*3.2. Analysis of Differentially Expressed Genes*

To get a closer look at the differentially expressed genes, we mapped volcanoes (Figure 1). In the volcano maps, red represents significantly upregulated differently expressed genes, blue represents significantly downregulated differently expressed genes and gray represents non-significant differently expressed genes.

FPKM (fragments per kilobase of exon model per million mapped fragments) was used to count the expression abundance of known genes in different samples. In this experiment, we used the difference multiplier FC ≥ 2 or FC ≤ 0.5 (i.e., the absolute value of log2FC ≥ 1) as the threshold of change and a *p*-value <0.05 as the criterion for screening differential genes. The number of differentially expressed genes in each comparison group was counted, and a bar chart (Figure 2) was used to visualize the number of significantly differentially expressed genes in different comparison groups, as well as the specific changes (up- and downregulation). Compared to the control, 227 genes were upregulated and 201 genes were downregulated in the low-iron-treated leaves (LF), whereas the number of differentially

expressed genes was higher in the root system, where 933 genes were upregulated and 1199 genes were downregulated, which indicated that the low-iron treatment had a more profound effect on transcription in the root system than in the leaves. Again, compared to the LF treatment, 606 genes were upregulated in the LF + Spd-treated leaves, and 302 genes were downregulated, while 422 genes were upregulated and 619 genes were downregulated in the root sample.

**Figure 1.** Volcano maps of expression differences. The horizontal coordinate represents the different expression fold changes of the gene in different samples, and the vertical coordinate represents the statistical significance of the difference in the gene expression change. LF\_L vs. CK\_L, Low Fe\_Leaf sample vs. Control\_Leaf sample; LF\_R vs. CK\_R, Low Fe\_Root sample vs. Control\_Root sample; LFS\_L vs. LF\_L, Low Fe + Spd\_Leaf sample vs. Low Fe\_Leaf sample; LFS\_R vs. LF\_R, Low Fe + Spd\_Root sample vs. Low Fe\_Root sample.

Then, we performed Venn diagram analysis for Control vs. Low Fe (CK vs. LF) and Low Fe vs. Low Fe + Spd (LF vs. LFS), gene ontology (GO) enrichment for Low Fe+ Spd\_Leaf sample vs. Low Fe\_Leaf sample (LFSL vs. LFL) and Low Fe + Spd\_Root sample vs. Low Fe\_Root sample (LFSR vs. LFR) and KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway enrichment analysis for differently expressed genes as influenced by Spd treatment.

The Venn diagram can visualize not only the number of differently expressed genes in the different treatment groups but also the number of genes that are differently expressed in each treatment group in total. As shown in Figure 3, a total of 104 differently expressed genes were co-expressed among 428 differentially expressed genes in the treatment group CK vs. LF, and there were 908 differentially expressed genes in the treatment group LFS vs. LF in the leaves. In the case of the root sample, a total of 677 differently expressed genes were co-expressed among 2132 differently expressed genes in the treatment group CK vs. LF, and there were 1041 differently expressed genes in the treatment group LFS vs. LF.

**Figure 2.** Number of significantly differentially expressed genes in different treatments. LF\_L vs. CK\_L, Low Fe\_Leaf sample vs. Control\_Leaf sample; Spd\_L vs. CK\_L, Spd\_Leaf sample vs. Control\_Leaf sample; LFS\_L vs. LF\_L, Low Fe + Spd\_Leaf sample vs. Low Fe\_Leaf sample; LFS\_L vs. Spd\_L, Low Fe + Spd\_Leaf sample vs. Spd\_Leaf sample; LF\_R vs. CK\_R, Low Fe\_Root sample vs. Control\_Root sample; Spd\_R vs. CK\_R, Spd\_Root sample vs. Control\_Root sample; LFS\_R vs. LF\_R, Low Fe + Spd\_Root sample vs. Low Fe\_Root sample; LFS\_R vs. Spd\_R, Low Fe + Spd vs. Spd\_Root sample.

