Defense Response of Pumpkin Rootstock To Cadmium

Cadmium pollution is severe in cucumber, although grafting is an effective method to improve its stress tolerance. Pumpkin is the commonly-used grafting rootstock for cucumber, and the breeding of rootstock with cadmium tolerance plays a vital role in the safe production of cucumber. However, there are no reports on rootstocks specic for cadmium tolerance. In this study, the rootstock of a pumpkin cross combination and its parents were used for the study of cadmium stress. The results indicated that under the 24mg·L − 1 cadmium stress, the relative conductivity of cross combination decreased by 35.86%~36.31% compared with the parents. When the concentrations of cadmium stress were 8 mg·L − 1 and 16 mg·L − 1 , respectively, the peroxidase (POD) activity of cross combination was higher than those of the parents. The subcellular distribution of cadmium in the root systems of the cross and the 041 − 1 parent was in the cell wall rst, followed by the cytoplasm and organelle, while that in the root system of 360-3 parent was in the cell wall rst, followed by the organelle and cytoplasm. Under cadmium stress with the 24mg·L − 1 concentration, the transfer coecient of cross was signicantly lower than that of the parents. The cross initiated the activity of membrane protective enzyme POD under cadmium stress, relieved the damage to membrane, and reduced the toxicity of cadmium through the accumulation of cadmium in the cell wall that blocked its entrance to the cytoplasm. This study provides a theoretical foundation to breed cadmium-tolerant rootstocks for melon vegetables.


Introduction
As one of the eight most toxic heavy metals in soil pollution, cadmium has a half-life as long as 10 ~ 35 days, which cannot be degraded by microorganisms and exists in soil for long time (Li et al.2017;Zheng et al.2006). Previous research (Shang et al.2018) indicated that the point-location standard-exceeding ratio of heavy metals in the arable soil of ve major grain producing areas in China reached as high as21.49%, whereas the increase in proportion of Cd pollution was the most signi cant. It increased from 1.32-17.39% during twenty some years. Recently, greenhouse vegetables have been the leading industry for farmers. Chen et al. ( Chen et al.2012a) found that the cadmium pollution in soil of greenhouse vegetable elds in Xinxiang city had reached as high aslevel6, which had already been severely polluted.
The soil of vegetable elds in the regions that included Beijing (Xu et al.2017;Suo et al.2016), Nanjing (Chen et al. 2013) and Hebei  were polluted by heavy metals to differing extents, whereas the cadmium pollution was most serious. Cadmium-polluted soil can not only affect the normal growth of crops, which leads to a decrease in crop yields and quality but can also be absorbed by the human body through food chain, which results in a serious threat to national food safety and human health.
Cucurbit vegetables are not only nutritious and delicious but also improve the diet and human health (XIA et al. 2010). However, when heavy metals are severe or exceed the standard for cultivated soil, the edible parts of melons easily accumulate heavy metals (Wang et al. 2018). The content of cadmium in cucumbers in China has reached as high as0.43mg·kg − 1 , far higher than the limiting each plant was 10mL, which was watered near the plant root system using a pipette, and the various indices were determined on August 10.

The Determination Method of Test Items
The determination of plant height, stem diameter and biomass Three plants were randomly selected in each treatment and used to determine the plant height and stem diameter. A tape was used to measure the aboveground height (from the plant base to growing point), and a Vernier caliper was used to measure the stem diameter (from the stem base to the half part of two cotyledons). The seedlings were washed with tap water and then three times with distilled water. The plants were divided into the aboveground and below ground parts (all parts contained root hairs) using scissors, dried for 30 minutes at105℃to remove the water and then dried at 70℃ until a constant weight was reached. The dry weight was then measured.
Measurement of relative conductivity, the activities of superoxide dismutase, POD and catalase, as well as the content of malondialdehyde in pumpkin seedling leaves The measurement on relative conductivity was conducted as described by Dresler et al.(2014). The activities of superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT), as well as the content of malondialdehyde (MDA) were measured as described by Li et al.(2000), which their concentrations had increased slightly.

