Effects of Iron De ciency Stress on Plant Growth and Quality in Flowering Chinese Cabbage and Its Adaptive Response

Iron (Fe) plays an important role in the growth and development of the human body and plants. The effects of different Fe concentrations, 1-aminocyclopropane-1-carboxylic acid (ACC), and cobalt chloride (Co 2+ ) treatments on plant growth, quality and the adaptive response to Fe deciency stress were investigated in owering Chinese cabbage. The results revealed that Fe deciency stress inhibited plant growth. The content of vitamin C (Vc), soluble protein, and soluble sugar in leaves and stalks were signicantly reduced under Fe deciency stress, while the content of cellulose and nitrate was increased. Meanwhile, Fe deciency stress obviously reduced the net photosynthetic rate and nitrate reductase (NR) activity of leaves. The balance system of active oxygen metabolism was destroyed due to Fe deciency, resulting in the decrease of catalase (CAT) activity, superoxide dismutase (SOD) activity of roots and leaves, and peroxidase (POD) activity of leaves, while POD activity in roots and malonaldehyde (MDA) content were signicantly increased. The treatments of Fe deciency and ACC signicantly reduced pH value of the root medium, promoted release of ethylene, and increased Fe 3+ reductase activity, while Co 2+ treatment showed the results opposite to those of Fe deciency and ACC treatments. Thus, Fe deciency stress could induce nitrogen metabolism, photosynthesis, reactive oxygen metabolism, pH of root medium, and Fe 3+ reductase activity that was related to physiological adaptive response and tolerance mechanisms. We also found that ethylene could involve in regulating the adaptive response to Fe deciency stress and improve the availability of Fe in owering Chinese cabbage. both roots and leaves increased signicantly. Based on Fe deciency stress with the increase of Fe concentration, ACC treatment could signicantly increase the active Fe content in plants, while the Co 2+ treatment reduced the active Fe content. These results indicated that Fe deciency stress or Co 2+ treatment inhibited the Fe 3+ reductase activity, leading to the decrease of active Fe content, while ACC treatment increased the Fe 3+ reductase activity, thus increasing the supply of available Fe of owering Chinese cabbage. were reduced in both roots and stems under low Fe deciency stress in three apple stocks. Thus, the antioxidant enzyme activities of different plants differed in response to Fe deciency. Our present research showed that Fe deciency stress led to disruption of the reactive oxygen metabolic system in owering Chinese cabbage, causing a signicant increase in POD activity of roots, but a signicant decrease in SOD and CAT activity of the roots and leaves, and POD activity in the leaves, and resulting in a large accumulation of MDA in plant. These ndings indicated that Fe deciency stress caused a decrease in the ability to scavenge reactive oxygen species and led to an increase of membrane lipid peroxidation, thus affected the normal growth of owering Chinese cabbage.