**Figure 3.** Venn diagram of significantly differentially expressed genes in different treatment comparisons. LFSL. vs. LFL, Low Fe+ Spd\_Leaf sample vs. Low Fe\_Leaf sample; CKL. vs. LFL, Control\_Leaf sample vs. Low Fe\_Leaf sample; CKR.VS.LFR, Control\_Root sample vs. Low Fe\_Root sample; LFSR. vs. LFR, Low Fe+ Spd\_Root sample vs. Low Fe\_Root sample.

The GO enrichment analysis of LFS vs. LF showed that enrichment in biological processes (BP) was mostly in functions such as transcriptional regulation with DNA as the template, protein phosphorylation, defense responses, redox processes, signal transduction processes, ethylene-activated signaling pathways, defense responses against fungi, and protein ubiquitination (Figure 4). In cellular components (CC), differentially expressed genes were involved in biological functions such as those of the nucleus, plasma membrane, membrane components, cytoplasm, chloroplast and extracellular regions. In molecular functions (MF), they were mainly enriched in sequence-specific protein-binding, specific DNA sequence-binding transcription factor activity, ATP-binding, DNA-binding, protein serine/threonine kinase activity, and metal ion-binding.

**Figure 4.** GO enrichment analysis of differently expressed genes. The abscissa represents different GO terms, blue represents biological processes, green represents cellular components, orange represents molecular functions and ordinate represents the number of differentially expressed genes. LFSL vs. LFL, Low Fe + Spd\_Leaf sample vs. Low Fe\_Leaf sample; LFSR vs. LFR, Low Fe + Spd\_Root sample vs. Low Fe\_Root sample.

To further explore the most important biochemical/metabolic pathways and signal transduction pathways involved in differentially expressed genes due to Spd treatment in low-iron-supplied tomato plants, the top-20 significantly enriched pathways were screened for KEGG enrichment analysis by the number of genes enriched in this pathway, and the enrichment results are presented in the form of bubble plots (Figure 5). KEGG enrichment analysis was performed on 908 differentially expressed genes in leaves and 1041 differentially expressed genes in roots, comparing the low-iron treatment and combined treatment of Spd and low iron.

The results showed that a total of 707 differently expressed genes in leaves were significantly enriched in 113 KEGG metabolic pathways, concentrated in metabolic pathways such as plant-pathogen interaction (79), phytohormone signaling (61), cytokinesis (30), amino and nucleotide sugar metabolism (29), phenyl propane biosynthesis (23) and starch and sucrose metabolism (23). A total of 867 differently expressed genes were significantly enriched in 117 KEGG metabolic pathways in the root system, concentrated in metabolic pathways such as plant-pathogen interaction (58), phytohormone signaling (56), benzyl propane biosynthesis (40), starch and sucrose metabolism (28), amino and nucleotide sugar metabolism (23) and carbon metabolism (22). It was found that the differentially expressed genes of iron-deficiency stress were mainly enriched in the pathways of phytohormone signaling, starch and sucrose metabolism and phenyl propane biosynthesis in both leaves and roots.

The results of GO enrichment analysis and KEGG pathway enrichment analysis showed that the differentially expressed genes in plant hormone signaling processes and starch and sucrose metabolism were significantly affected by low-Fe stress. Therefore, we performed a heat map analysis of differentially expressed genes in phytohormone signaling pathways and starch and sucrose metabolism. The results showed that a total of nine differently expressed genes in the phytohormone signal transduction pathway— *Solyc00g174330.3(PR1)*, *Solyc05g009610.1(GID1)*, *Solyc09g007010.1(PR1)*, *Solyc06g062460.3 (PIF3)*, *Solyc07g056000.2(TCH4)*, *Solyc09g089930.2(ERF1)*, *Solyc12g036470.2(PIF3)*, *Solyc01g1 07400.2(GH3)* and *Solyc03g093080.3(TCH4)*—were common to leaves in the comparison groups of LF vs. CK and LFS vs. LF (Figure 6). The expression of oleuropein sterol regulatory protein EBRU1 precursors *Solyc07g056000.2(TCH4)* and *Solyc03g093080.3(TCH4)* was downregulated under low-iron treatment, while all other genes were upregulated. In contrast, all nine differently expressed genes were upregulated in leaves after spraying with Spd under low-iron treatment. A total of 37 differently expressed genes were expressed in the root system, mostly concentrated in the growth hormone and ethylene metabolic pathways. The expression of *Solyc03g082510.1(SAUR)* and *Solyc10g076790.2(AUX1)* in the growth hormone metabolic pathway and *Solyc08g066660.1(ERF1)* and *Solyc03g114310.3(CTR1)* in the ethylene metabolic pathway were downregulated under low-Fe stress, while their expression with Spd treatment under low-iron stress was upregulated. It appears that differentially expressed genes related to hormone metabolism showed different trends in leaves and roots. For the upregulated genes, in leaves, Spd foliar-spray treatment could further upregulate gene expression, whereas, in roots, Spd foliar treatment downregulated genes to the control level. Of the 18 differentially expressed genes in the root system for starch and sucrose metabolic processes, seven differently expressed genes were downregulated and 11 differently expressed genes were upregulated, and the expression of genes related to hormone signaling was consistent with the Spd treatment; nonetheless, all of these were backregulated to the control level in the root sample.