Measurement ofthe morphological indices and activity of root system
The morphological indices of root system were measured as follows: Three seedlings were randomly selected in each treatment, and their root systems were washed and cleaned with tap water. The roots were placed on root plate of a root scanner (EPSON perfection 4990 PHOTO, EPSON Co., Ltd., Beijing, China) for scanning. After processing the photos of scanned root system, various parameters, including the total root length, total projected area, root surface area, the average diameter of root system, total root volume and root tip number, were obtained through an analysis using professional root analytics software (Win RHIZO Pro 2007, Regent Instruments, Quebec City, Canada) on March 13, 2007. The TTC method was applied to measure the activity of root system as previously described (Li H S.2000).
Measurement ofthe photosynthetic characteristics of pumpkin seedling leaves A LI-6400 portable photosynthesis measurer (LI-COR, Lincoln, NE, USA) was utilized tomeasurethe photosynthetic characteristics of pumpkin seedling leaves.
Measurement of the cadmium contents in each organ of pumpkin seedling.
Three pumpkin seedlings were randomly selected in each treatment. Dried samples of approximately 0.2g of the stems, leaves and root systems were weighed and ground in a mortar. A volume of 7ml of concentrated nitric acid and 2ml of hydrogen peroxide were added in a digestion tank and placed in a digestion instrument at 165℃at 5w for 30 -60 minutes until the solution was clari ed, and no impurities remained. The digested solution was transferred to a 50ml polytetrachloro ethylene beaker, and the acid was removed on a 170ºC hot plate. The chlorine was removed until it was nearly dry, and 0.5% nitric acid was used to dilute the sample to 10ml in a centrifuge tube. The mass concentration of cadmium in the samples was measured using an Optima 2100 DV inductively coupled plasma atomic emission spectrometer (ICP-AES) (Perkin Elmer, Waltham, MA, USA) ( Chen et al. 2012b).
Transfer coe cient % = heavy metal content in different part/heavy mental content in root system * 100 (Wassenaar et al. 2020) Isolation and analysis of the subcellular components in pumpkin seedling root system The method of Song et al. (Song et al. 2011) was utilized and slightly improved. In each treatment, the root systems of three seedlings were randomly selected, cut into pieces and mixed. A total of 0.3g fresh samples were taken and ground in a mortar at 4ºC. A volume of 5mL of homogenate was added, and the components consisted of 250 mmol·L -1 sucrose, 50 mmol·L -1 Tris-HCl (pH 7.5) and 1 mmol·L -1 DTT), transferred into a centrifuge tube and centrifuged at 3 000 r·min -1 for 1 minute. The precipitate comprised the cell wall components. The supernatant was taken and centrifuged at 14500 r·min -1 for 45 minutes, and the precipitate was organelles, while the supernatant was cytoplasm. The centrifuged cell wall and organelle components were digested again. Diluted nitric acid with a mass ratio of 0.5% was used to dilute the sample to 10mL, and the cytoplasm components were directly diluted in avolumetric ask for measurement.

Statistical analysis
The experimental results were analyzed used Data Processing system 7.55 software (DPS7.55) and Microsoft Excel 2007 (Redmond, WA, USA), and a Duncan analysis was applied for difference analysis.
All experiments were performed and analyzed separately with at least three biological replicates.

