*3.3. Exogenous Spd Improved the Growth and Photosynthetic Efficiency of Tomato Plants under Low-Iron Stress*

The growth of tomato seedlings was significantly inhibited by low-Fe stress, along with significantly decreased dry and fresh weights by 28.57 and 27.91%, respectively. However, plant biomass was significantly increased by Spd foliar treatment under low-iron stress (Table 2). Likewise, root growth was significantly affected by low-iron stress, but Spd foliar treatment under low-Fe conditions increased the total root length, total root surface area and total root volume by 78.63, 41.35 and 40.91%, respectively, compared to the low-iron treatment (Table 2). It is evident that exogenous Spd-spraying has a mitigating effect on the growth of tomato seedlings under low-iron stress.

**Table 2.** Effects of exogenous Spd on tomato biomass under low-iron stress.


CK, control; LF, Low Fe; Spd, spermidine; LF + Spd, Low Fe plus spermidine. Data are shown as mean ± SD. Within each column, entries followed by the same lowercase letters are not significantly different according to Duncan's test at *p* ≤ 0.05.

Moreover, root vigor and Fe3+ reductase activity were significantly increased by either low-iron stress or Spd-spraying (Supplementary Figure S2). When compared to the low-Fe treatment, root vigor and Fe3+ reductase activity were further increased by 23.21 and 21.35%, respectively, after spraying with Spd under low-Fe stress.

The photosynthetic pigment content of tomato leaves was repressed by low-iron stress. However, the chlorophyll a, chlorophyll b and chlorophylla+b contents were significantly increased by 23.58, 12.50 and 21.58%, respectively, in Spd treatment under low-Fe stress compared to low-iron stress only, though the carotenoid content was affected by Spd treatment under low-iron stress (Table 3).

**Table 3.** Effects of exogenous Spd on chlorophyll content in tomato leaves under low-iron stress.


CK, control; LF, Low Fe; Spd, spermidine; LF + Spd, Low Fe plus spermidine. Data are shown as mean ± SD. Within each column, entries followed by the same lowercase letters are not significantly different according to Duncan's test at *p* ≤ 0.05.

In line with the photosynthetic pigment concentrations, the net photosynthetic rate was inhibited by 49.07% in leaves under low-Fe stress, and exogenous foliar-spraying of Spd alleviated the reduction of gas-exchange parameters in tomato leaves caused by low-Fe stress, and increased Pn, Tr, Gs and the intercellular CO2 concentration (Ci) (Table 4).


**Table 4.** Effects of exogenous Spd on photosynthetic parameters in tomato leaves under lowiron stress.

CK, control; LF, Low Fe; Spd, spermidine; LF + Spd, Low Fe plus spermidine. Data are shown as mean ± SD. Within each column, entries followed by the same lowercase letters are not significantly different according to Duncan's test at *p* ≤ 0.05.

Under low-Fe stress, chlorophyll fluorescence parameters such as the maximum photochemical efficiency of PSII (Fv/Fm), electron transfer efficiency (ETR), actual photochemical quantum yield of PSII (ΦPSII) and photochemical quenching coefficient (qP) of leaves significantly decreased by 7.47, 37.21, 37.32 and 35.47%, respectively, and the non-photochemical quenching coefficient (NPQ) increased by 85.94%. However, all these indicators, except for qP, increased significantly after spraying with Spd, suggesting that exogenous foliarspraying with Spd under low-iron stress had a strong ameliorative effect on leaf chlorophyll fluorescence characteristics (Table 5).

**Table 5.** Effects of exogenous Spd on fluorescence parameters in tomato leaves under low-iron stress.


CK, control; LF, Low Fe; Spd, spermidine; LF + Spd, Low Fe plus spermidine. Data are shown as mean ± SD. Within each column, entries followed by the same lowercase letters are not significantly different according to Duncan's test at *p* ≤ 0.05.