Introduction
Iron is an essential mineral nutrient element for human health and plant growth and development, not only is an essential element for all living organisms as it participates in a wide variety of metabolic processes, including oxygen transport and deoxyribonucleic acid synthesis, but also play signi cant roles in the physiological processes of photosynthesis, respiration, nitrogen metabolism, redox system of the plasma membrane, protein and nucleic acid synthesis in plant (Connorton et al. 2017).
Although Fe is a relatively abundant element in soil, the contents of the soluble Fe are remarkably poor, and hence poor solubility impairs its effectiveness, which often becomes a limiting nutrient element for plant growth and development. Indeed, one-third of the cultivated areas in the world are made of calcareous soils, which are the primary cause of Fe de ciency and about 40% of soil occur Fe de ciency. Thus, Fe de ciency is a common problem affecting the yield of crop (Mori 1999). Fe de ciency had a detrimental effect on plant growth and development, inducing various metabolic disorders, resulting in the abnormalities of chloroplast morphology and structures, reducing chlorophyll contents and photosynthetic rate, and diminishing respiratory of plants, which severely reduced plant yield and quality (Dey et al. 2020;Riaz et al. 2021).
The adaptive mechanisms of higher plant activation of Fe are divided into strategy I (Reduction Strategy) and strategy II (Chelation Strategy) (Romheld et al. 1986). For example, cucumbers, tomatoes, and soybeans are dicotyledons, their adaptive mechanisms induced by Fe de ciency all belonged to the strategy I. However, their speci c responses to Fe de ciency stress are different. Cucumber and tomato with Fe de ciency mainly show enlargement and coarseness near the root tip, increased root hairs, developed into a characteristic of metastatic cells, actively excrete large amounts of H + that signi cantly enhanced the Fe 3+ reduction ability of roots, which improved the availability of Fe in the rhizosphere. Soybean reduced the insoluble Fe 3+ mainly by the swelling and thickening of root tips and the accumulation of a large number of phenolic substances in the epidermis and cortex (Yi et al. 1998). Therefore, different plants show different response mechanisms to Fe de ciency stress.
Brassica campestris L. ssp. chinensis var. utilis Tsen et Lee, known as owering Chinese cabbage, belongs to the genus Brassica, which is a popular cruciferous vegetable commercially distributed in China, especially is one of the annually-produced vegetables with the largest cultivation scale and consumption in South China (Kang et al. 2014). The lack of Fe nutrition gradually becomes the key factor limiting the yield of owering Chinese cabbage due to the continuous increase of multiple cropping index and the higher and higher degree of soil alkalization. Our previous study indicated that mineral nutrition has obvious effects on the growth, yield, quality, and disease resistance of owering Chinese cabbage (Chai et al. 2013;Yang et al. 2000Yang et al. , 2004Yang et al. , 2012.
However, the effects of Fe de ciency on the growth and development of owering Chinese cabbage and its related physiological characteristics are still lacking in an investigation. In this study, we aim to investigate the effects of Fe de ciency stress on the growth, yield, and quality, and to investigate its adaptive physiological mechanism to Fe de ciency stress through different Fe concentrations, ACC, and Co 2+ treatments in owering Chinese cabbage.

Materials and reagents
This experiment was carried out in greenhouse of South China Agricultural University with owering Chinese cabbage '60-day' as experimental cultivar, Guangzhou, China. The seeds were sown in the nutrient bowl containing perlite on April 13, with 1/4 Hoagland nutrient solution were sprayed every 3 days at the seedling stage. They were transplanted in a plastic box (61 cm×42 cm×15 cm) with 20 L Hoagland nutrient solution for hydroponic cultivation on May 2, while the seedlings had 3-4 true leaves.

Determination of growth and quality index
The plants were collected on May 25. The plant height, stem diameter, fresh weight of roots, overground part of the plant, and whole plant as well as the quality of ower stalk of each 10 plants as a treatment group were measured. The ower stalk was the part above the third leaves of the plant. The stalk was the lea ess part of the ower stalk. The quality index of the ower stalk was determined after separating it into the leaves and the stalks. Vc content was measured by molybdenum blue colorimetric method. Soluble sugar content and soluble protein content were determined by following the method of Chai et al. (2013) and Bradford (1976) respectively. Nitrate content was determined by the derivate spectrophotometric method (Lastra 2003), while cellulose content was determined by the anthrone colorimetric method (Gao 2006). Active Fe content was measured by the method of Pierson et al. (1984).

Determination of photosynthetic characteristics
The chlorophyll content was determined by the method of Arnon et al. (1949). The photosynthetic rate, transpiration rate, intercellular CO 2 concentration, and stomatal conductance were performed on a portable automatic photosynthesizer (LI-6400, LI-COR, USA).

Determination of reactive oxygen metabolism
The reactive oxygen metabolism was determined using our previously reported method (Yang et al. 2000). In brief, each 0.5 g sample was precooled in a mortar, and 5 mL of 0.05 mol·L −1 pH 7.0 phosphate buffer solution and 0.3 g polyvinylpyrrolidone were added and grind, then centrifugated at 4℃ with 3000 r·min −1 rates for 10 min, and the supernatant was collected for the determination of the POD (EC 1.11.1.7), CAT (EC 1.11.1.21) and SOD (EC 1.15.1.1) activities and MDA content.