Results
The effects of cadmium stress to the growth and development of pumpkin cadmium-tolerant rootstock resource and cross combination The plant seedlings had a sensitive response to cadmium stress, and the phenomena included a physiological metabolic disorder, as well as slowed growth and development . Table 1 shows that with the increased mass concentration of cadmium stress, the growth of 360-3×041 − 1 and its parents were suppressed to different levels. Only under conditions without the addition of cadmium, was the plant height of 360-3×041-1signi cantly lower than that of the male parent. With the increase in mass concentration of cadmium stress, there was a non-signi cant difference on the seedling growth compared with those of the parents.   The effects of cadmium stress on membrane lipid peroxidation and membrane protective enzyme activity in pumpkin cadmium-tolerant rootstock resource and cross combination Figure 1 shows that the conductivity of 360-3×041 − 1 and its parent leaves rst increased, decreased and then increased again with the increase in mass concentration of cadmium stress. Under the same concentration of cadmium stress, there were signi cant differences between various varieties. When the mass concentration of cadmium stress reached its maximum, the relative conductivity of 360-3×041 − 1 was signi cantly lower than those of the parents, which decreased by 35.86%~36.31%. Figure 2 shows that with the increase in concentration of cadmium stress, the content of MDA in seedling leaves of 360-3×041 − 1 and its parents generally increased. This indicated that cadmium stress resulted in damage to the leaves of three materials to some extent. The content of MDA in 360-3×041-1was only signi cantly higher than that of the female parent under conditions without cadmium stress, while under the cadmium stress with mass concentration of 8 ~ 24mg·L − 1 , there was a non-signi cant difference in the content of MDA between 360-3×041 − 1 and its parents.
As membrane protective enzymes, SOD, POD and CAT can oxidize and decompose the reactive oxygen species (ROS) into non-toxic water and oxygen, thus, reducing the toxicity of heavy metals to plants . Figure 3 shows that under the cadmium stress with a higher mass concentration of 24mg·L − 1 , SOD activity of 360-3×041 − 1 was signi cantly higher than that in the male parent 041 − 1 (Fig. 3A).
Under the cadmium stress with mass concentrations of8mg·L − 1 and 16mg·L − 1 , the POD activity of 360-3×041 − 1 was higher than those of the parents, whereas at the concentration of 16mg·L − 1 , it differed signi cantly compared with the male parent 041 − 1 (Fig. 3B). Under the cadmium stress with different mass concentrations, CAT activity of 360-3×041 − 1 was always between that of the parents, which had not reached signi cant differences (Fig. 3C).
The effects of cadmium stress on the root system growth of pumpkin cadmium-tolerant rootstock resources and cross combination The plant root system is the organ that suffers rst from cadmium toxicity, and the activity of root system is one of the key indices that represents the growth status and activity level of pumpkin seedling root systems (Ba et al. 2017). Figure 4 shows that under cadmium stress with different mass concentrations, there were signi cant differences on the root system activity between 360-3×041 − 1 and the parents. With the increase in mass concentration of cadmium stress, the root systems of 360-3 and 041 − 1 were all damaged to differing levels, and the root system activities also decreased. However, the root system activity of 360-3×041-1increased with the increase in mass concentration of cadmium stress. When the mass concentration of cadmium stress reached 16mg·L − 1 and 24mg·L − 1 , the root system activity of 360-3×041 − 1 was signi cantly higher than those of the parents and increased by 18.80%~22.05% and 89.85 91.45% compared with those of the parents, respectively.
After scanning with the root system scanner, the root system pictures were used to observe the root hair numbers of 360-3×041 − 1 and its parents under cadmium stress with different concentrations. It was found that with the increase in concentration of cadmium stress, the root hair numbers of 360-3 and 041-1obviously decreased. The root hair numbers of 360-3×041-1decreased under the stress with a concentration of 8mg·L − 1 , while it increased under the cadmium stress with higher concentrations, and it was higher than those of the parents (Fig. 5).