#### *3.4. Effect of Exogenous Spd on ROS Accumulation, Antioxidant System and Osmoregulatory Substances in Tomatoes under Low-Iron Stress*

Low-iron stress increased the accumulation of intracellular O− <sup>2</sup> and H2O2, leading to increased membrane permeability, and disruption of plant cell membranes as evidenced by a significant increase in the relative electrolyte leakage and MDA content in the root sample. However, exogenous spraying of Spd decreased the O− <sup>2</sup> and H2O2 contents, which, in turn, reduced the levels of MDA and relative conductivity, thereby effectively alleviating the deleterious effects of low iron on the cell membrane (Figure 8, Supplementary Figure S3).

Next, we observed the ultrastructure of tomato leaves to reveal the effect of low-ironinduced oxidative stress on the plant cell structure. Figure 9 shows that under low-iron stress, the cell exhibited the phenomenon of plasma-wall separation, the cell membrane was damaged, the chloroplast and starch granules in the leaf were deformed, the chloroplast was irregularly spherical and the starch granule swelled obviously. However, with Spd foliar treatment under low-Fe stress, chloroplast deformity was recovered to some extent with elliptical bands, and the shape of starch grains was restored.

To study whether the alleviation of low-iron stress by exogenous Spd was related to the change in antioxidant enzyme activity in tomatoes, we analyzed the activities of SOD, POD and CAT in leaves and roots. The results, shown in Figure 10, revealed that the activities of SOD, POD and CAT decreased in leaves and roots under low-iron stress, which potentially indicated a weakened ROS scavenging ability. However, foliar-spraying with Spd increased the activities of SOD, POD and CAT to varying degrees, thereby effectively alleviating the ROS-induced damage to the cell membrane.

**Figure 8.** Effect of exogenous Spd on superoxide anion (O− <sup>2</sup> ) content and hydrogen peroxide (H2O2) content under low-iron stress in tomato plants (**A**,**B**). Means denoted by the different lower case letters are significantly different according to Duncan's multiple range test (*p* ≤ 0.05); the mean represents the average of three replicates and the vertical bar indicates ± standard deviation (SD).

**Figure 9.** Ultra-structure of leaf cells revealed by transmission electron microscopy. CW, cell wall; Va, vacuole; Chl, chloroplast; Mi, mitochondria; S, starch grain.

We also analyzed the levels of osmoregulatory substances such as proline, sugars and proteins, which are vital for osmotic regulation under stressful conditions in plants. The proline contents in both leaves and roots significantly increased by 40.91 and 32.05%, respectively, and the free amino acid content significantly decreased by 31.20 and 14.79%, respectively, under low-Fe stress when compared with the control. Interestingly, the proline and free amino acid contents in leaves and roots increased with Spd foliar treatment under low-Fe stress compared to low-Fe stress only. The soluble protein content decreased in leaves and roots under low-Fe stress; however, it increased by 13.45% in leaves and 31.16% in roots after Spd foliar treatment under low-Fe stress. The soluble sugar content in leaves significantly decreased by 38.62% under low-Fe stress, while there was no significant change in this in roots. However, compared to low-Fe stress alone, treatment with Spd and low-Fe stress increased the soluble sugar content significantly in both leaves and roots (Figure 11).

**Figure 10.** Effect of exogenous Spd on antioxidant enzyme activity under low-iron stress in tomatoes. The first row represents the leaves and the second row represents the roots. Means denoted by the different lower case letters are significantly different according to Duncan's multiple range test (*p* ≤ 0.05); the mean represents the average of three replicates and the vertical bar indicates ± standard deviation (SD).

**Figure 11.** Effect of exogenous Spd on proline content, free amino acid content, soluble protein content and soluble sugar content under low-iron stress in tomato plants (**A**–**D**). Means denoted by the different lower case letters are significantly different according to Duncan's multiple range test (*p* ≤ 0.05); the mean represents the average of three replicates and the vertical bar indicates ± standard deviation (SD).