Determination of ethylene release from roots
The content of ethylene release from roots was measured for consecutive 6 days using the modi ed method (Romera et al. 1999). Brie y, each of ve roots and 200 µL water was added in a 25 mL tube, sealed and kept in dark conditions for 2 h, then 1 mL extracted gas from the tube for the determination of ethylene release were carried out using a Shimazu gas chromatography (GC-14C).

Measurement of pH of root medium
First, the pH value of the initial solution of the root medium was adjusted to be 6.26 and treated with different Fe concentrations, ACC and Co 2+ , then the pH of the root medium was measured for consecutive 6 days (at 9 am each day) using a Leici PHS-3B precision pH meter.

Determination of the Fe 3+ reductase activity of roots
The Fe 3+ reductase (EC 1.16.1.7) activity was evaluated for consecutive 6 days using a 2,2-bipyridine method (Gogorcena et al. 2000). In brief, the samples were put in the saturated CaSO 4 solution and soaked for 5 min, then washed using deionized water, then placed in a nutrient solution containing 0.1 mM Fe ( )-EDTA and 0.4 mM 2,2'-bipyridine with Fe ( ). After a 2-hour reaction under continuous ventilation with light illumination, the roots were weighted and the absorbance of the reactive liquid at 520 nm was measured.

Data processing and analysis
Data from experiments with triplicate or sextuplicate were statistically analyzed using SAS 13.0 software (SAS Institute, Inc, Cary, NC, USA) by Duncan's multiple range. The difference was considered to be statistically signi cant at P<0.05. The gures were generated using Origin 2018c.

Effects of Fe de ciency stress on the plant growth
Our results indicated that Fe de ciency stress obviously inhibited the plant growth (Table 1 and Figure 1). From the plant phenotype, Fe de ciency treatment had poor growth, dwarf, and yellowed heart leaves. The 1/2Fe or 1Fe treatment showed plant fresh weight, shoot fresh weight, stem height, and stem diameter signi cantly better than those of Fe de ciency treatment, Compared with the 1Fe treatment, the plant fresh weight in the Fe de ciency stress and 1/2Fe treatments decreased by 33.6% and 9.2%; the root fresh weight in the Fe de ciency stress and 1/2Fe treatments decreased by 43.7% and 39.8%; the plant height in the Fe de ciency stress and 1/2Fe treatments decreased by 21.4% and 1.9%; the stem diameter in the Fe de ciency stress and 1/2Fe treatments decreased by 22.8% and 6.4%; while the 1Fe treatment displayed the plant fresh weight, root fresh weight, stem height, and stem diameter slightly better than that of 1/2Fe treatment with no signi cant difference. The root fresh weight, and root shoot ratio of 1Fe treatment were signi cantly higher than those of 1/2Fe or Fe de ciency treatment, while those of 1/2Fe and Fe de ciency treatments showed no signi cant difference. Thus, these results indicated that Fe de ciency stress could signi cantly inhibit the growth of aboveground and roots and the expansion of ower stalk, which resulted in a signi cant decrease in fresh weight of owering Chinese cabbage. 3.2. Effects of Fe de ciency stress on the quality of ower stalk Fe de ciency stress obviously reduced the contents of Vc, soluble protein, and soluble sugar in leaves and stalks of the ower stalk, while increased the content of cellulose, and promoted the accumulation of nitrate in leaves and stalks of the ower stalk. Compared with the 1Fe treatment, the Vc content in leaves of the ower stalk in the Fe de ciency stress and 1/2Fe treatments decreased by 22.5% and 14.6%; the soluble protein content in leaves of the ower stalk in the Fe de ciency stress and 1/2Fe treatments decreased by 15.1% and 3.5%; the cellulose content in leaves of the ower stalk in the Fe de ciency stress and 1/2Fe treatments increased by 24.5% and 2.4%; the nitrate content in leaves of the ower stalk in the Fe de ciency stress and 1/2Fe treatments increased 49.8% and 17.8%. The contents of nitrate and cellulose of ower stalk in the 1/2Fe treatment were higher than those in the 1Fe treatment, while the Vc content of the 1/2Fe treatment was lower than that of the 1Fe treatment in stalks of the ower stalk. Compared with the 1Fe treatment, the Vc content in stalks of the ower stalk in the Fe de ciency stress and 1/2Fe treatments decreased by 56.2% and 30.3%; the soluble protein content in stalks of the ower stalk in the Fe de ciency stress and 1/2Fe treatments decreased by 11.7% and 5.0%; the cellulose content in stalks of the ower stalk in the Fe de ciency stress and 1/2Fe treatments increased by 43.1% and 11.9%; the nitrate content in stalks of the ower stalk in the Fe de ciency stress and 1/2Fe treatments increased 14.9% and 11.5%. There was no signi cant difference in the contents of soluble protein, soluble sugar, and Vc in leaves and stalks of the ower stalk between 1/2Fe and 1Fe treatment. Thus, it could be seen that Fe de ciency stress affected the formation of the quality of the ower stalk, leading to the decline of the quality of owering Chinese cabbage (Table   2).  Table 3, the chlorophyll content in the Fe de ciency treatment was signi cantly lower than that in 1/2Fe and 1Fe treatments, compared with the 1/2Fe and 1Fe treatments, the chlorophyll contents were signi cantly decreased by 21.0% and 20.5%, respectively, while there was no signi cant difference between the 1/2Fe and 1Fe treatment.
Fe de ciency did not affect the transpiration rate of leaves but had signi cant effects on stomatal conductance, photosynthetic rate, and intercellular CO 2 concentration (Table 3). Fe de ciency stress signi cantly reduced the net photosynthetic rate and stomatal conductance but signi cantly increased the intercellular CO 2 concentration in leaves. The net photosynthetic rate and stomatal conductance in the 1/2Fe treatment were not signi cantly different except that the intercellular CO 2 concentration was higher than that in the 1Fe treatment. Thus, these results showed that Fe de ciency stress signi cantly inhibited the formation of chlorophyll and destroyed the photosynthetic characteristics, which resulted in the decrease of photosynthetic rate in owering Chinese cabbage.

