O 2 content induced the down-regulation of PGM1 expression and reduced the starch content.This altered the gene expression pattern of the downstream calcium signaling and phytohormone signaling pathways.We hypothesized that phytohormone signaling may regulate the down-regulation of the cell wall extensin EXPA/XTH gene and the increase in lignin accumulation, while the application of CaCl 2 could alleviate the inhibition of pea primary roots toward gravitropism by exogenous H 2 O 2 .In the present study, CaCl 2 may have alleviated the inhibition of pea primary root gravitropism by H 2 O 2 by regulating the level of oxidative stress in primary roots (a decrease in POD activity and an increase in SOD activity), increasing the endogenous H 2 O 2 and starch content, and decreasing the lignin accumulation to mitigate the inhibition of pea primary root gravitropism by H 2 O 2 .Black text: body content (signaling molecules, signaling pathways, metabolites, etc.) as obtained from the summary of this study.Black arrows: regulatory pathways.Black dashed arrows: possible regulatory pathways.Purple arrows: the regulatory pathways of H 2 O 2 .Red arrows: the regulatory pathway of CaCl 2 .

Table 1 .
Concentrations of the H 2 O 2 and CaCl2 treatments.