#### *3.5. Effect of Exogenous Spd on the Organic Acid Content in Roots and the Polyamine Content in Leaves under Low-Iron Stress in Tomato Plants*

Oxalic, malic, acetic and citric acids in the root system responded differently to low-Fe stress (Table 6). The oxalic acid level was not significantly altered by low-Fe stress compared to the control; however, the citric and malic acid contents increased by 78.12 and 69.58%, respectively, and the acetic acid content decreased by 49.76% in tomato roots under low-Fe stress. Spd treatment under low-Fe stress significantly increased the contents of citric (49.15%), malic (172.76%) and acetic acids (310.88%) compared to low-Fe stress alone, suggesting that exogenous Spd treatment-induced increased secretion of organic acids from the root potentially enhanced the Fe transport capacity.

**Table 6.** Effects of exogenous Spd on organic acid content in tomato roots under low-iron stress.


CK, control; LF, Low Fe; Spd, spermidine; LF + Spd, Low Fe plus spermidine. Data are shown as mean ± SD. Entries within each column followed by the same lowercase letters are not significantly different according to Duncan's test at *p* ≤ 0.05.

Meanwhile, under low-Fe stress, soluble and bound Put, Spd and Spm concentrations increased in the leaves, while free Put decreased and free Spd and Spm did not significantly change (Table 7). It is likely that free polyamines were converted to bound polyamines, which increased the bound polyamines under stress conditions. However, all three forms of polyamines, except for bound Spd, increased to different degrees after Spd foliar-spraying under low-Fe stress. This showed that exogenous Spd treatment could improve the biosynthesis and interconversion of endogenous polyamines to increase the plants' ability to withstand stress.

**Table 7.** Effects of exogenous Spd on the polyamine content in tomato leaves under low-iron stress.


CK, control; LF, Low Fe; Spd, spermidine; LF + Spd, Low Fe plus spermidine. Data are shown as mean ± SD. Entries within each column followed by the same lowercase letters are not significantly different according to Duncan's test at *p* ≤ 0.05.

#### *3.6. Effect of Exogenous Spd on Sugar Metabolism in Tomato Leaves under Low-Iron Stress*

In addition to being a source of energy for plant metabolism, sucrose has also been identified as a signaling molecule involved in the regulation of Fe deficiency. The sucrose content in leaves increased by 41.52, 28.24 and 48.57% with time after low-Fe treatment, and was higher than the control. Spd-spraying under low-Fe stress significantly reduced the sucrose content in the leaves compared to the low-Fe treatment (Figure 12).

**Figure 12.** Effects of exogenous Spd on the sucrose content in tomato leaves under low-iron stress. Means denoted by the different lowercase letters on the same color bars are significantly different according to Duncan's multiple range test (*p* ≤ 0.05); the mean represents the average of three replicates and the vertical bar indicates ± standard deviation (SD).

The results of the measurement of enzymes' activities related to sugar metabolism showed that under low-iron stress, the activities of both SS and SPS enzymes decreased, while the activities of two conversion enzymes, NI and AI, increased on day 10 after low-Fe treatment, although the sucrose content increased rather than decreased. This shows that the catabolic direction of SS and SPS enzyme activities was greater than the synthetic direction under low-Fe stress, and with the decrease in enzyme activities, the transport of photosynthetic products was blocked, causing the accumulation of sucrose in leaves, while the degradation and utilization of sucrose were weakened, which, in turn, stimulated the activities of two converting enzymes, NI and AI, and maintained the stability of sucrose anabolism. Exogenous Spd treatment significantly increased the SS activity and decreased the sucrose content, indicating that Spd promotes the degradation of sucrose, accelerates the consumption of sucrose transported from the leaves, promotes the transfer of photosynthetic products from the source to the reservoir and prevents the inhibitory effect of sucrose accumulation on photosynthetic efficiency (Supplementary Table S4).