Effects of Fe de ciency stress on the NR activity
The NR activity of leaves decreased signi cantly. Compared with the 1Fe treatment, the NR activity of leaves in the Fe de ciency stress and 1/2Fe treatments decreased by 36.9% and 24.2%, respectively (Table 3). These results suggested that Fe de ciency stress caused the decrease of NR activity of leaves, which resulted in the decrease of nitrogen metabolism and the accumulation of nitrate in leaves, and inhibited the normal plant growth of owering Chinese cabbage. Fe de ciency stress affected the balance of the reactive oxygen metabolism system. As shown in Figure 2a, POD activity in roots and leaves had different trends under the different Fe concentrations. Compared with 1Fe treatment, POD activity of roots in Fe de ciency stress and 1/2Fe treatments was signi cantly increased by 6.1% and 5.7%, respectively, but there was no signi cant difference between Fe de ciency stress and 1/2Fe treatment. Compared with 1Fe treatment, POD activity of leaves in Fe de ciency stress and 1/2Fe was signi cantly decreased by 21.5% and 35.2%, respectively, but there was no signi cant difference between the Fe de ciency stress and 1/2Fe treatment. Moreover, we could also see that the POD activity of roots was nearly 100 times higher than that of leaves. The increase of POD activity in roots under the Fe de ciency condition may be due to the root resistance to the production of reactive oxygen caused by Fe de ciency stress and induces a root resistance reaction to damage of cell membrane system caused by the reactive oxygen, which prevents the further damage to the plant.
Compared with the 1Fe treatment, CAT activity of roots in Fe de ciency and 1/2Fe treatments was decreased by 28.5% and 21.0%, respectively, and CAT activity of leaves decreased by 66.1% and 38.8%, respectively (Figure 2b). In the 1Fe and 1/2Fe treatments, the SOD activity in roots or leaves was signi cantly higher than that in the Fe de ciency stress treatment, but the difference was not signi cant ( Figure 2c). These results indicated that Fe de ciency stress could reduce the CAT and SOD activities of roots and leaves.
The root of MDA content increased signi cantly with the decrease of Fe concentrations. Compared with the 1Fe treatment, MDA content in the Fe de ciency stress and 1/2Fe treatments increased by 97.1% and 26.8%, respectively. Compared with the 1Fe treatment, the leaves of MDA content in Fe de ciency treatment was increased, but the difference was not signi cant. While the MDA content of leaves in the 1/2Fe treatment was lower than those in the Fe de ciency and 1Fe de ciency treatments, but there was no signi cant difference between 1/2Fe and 1Fe treatments (Figure 2d). These results indicated that Fe treatment with appropriate concentration could inhibit membrane lipid peroxidation in leaves and roots, while Fe de ciency stress increased membrane lipid peroxidation in leaves and roots. In addition, the effect of Fe de ciency stress on membrane lipid peroxidation of roots was different from that on leaves, the effect of Fe de ciency stress on membrane lipid peroxidation in roots was greater than that in leaves. While in the 1Fe treatment, the membrane lipid peroxidation level of leaves increased, which might be due to the stress caused by excessive absorption of Fe in leaves under the condition of rich Fe nutrition.
The above results indicate that Fe de ciency stress could cause the metabolic system of reactive oxygen to be disturbed. Fe de ciency stress signi cantly increased the POD activity of roots, but the activity of SOD and CAT in roots and leaves and POD in leaves decreased signi cantly, and MDA content in roots and leaves was greatly accumulated, which indicated that Fe de ciency stress decreased the scavenging capacity of reactive oxygen species and increased the membrane lipid peroxidation level, thus affecting the normal plant growth and development of owering Chinese cabbage.
3.6. Effects of Fe de ciency stress, ACC and Co 2+ on endogenous ethylene release from roots Fe de ciency stress had a great effect on ethylene release from roots ( Figure 3a). With the prolongation of treatment time, the release of ethylene in the Fe de ciency stress, 1/2Fe, and 1Fe treatments showed a single peak curve, which rst increased and then decreased, and their peak value came out on the fourth day. With the increase of Fe concentrations, the ethylene release from roots decreased continuously. In short, Fe de ciency stress could promote the release of ethylene from roots, which indicated that ethylene was involved in the response to Fe de ciency stress.
ACC is a precursor of ethylene biosynthesis and Co 2+ is an inhibitor of ACC synthase. To verify the role of ethylene in the response to Fe de ciency stress, ACC and Co 2+ were added to study the effects of ethylene release from roots based on Fe de ciency stress. The results showed that ACC could obviously improve the release of ethylene in roots, while Co 2+ led to inhibition under the condition of Fe de ciency (Figure 3b). It is further suggested that ethylene was involved in the regulation of the response to Fe de ciency, and Fe de ciency stress led to increasing the release of ethylene from roots might be an adaptive response to Fe de ciency stress in owering Chinese cabbage.