With the increase in mass concentration of cadmium stress, the total root length, total projected area, root surface area, total root volume and root tip number of 360-3×041 − 1 and its parents decreased, while the average diameter of root system tended to rst increase and then decrease (Fig. 6A-F). Under conditions without cadmium stress, the total root length, total projected area, root surface area, total root volume and root tip number of 360-3×041 − 1 were between those of the parents, and the differences were signi cant (Fig. 6A-F). With the increase in mass concentration of cadmium stress, with the exception that the total root length of 360-3×041 − 1 was lower than those of the parents at a concentration of 8mg·L − 1 (Fig. 6A), the total root length, total projected area, root surface area, total root volume and root tip number were all higher than those of the parents under cadmium treatment with other mass concentrations. However, the differences were not signi cant ( Fig. 6A-F), and the root system growth showed super-parent heterosis.
The effects of cadmium stress on the photosynthetic characteristics of pumpkin cadmium-tolerant rootstock resources and cross combination Figure 7 shows that under cadmium stress with different mass concentrations, the net photosynthetic rate of 360-3×041 − 1 and its parents tended to rst decrease and then increase, and the net photosynthetic rate of 360-3×041 − 1 was always higher than those of its parents (Fig. 7A). However, the differences were not signi cant. The stomatal conductance of 360-3×041 − 1 and its parents reached their maximum under cadmium stress with a concentration of 8mg·L − 1 .With the increased mass concentration of cadmium stress, the stomatal conductance of 360-3×041 − 1 was always higher than those of its parents and signi cantly higher than that of its male parent 041 − 1 (Fig. 7B). The transpiration rate of 360-3×041 − 1 was always lower than those of its parents, except that under conditions without cadmium stress, it was signi cantly lower than that of 041 − 1.There were nonsigni cant differences compared with those of the parents under the cadmium treatment with other mass concentrations (Fig. 7C). With the increase in mass concentration of cadmium stress, the intercellular carbon dioxide of 360-3×041 − 1 was higher than those of its parents, whereas it was signi cantly higher than that of the male parent 041 − 1 at the concentrations of 16mg·L − 1 and 24mg·L − 1 (Fig. 7D).
The effects of cadmium stress on cadmium accumulation characteristics and subcellular distribution of pumpkin cadmium-tolerant rootstock resources and cross combination As shown in Fig. 8, under the cadmium stress with different mass concentrations, the root system of 360-3×041 − 1 and its parents were the part that had the highest accumulation of cadmium, followed by the stems and leaves. The cadmium distribution rule in each organ of 360-3×041 − 1 and 360-3 was root > stem > leaf, while in 041 − 1, it was root > leaf > stem. Compared with the parents, with the exception that the accumulation of cadmium in the root system of 360-3×041 − 1 was lower than those of its parents under stress with a concentration of 8mg·L − 1 , it was higher than those of both parents under the cadmium treatments at other mass concentrations. Under the cadmium stress with higher mass concentrations, the accumulation in root system of 360-3×041-1increased.The proportions that were distributed in the in stems and leaves were lower than those of its parents. Simultaneously, the accumulation of cadmium in leaves was also reduced.
In Table 3, the subcellular distribution of cadmium mass concentration in pumpkin seedling roots under cadmium stress with different mass concentrations was analyzed. Table 3 shows that there were signi cant differences in the subcellular distribution of the mass concentration of cadmium in the root systems between different materials. In 360-3×041 − 1 and its parents, most cadmium accumulated in the cell walls, followed by the cytoplasm and organelles. In contrast, the subcellular distribution rule of cadmium mass concentration in the root system of 360-3×041 − 1 and 041 − 1 seedlings was cell wall > cytoplasm > organelle, while it in the 360-3 seedlings, it was cell wall > organelle > cytoplasm. The effects of cadmium stress on cadmium transfer coe cient of pumpkin cadmium-tolerant rootstock resource and cross combination Figure 9 shows that with the increase in mass concentration of cadmium stress, the transfer coe cient of 360-3 tended to decrease rst and then increase, while both041-1 and 360-3×041-1exhibited a tendency to decrease. In addition, the decrease in 360-3×041 − 1 was more signi cant, whereas it was obviously lower than 360-3 with the maximum mass concentration of cadmium stress. Under the lower mass concentration of cadmium stress, the transfer capacities to the aboveground parts in 041 − 1 and 360-3×041 − 1 were much higher, while both decreased under a higher mass concentration of cadmium stress. The cadmium transfer capacity in 360-3×041 − 1 was signi cantly lower than those of its parents.
Most cadmium was xed in the roots, which limited its transport to the aboveground parts.