#### **4. Discussion**

Iron is a vital element for the metabolism, growth and development of plants. Nevertheless, the lack of adaptive mechanisms to combat iron deficiency severely impairs plant biomass accumulation. Biomass is a direct manifestation of plant growth variation and can be an important basis for assessing the degree of plant injury due to stress [45]. Roots not only provide structural support to the above-ground parts of the plant but also provide nutrients and water. Therefore, the survival of a plant depends on its proper growth, development and root function [46]. Under low-iron stress, a decrease in above-ground and below-ground biomass (Table 2), and a suppressed total root length, total root surface area and total root volume of seedlings were observed (Supplementary Table S3). Morphological inhibition is one of the adverse effects caused by low-iron stress, and our results were consistent with earlier accounts of iron-deficiency effects on crop plants [47]. This is because adverse stress conditions inhibit both the division and growth of root cells, causing a significant decline in root biomass [48]. However, foliar-spraying with Spd increased not only above-ground and below-ground biomass but also the total root length, total root surface area, total root volume, root vigor and Fe3+ reductase activity, which potentially improved nutrient acquisition and alleviated low-iron stress in tomato seedlings.

Since photosynthesis is the most essential plant process, its efficiency has a significant influence on growth, yield and stress resistance in plants [49]. In this study, the photosynthetic pigment content of tomato leaves was significantly inhibited under low-iron stress and leaf photosynthetic activity was drastically reduced (Tables 3 and 4), which is consistent with the findings of Yao et al. [50]. This is because iron-deficiency stress hinders chlorophyll synthesis in tomato seedlings, leading to a reduction in chloroplast lamellae and disruption of the chloroplast structure. However, the chlorophyll contents in tomato leaves increased significantly after Spd foliar-spraying. Such effects support the hypothesis that the ability to capture and convert light energy was restored, and the exogenously sprayed Spd could safeguard chloroplasts and protect the photosynthetic mechanism from the adverse effects of environmental stress [51]. Moreover, chlorophyll fluorescence parameters such as Fv/Fm, PSII, ETR, etc., decreased significantly and NPQ increased under low-iron stress (Table 5), which was in agreement with the previous findings [52]. This is because damage to the photosystem II reaction centered on low-iron stress-inhibited PS II photochemical activity, reduced PS II primary light energy conversion efficiency and hindered the photosynthetic electron transfer process. Consistent with the previous reports in Sweet Corn [53], exogenous Spd increased the chlorophyll content and stabilized the photosynthetic system in tomato seedlings, thus alleviating the damage to the photosystem and enhancing or restoring photosynthetic efficiency. It can be inferred that exogenous Spd-spraying is crucial to improve the photosynthetic efficiency of tomato seedlings, leading to increased biomass and dry matter accumulation.

Polyamines protect plants from environmental stress by regulating the accumulation of sugar, proline and other osmotic substances [54]. Proline is an important osmotic adjustment substance in plants that functions in maintaining the membrane structure and is used as a physiological and biochemical indicator for the plant stress response [55]. Du [56] showed that the proline content in plants under stress increased, and was further increased by Spd treatment, which is in agreement with our results showing that proline content in leaves and roots of tomato seedlings under low-iron stress increased significantly compared to the control, and were further significantly increased after foliar-spraying of Spd under low-iron stress compared to LF treatment. The proteins synthesized and stored during plant growth are degraded to free amino acids for biosynthesis to maintain normal plant life activities [57]. When plants are subjected to stress, particularly osmotic stress, the soluble sugar content increases, which can improve the osmoregulatory capacity of leaves and provide carbon and nitrogen sources for plant organic matter synthesis [58]. The soluble sugar content in leaves and roots of tomato seedlings decreased under low-iron stress; however, exogenous Spd treatment increased the soluble sugar content in tomato seedlings under low-iron stress, suggesting that Spd improves the ability of plants to synthesize sugars [59]. To improve the plant tolerance to iron deficiency, roots can reduce the inter-root pH by secreting organic acids and increasing Fe3+ solubility [60]. Exogenous spraying with Spd significantly increased the content of citric and malic acids in the root system, which indicates that Spd potentially increases the secretion of organic acids in the root system, thus enhancing the iron transport in plants [61].