Effects of Fe de ciency stress and ACC and Co 2+ treatment on the pH value in root medium
Fe de ciency stress had a signi cant effect on the pH value of root medium (Figure 3c). After Fe de ciency stress treatment, the pH value of root medium decreased gradually, reached a low peak on the third day, and then increased gradually. The pH value of root medium of 1/2Fe and 1Fe treatments decreased on the rst day and then increased linearly. In general, the pH value of root medium with 1/2Fe treatment was the highest, followed by 1Fe treatment, and the Fe de ciency treatment was the lowest. It indicated that the acidi cation capacity of the plant root was signi cantly improved under the condition of severe Fe de ciency, thus improving the availability of Fe in the root medium.
Under the condition of Fe de ciency, ACC treatment could decrease the pH value of the root medium with the extension of treatment time, and the pH value decreased to the trough on the third day and gradually increased, and then decreased again on the fth day. Moreover, under the condition of Fe de ciency, Co 2+ treatment also decreased the pH value of root medium with the extension of treatment time, and it decreased to the trough on the third day and then kept rising gradually. Among different treatments, the pH value of the root medium with ACC treatment was the lowest, while that of Co 2+ treatment was the highest (Figure 3d). The results suggested that ACC treatment based on Fe de ciency stress could enhance the acidi cation capacity of the root medium, while the Co 2+ treatment showed an opposite result, which might be because the proton pump controlled by ATPase on the protoplasm membrane of the root cells was induced by ACC, and the number of protons pumped out of the plasma membrane increased, resulting in a signi cant decrease in the pH value of the root medium of owering Chinese cabbage.
3.8. Effects of Fe de ciency stress and ACC and Co 2+ on the Fe 3+ reductase activity in roots Different Fe concentration treatments had signi cant effects on the Fe 3+ reductase activity of roots (Figure 3e). With the increases in treatment time, the Fe 3+ reductase activity treated different Fe concentrations showed an increasing trend. On the fth day of treatment, the Fe 3+ reductase activity in the Fe de ciency stress and 1/2Fe treatments decreased, but continued to increase in the 1Fe treatment. In a word, Fe de ciency stress or rich Fe nutrition could increase the Fe 3+ reductase activity of roots in owering Chinese Cabbage, especially the Fe 3+ reductase activity in the Fe de ciency stress treatment was the highest.
Based on Fe de ciency stress, ACC and Co 2+ treatments also had a great impact on the Fe 3+ reductase activity in roots (Figure 3f).
ACC treatment could rapidly increase the Fe 3+ reductase activity in roots, and the line shows an "M" shape in the process of treatment. Co 2+ treatment could rapidly decrease the Fe 3+ reductase activity in roots, which decreased a small trough on the second day, then gradually increased, and reached a peak on the fth day, and gradually decreased at last. In the process of treatment, Fe 3+ reductase activity of roots in the ACC treatment was always the highest, while in the Co 2+ treatment was always the lowest. Thus, the result indicated that ACC could induce the increase of Fe 3+ reductase activity of roots in owering Chinese cabbage, while Co 2+ was the opposite.
In summary, the above results showed that Fe was related to the Fe 3+ reductase activity of roots in owering Chinese Cabbage.
And Fe de ciency stress led to the improvement of the Fe 3+ reductase activity in roots, ACC was further induced to increase the Fe 3+ reductase activity of roots, while Co 2+ was inhibited.
3.9. Effects of Fe de ciency stress and ACC and Co 2+ on active Fe content Fe de ciency stress, ACC, and Co 2+ treatments had signi cant effects on the active Fe content (   (Dey et al., 2020;Riaz et al. 2021), which caused a signi cant yield reduction in leaf vegetable, as well as affected quality, such as color, hardness, and acidity (Alvarez-Fernandez et al. 2003). Ding et al. (2007) found that Fe de ciency stress inhibited the growth of pakchoi and reduced the content of nutrient, such as soluble protein and Vc, leading to an undesirable quality. Fe acted as an activator of sucrose phosphate synthase and was involved in sucrose synthesis, thus the Fe de ciency also led to a reduction in sucrose content and inhibition protein synthesis (Yarnia et al. 2008). Our present results showed that Fe de cient plants with yellowing of heart leaves, dwarf, signi cantly decreased in plant height, stem thickness, aboveground weight, plant weight, leaf chlorophyll content, and decreased in the content of Vc, soluble protein, and soluble sugar in the leaves and stalks of the ower stalk, while the contents of cellulose and nitrate in the leaves and stalks of the ower stalk were signi cantly increased. Therefore, Fe de ciency stress inhibited the plant growth, which resulted in a signi cant decrease in the quality of owering Chinese Cabbage.