Discussion
Cd in the soil can enter into a plant through absorption by the roots, which generates toxicity to plants and thus, affects their growth. There are some differences on the cadmium tolerance in different varieties even in the different tissues of the same plant, and the growth of more tolerant plants was less affected by toxicity (Zhao et al. 2015 This indicated that all three materials had a stronger tolerance to cadmium, and there was a non-signi cant difference on the growth between different materials, which might be related to parents that also had a higher tolerance to cadmium. The heavy metal stress with a high mass concentration will lead to the generation of abundant reactive oxygen species (ROS) in the plant cells, which will result in membrane lipid peroxidation caused by unsaturated acids in the plasma membrane (PM)and thus, increased its permeability Lei et al. 2018) The cell PM is the main part of the plant that is damaged by stress. Generally, the relative conductivity, namely the permeability of PM, is used to re ect the extent of plant injury under stresses (Xia et al. 2009). As the nal product of membrane lipid peroxidation, MDA is an important index that re ects its effects. It can cause damage to proteins by generating covalent complexes, which could be involved in the damage to tissues during aging (Traverso et al. 2004;Hodges et al. 2009). Zhao(2015)found that with the increase in mass concentration of cadmium stress, as well as the extension of its duration, the membrane permeability of corn leaves was increased, which resulted in an increase in the relative conductivity in leaves and thus, affected the growth and development of corn. Heavy metal stress also leads to an increase in the content of MDA in many plants, which caused the  . 2015). Thus, this process will reduce the damage of membrane lipid peroxidation. In this study, with the increase in mass concentration of cadmium stress, the conductivity in leaves of 360-3×041 − 1 and its parents all tended to rst increase, then decrease and increase again. Under the cadmium stress with the highest mass concentration of 24 mg·L − 1 , the relative conductivity in 360-3×041 − 1 was signi cantly lower than those of its parents. Under cadmium stress with different mass concentrations, the content of MDA tended to increase. Under cadmium stress with amass concentration of 8 ~ 24mg·L − 1 , there were non-signi cant differences in the content of MDA between 360-3×041 − 1 and its parents. With the increase in mass concentration of cadmium stress, the activity of SOD tended to rst decrease and then increase. Under cadmium stress with a mass concentration of 24mg·L − 1 , the activity of SOD in 360-3×041 − 1 was signi cantly higher than that of the male parent, while it had a nonsigni cant difference compared with the female parent. At the concentration of 8mg·L − 1 and 16mg·L − 1 , the activity of POD in 360-3×041 − 1 was higher than those of its parents, while it had a signi cant difference compared with that of the male parent at 16mg·L − 1 . Under cadmium stress with different mass concentration, the activity of CAT in 360-3×041 − 1 had non-signi cant differences compared with its parents. The increase in SOD activity initiates and enhances the protective capability of the PM, which can transform highly toxic ROS to H 2 O 2 , which has a weaker oxidizing capacity, and further stimulate the increase in activity of POD (He et al. 2015). This indicated that the capacity to eliminate oxygen radicals in 360-3×041 − 1 was higher than those of its parents, where as POD played a key role in relieving cadmium stress, which was consistent with the results of Wan et al.( 2015).
The effects of soil stresses to root system are the most direct. The disruption in growth of the root system will directly affect the supply of nutrients and water to the aboveground parts of plants. indicated that under stresses, the growth of plant roots was suppressed; the root activity was decreased, and the changes in root system included the inhibitory effects on total root length, total root surface area, root volume and root tip number. In this experiment, under cadmium stress with different mass concentrations, the root growths of 360-3, 041 − 1 and 360-3×041 − 1 were suppressed to differing extents. With the increase in mass concentration of cadmium stress, the root activities of parents 360-3 and 041-1gradually decreased, while that of 360-3×041-1tended to increase, which was signi cantly higher than those of its parents under cadmium stress at higher mass concentrations. Through the observation of root hair numbers and analysis of root morphological indices in 360-3×041 − 1 and its parents, this indicated that with the increase in degree of cadmium stress, the root hair numbers in 360-3 and 041-1decreasedsigni cantly.The root hair numbers in 360-3×041-1increased under cadmium stress with a higher concentration, and it was greater than those of the parents. The total root length, total projected area, root surface area and root tip numbers in 360-3×041 − 1 seedlings were all higher than its parents. This indicated that under cadmium stress, the growth of pumpkin seedling roots was suppressed. The degree of inhibition in 360-3×041 − 1 was lower than those of its parents 360-3 and 041 − 1, and the growth of root system presented super parent heterosis.
Photosynthesis has a close relationship with crop growth and yield (Zhou et al. 2015), which renders it one of the important indices to evaluate plant productivity and adapt ability (Suo et al. 2020). The net photosynthetic rate (Pan R Z .2012)is one of the important indices to evaluate photosynthesis and is represented as the accumulation of dry matter in plant leaves. In this study, under the cadmium stress with different mass concentrations, the net photosynthetic rate of 360-3×041-1was always higher than that of its parents and presented super parent heterosis, which indicated that its photosynthetic capacity was higher than that of its parents. The stomatal conductance in 360-3×041 − 1 was higher than its parents under cadmium stress with higher mass concentrations, which had signi cant differences compared withthe male parent 041-1and increased by 3.37%~24.32% and 6.45%~28.57%, respectively, compared withboth parents. The transpiration rate in 360-3×041 − 1 was always lower than those of its parents, and the differences compared with the parents were not signi cant. such as a developed root system, whereas the root is the key organ for cadmium accumulation. With the increase in cadmium mass concentration, the absorption of cadmium in root system was signi cantly higher than that in the aboveground parts. However, the cell wall of subcellular components in root system plays a key role in the inhibition of transport of cadmium from the roots to stems (Xue et al. 2014). In this experiment, under cadmium stress with different mass concentrations, the distribution rule of cadmium in the 360-3 and 360-3×041 − 1 seedlings was root > stem > leaf, while in the041-1 seedlings, it was root > leaf > stem. The subcellular distribution rule of cadmium mass concentration in 360-3×041 − 1 and 041 − 1 seedling roots was cell wall > cytoplasm > organelle, while in the 360-3 seedling roots, it was cell wall > organelle > cytoplasm. This indicated that the root system was the main organ for cadmium accumulation in 360-3×041 − 1 and its parents, and most was absorbed by cell walls in the root system.