Plant performance under multiple abiotic stresses is linked to the accumulation of Put, Spd and Spm [62]. In this study, the content of all three forms of polyamines increased to different degrees after Spd-spraying, which is consistent with the results of Shan et al. [61]. It is highly likely that exogenous Spd treatment potentially improves the biosynthesis of endogenous polyamines and significantly enhances the ability of plants to withstand adversity. Moreover, the study also found that Spd treatment significantly increased SS enzyme activity, reduced sucrose content, promoted sucrose degradation, accelerated sucrose consumption, facilitated the transfer of photosynthetic products from source to sink and prevented the inhibitory effect of sucrose accumulation on photosynthetic efficiency in tomato seedlings [63], which is consistent with the results of our study.

Under stress conditions, reactive oxygen species (ROS) are profusely generated in plants, causing oxidative stress and damage to important molecules in plants [55,64]. The cell membrane is a barrier that maintains the relative stability of plant cells. Under stress conditions, the degree of membrane lipid peroxidation intensifies due to excessive accumulation of ROS, which changes the membrane permeability and affects the normal physiological and biochemical reactions [65]. In this study, low-iron stress reduced SOD, POD and CAT activities in tomato plants and weakened their ability to scavenge ROS, resulting in excessive intracellular O− <sup>2</sup> and H2O2 accumulation, increased membrane permeability and disruption of plant cell membranes (Figure 8). This relies on the fact that iron acts as a component of enzymes such as SOD, POD and CAT, and the three enzymes' activities were significantly inhibited when plants were subjected to a low-iron environment. After exogenous spraying of Spd treatment, the SOD, POD and CAT activities increased to different degrees and O− <sup>2</sup> , H2O2, MDA and the relative conductivity decreased, indicating that Spd effectively alleviated the extent of cell membrane disruption.

In iron-chelating reductase *FRO7* mutant plants, the iron content in chloroplasts and the activity of iron reductase are significantly lower than in wild-type plants, and the electron transport chain in the photosystem is interrupted, causing impaired photosynthesis [66]. Moreover, *FRO7* mutant plants show a severe yellowing phenotype, along with the occurrence of seedling lethality, indicating that the *FRO7* gene is important for maintaining iron homeostasis in chloroplasts and for the proper performance of photosynthesis in the plant [66]. In the present study, Spd treatment under low-iron stress upregulated the expression of the *FRO* gene and related Fe transporter genes *IRT1* and *IRT2* in the root, which is consistent with the results of a previous study in *Pyrus betulaefolia* [67].

Previous studies established that IAA plays an important role as a signaling molecule in the response to iron deficiency in plants, and that the local iron supply affects the plant lateral root growth and development by inducing the growth hormone AUX-1 transporter [68]. The strategy-I plants induce ethylene synthesis in response to iron-deficiency stress, and ethylene positively regulates the iron-deficiency response [69]. The ethylene response factor *ERF4/ERF72* is involved in iron-deficiency response in apple rootstocks, and interference with these two genes results in upregulated expression of iron-uptake genes in *Ziziphus jujube* roots, promoting iron uptake by the roots [70]. Accordingly, we also found that transcript levels of *ERF1* and *ERF2* genes were upregulated in the root system under low-iron stress, and exogenous spraying of Spd treatment further upregulated the expression of *ERF1* genes in the leaves, while it downregulated them in the root. Differential expression of these genes related to growth hormones and ethylene, together with the expression of downstream *FRO* and *IRT1* genes, potentially contributed to improved iron acquisition and transport under low-iron stress.