Effect of Fe de ciency stress on nitrogen assimilation and photosynthesis
About 80% of the Fe in plants is found in photosynthetic cells. Fe de ciency negatively affected the constituent proteins of chloroplasts such as Cyt/b6f protein complex and ferredoxin, reducing photosynthetic pigment synthesis and chlorophyll content, making plant leaves chlorotic and reducing photosynthetic e ciency (Roncel et al. 2016). We found that Fe de ciency stress resulted in a signi cant decrease in chlorophyll content and an obvious impact on photosynthetic performance, with a signi cant decrease in photosynthetic rate and stomatal conductance, and an increase in intercellular CO 2 concentration in owering Chinese cabbage. Additionally, Fe is also an important component of NR and nitrite reductase. NR is a core enzyme in plant nitrogen metabolism regulated by NO 3− , has a great impact on plant nitrogen assimilation and utilization (Faure et al. 1991). Kaya et al. (2020) suggested that Fe de ciency caused an increase in NR activity, but Zou et al. (1998) insisted that NR activity was reduced in maize when it was de cient. Our present results indicated that Fe was closely related to the nitrogen metabolism of owering Chinese Cabbage, and Fe de ciency stress caused a decrease in NR activity, resulted in weakened nitrogen metabolism of leaves, caused a large accumulation of nitrate in leaves, and affected the availability and utilization of nitrogen nutrients, which in turn inhibited plant growth. Our present results agreed with the ndings of the peanut investigated by Zou et al (1998) and Song et al. (2016). It could be seen that Fe de ciency stress had different effects on nitrogen assimilation in different plants.