Conclusions
The cross combination (360-3×041 − 1) had a higher defense capability compared withits parents under cadmium stress with a certain mass concentrations.Cadmium accumulated in the roots andto a high extent in the cell walls of root system. The transfer capability to aboveground parts was lower than those of its parents, and the cadmium toxicity to aboveground parts was lower than those of its parents. The net photosynthetic rate was always higher than its parents and presented super parent heterosis. The capacity of eliminating reactive oxygen species in 360-3×041 − 1 was higher than those of its parents, whereas the activity of POD played a keyrole in relieving the stress owing to cadmium. Data availability: All data generated or analyzed during this study were included in this manuscript.

Declarations
Compliance with ethical standards Con ict of interest: The authors declare that they have no con ict of interest.
Ethical approval: All authors declared that they had no known competing nancial interests or personal relationships that seemed to affect the work reported in this article. All authors followed the ethical responsibilities of this journal.
Consent to participate and publish: All authors participated and approved the nal manuscript to be published.  The effects of cadmium stress on photosynthetic characteristics of pumpkin seedlings. Bars with different letters indicate signi cant differences at p 0.05. Mean values and SDs for three replicates are

References
shown.

Figure 8
The proportion for mass concentration of cadmium in roots, stems and leaves of pumpkin seedlings under cadmium stress.