Meanwhile, sucrose accumulation in leaves increased under low-iron stress, which indicated that the translocation capacity of sucrose to the root system was possibly reduced; nonetheless, sucrose could act as a long-range signal to regulate the response of plants to Fe deficiency [71]. It is worth noting at this point that the expression of genes such as *COX15* in chlorophyll metabolism was downregulated under low-iron stress, indicating that the transport of sucrose to the lower part of the ground was inhibited [72]. The upregulated expression of genes such as *CHLP*, *PetF* and *CAT*, which are involved in chlorophyll synthesis and antioxidant enzyme activities, as well as significantly upregulated *SUS* and *TPS* gene expression and significantly increased sucrose synthase activity after Spdspraying, indicated that Spd treatment also affected sugar metabolism to confer tolerance to low-iron stress in tomato plants.

#### **5. Conclusions**

Iron (Fe) deficiency severely limits agricultural crop yield due to its low availability, particularly in soils with a high pH. The success of iron fertilization largely depends on soil pH management, which is very challenging in field conditions. In this study, we showed that foliar application of exogenous plant growth regulator Spd could improve plant tolerance to low-iron stress. Briefly, the transcriptomic analysis revealed that exogenous Spd could regulate the plant response to low-iron stress by modulating the expression of genes involved in the processes of hormone metabolism, sucrose metabolism, antioxidant defense system, photosynthesis, chlorophyll metabolism and Fe uptake and transport. Besides

this, biochemical and physiological analyses revealed that low-iron stress-induced suppression, in photosynthesis and growth of tomato seedlings, were significantly alleviated by exogenous Spd treatment, which was closely associated with differential modulation of photosynthetic pigment contents, gas exchange, chlorophyll fluorescence capacity, proline content, sucrose content, root vigor, citric and malic acid contents, ROS metabolism and polyamine synthesis and interconversion. Overall, this study reveals the critical mechanism of exogenous Spd-induced enhanced tolerance to low-iron stress in tomatoes and provides a novel characterization of the key traits associated with the adaptation of tomatoes to a low-iron environment. Traits associated with changes in low-iron-tolerance genes can potentially be used to improve yields of greenhouse tomatoes in low-iron environments. Nonetheless, large-scale experimentation is required to unveil and extend this knowledge, to develop better agricultural practices.

**Supplementary Materials:** The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/antiox11071260/s1, Figure S1: The relative expression of selected differentially expressed genes verified by qRT-PCR; Figure S2: Effect of exogenous Spd on (A) root vigor and (B) Fe3+ reductase activity of tomato under low-iron stress; Figure S3: Effect of exogenous Spd on lipid peroxidation (A) and ion leakage (B) in tomato roots under low-iron stress; Table S1: qRT-PCR test reaction system; Table S2: Primers used for qRT-PCR; Table S3: Effects of exogenous Spd on tomato root morphological indexes under low-iron stress; Table S4: Effects of Spd on enzyme activities related to sucrose metabolism in tomato leaves under low-iron stress.

**Author Contributions:** Y.S., conceptualization, methodology, formal analysis, investigation and writing—original draft. Y.Z. (Yihong Zhao), formal analysis, investigation and writing—original draft. Q.Y., formal analysis and investigation. F.L., methodology and funding acquisition. X.L., formal analysis and investigation. X.J., formal analysis and investigation. Y.Z. (Yi Zhang), conceptualization, supervision, resources, writing—original draft, funding acquisition and project administration. G.J.A., conceptualization, writing—review and editing, funding acquisition and project administration. All authors have read and agreed to the published version of the manuscript.

**Funding:** This work was supported by the National Key R&D Program of China (2019YFD1000300), Shanxi Province Key R&D Plan (201903D211011), the Basic Research Program in Shanxi (20210302123366) and the National Natural Science Foundation of China (3195041055, 31501750, 31550110201).

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Data are contained within the article and Supplementary Materials.

**Conflicts of Interest:** The authors declare no conflict of interest.

### **References**