Effect of Fe de ciency stress on the antioxidant
When plants faced with stress, reactive oxygen metabolism is dysregulated and the free radical balance is disrupted, resulting in the accumulation of reactive oxygen species, which triggers and exacerbates membrane lipid peroxidation and may lead to the destruction of cell membrane integrity (Shabala et al. 2014). The plant could balance reactive oxygen metabolism by increasing the activity of antioxidant system. Antioxidant enzymes such as POD, CAT, and SOD, which played a role in scavenging free radicals and preventing free radical formation, they were contributing to reducing peroxidative damage, stabilizing membrane structure and its function, and to improving cellular resilience (Yang et al. 2004). Additionally, Fe was a component of SOD, POD, and CAT, that affects their biological activities. In the Fe de cient plants, the SOD activity slightly increased in leaves and roots, but POD and CAT activities in roots decreased by 52.0% and 34.5%, respectively, as compared to the control, MDA content increased by 94.9% and 99.0% in leaves and roots as compared to the control (Song et al. 2016). Dey et al. (2020) pointed out that Fe de ciency caused an increase in SOD activity and MDA content, while CAT activity was decreased in black gram. However, Jia et al. (2018) concluded that SOD, POD, and CAT activities were reduced in both roots and stems under low Fe de ciency stress in three apple stocks. Thus, the antioxidant enzyme activities of different plants differed in response to Fe de ciency. Our present research showed that Fe de ciency stress led to disruption of the reactive oxygen metabolic system in owering Chinese cabbage, causing a signi cant increase in POD activity of roots, but a signi cant decrease in SOD and CAT activity of the roots and leaves, and POD activity in the leaves, and resulting in a large accumulation of MDA in plant. These ndings indicated that Fe de ciency stress caused a decrease in the ability to scavenge reactive oxygen species and led to an increase of membrane lipid peroxidation, thus affected the normal growth of owering Chinese cabbage.
4.4. The role of ethylene in the adaptive response to Fe de ciency Fe de ciency stress in plants will cause a series of physiological adaptive responses, such as morphological structure alteration of the root system, causing inter-root acidi cation and increasing capacity of Fe reduction. Under Fe de ciency stress, the increased acidi cation capacity of the root system and the increased Fe 3+ reductase activity of roots are important mechanisms for plants to improve Fe uptake in soil (Schmidt 2003). The dicotyledonous plants, cucumber, tomato, and soybean, their adaptive mechanisms induced by Fe de ciency all belong to the strategy I, but their speci c responses to adapt to Fe de ciency stress were different. In cucumber and tomato, Fe de ciency was mainly showed as enlargement and thickening near the root tip, root hairs increased and developed the features of transfer cells, and automatically secreted large amounts of H + , which resulted in a signi cant increase of Fe 3+ reduction ability in the root systems, and increased the effectiveness of Fe in roots (Yi et al. 1998). Our present research also con rmed that Fe de ciency caused acidi cation of the inter-root medium, meanwhile, increased the Fe 3+ reductase activity of the root system in owering Chinese cabbage.
Ethylene is one of the main plant hormones, which plays an important role in regulating plant metabolism, such as inhibiting growth, inducing fruit ripening, accelerating the abscission of leaves, owers, and fruits, and so on. The relationship between ethylene and Fe uptake of the plant is mainly manifested in two aspects, one is to in uence the redistribution of Fe by participating in the metabolism; the other is to act as a signalling substance to regulate the adaptive response to Fe de ciency. Plant with Fe de ciency induced the accumulation of ethylene in roots (Sabrina et al. 2009). We found that ethylene was involved in regulating the Fe de ciency response of owering Chinese cabbage, and Fe de ciency stress caused a signi cant increase of ethylene release in root. These results were in accordance with the ndings of the investigation carried out by Romera et al. (2004). However, to study if the release of ethylene is an adaptive response to Fe de ciency stress, researchers have been gradually carried out on the ethylene biosynthesis precursor ACC and the ACC synthase inhibitor Co 2+ . Waters et al. (2007) showed that the addition of ACC after 1 day of Fe de ciency treatment signi cantly enhanced the Fe 3+ reduction ability, whereas the addition of Co 2+ signi cantly reduced the Fe 3+ reduction ability, which indicated that ethylene was involved in the regulation of the adaptive response to Fe-de ciency. Our present study also found that ethylene was involved in regulating the adaptive response to Fe de ciency in owering Chinese cabbage. ACC treatment promoted the ethylene synthesis in roots, reduced the pH of the root medium, enhanced Fe 3+ reductase activity in roots as well as increased the active Fe content in roots and leaves under the Fe de ciency stress, while the Co 2+ treatment did the opposite results, which indicated that the inter-root environmental conditions were improved and the effectiveness of root Fe nutrition by ethylene induction in owering Chinese cabbage. However, there are different views on the role of ethylene in Fe de ciency stress. The role of Co 2+ in the adaptive response to Fe de ciency depended on the concentration of Fe and the genotype of plant. Fe 3+ reduction ability of Fe e cient mutant of pea under Fe de ciency conditions decreased signi cantly when the concentration of Co 2+ was 100 µmol·L −1 , while the Fe 3+ reduction ability in roots of parental increased gradually, and there was no obvious change under the concentration of Co 2+ was 1 µmol·L −1 or 10 µmol·L −1 (Cui et al. 1999). There was no regular change of Fe 3+ reduction ability in roots when using ethephon, and the results of direct ethylene collection assay also showed no direct correlation between root Fe 3+ reduction ability and ethylene release rate (Romheld et al. 1986). The reduction of Fe could stimulate the synthesis of ethylene, but there was no correlation between this stimulation and the Fe status of the cell. The Co 2+ reduced the synthesis of ethylene but did not affect the Fe reduction (Malerba et al. 1996). Consequently, the physiological adaptations in response to Fe de ciency are different in different crops, and more indepth studies on the relationship between ethylene and Fe reduction are needed.

Conclusion
Our present study showed that Fe de ciency stress treatment induced a series of physiological adaptive responses, caused a decrease in nitrogen metabolism, photosynthesis, and pH value of root media, promoted the release of ethylene and the increase in Fe 3+ reductase activity, and improved the effective supply of Fe nutrients to plant, but disrupted the balance of the reactive oxygen metabolism system, led to a decrease of CAT in roots and leaves, SOD and POD activities in leaves, and membrane lipid peroxidation product MDA accumulated in large quantities, all the changes led to poor plant growth and decreased yield and quality. ACC treatment signi cantly improved the pH environmental of roots, promoted the release of ethylene, increased the Fe 3+ Figure 1 The plant phenotype of different Fe concentration treatments for 9 days Figure 2 The activities of POD(a), CAT(b), SOD(c) and content of MDA(d) in owering Chinese cabbage treated with Fe de ciency stress. Data are mean ± SE (n=3). Different letters above the columns show signi cant differences (p<0.05) between treatments