**Figure 5.** Enrichment analysis of differentially expressed genes in the KEGG pathway. The horizontal axis indicates the degree of enrichment (Rich factor), and the vertical axis indicates the enriched KEGG pathway; the size of the dots indicates the number of differentially expressed genes enriched in a KEGG pathway; the color of the dots indicates different *p* values; the Rich factor indicates the number of differentially expressed genes belonging to a KEGG pathway/the total number of genes belonging to this KEGG pathway. The larger the Rich factor, the higher the enrichment of the KEGG pathway. LFSL vs. LFL, Low Fe + Spd\_Leaf sample vs. Low Fe\_Leaf sample; LFSR vs. LFR, Low Fe + Spd\_Root sample vs. Low Fe\_Root sample.

Then, we analyzed the expression of genes related to hormone signaling pathways and sucrose metabolism, as well as differentially expressed genes of other metabolic pathways, as shown in Figure 7. In leaf blades, *Solyc01g008620.3(GN1-2-3)* expression was upregulated in the starch and sucrose metabolic pathways, which potentially accelerated glucose synthesis; *Solyc02g071620.3(CHLP)* and *Solyc07g064720.3(CHLP)* expression were upregulated in porphyrin and chlorophyll metabolism, which in turn, potentially functioned in the synthesis of chlorophyll a and chlorophyll b, respectively. *Solyc07g024000.3(NOL)* expression was downregulated, thus, perhaps, inhibiting the conversion of chlorophyll b to hydroxy-chlorophyll a. In the photosynthetic pathway, *Solyc11g006910.2(PetF)* iron oxytocin gene expression was upregulated during photosynthetic electron transfer; in the peroxisome pathway, i.e., the antioxidant enzyme system, *Solyc12g094620.2(CAT)* expression was upregulated in the antioxidant enzyme system and so on. In the root system, more

genes are related to the expression of hormone metabolism, and among them, the expression of *Solyc10g076790.2(AUX1)* and *Solyc03g082510.1(SAUR)* was upregulated after Spd treatment under low iron, both of which are jointly involved in plant cell growth. However, *Solyc09g089610.3(ETR)*, *Solyc09g066360.1(ERF1)* and *Solyc04g071770.3(ERF2)* transcripts were downregulated, which potentially alleviated the effect of ethylene on cell senescence. Again, *Solyc12g038580.2(TPS)* expression was upregulated in the starch and sucrose metabolic pathways, which affected sugar synthesis, and *Solyc12g009300.3(SUS)* expression was downregulated, which might affect sucrose synthase activity. In the peroxisome pathway, the epoxidation process was promoted by upregulation of *Solyc01g066457.1(EPHX2)*. Upregulation of *Solyc01g058210.2(HMGCL)*, *Solyc10g007600.3(HAO)* and *Solyc12g099930.2 (AGXT)* contributed to amino acid metabolism, and upregulated expression of *Solyc12g094620.2 (CAT)* in hydrogen peroxide metabolism potentially increased redox levels. In addition to affecting the expression of related metabolic genes in each pathway, Spd-spraying under low iron upregulated the expression of *Solyc02g069200.3(IRT1)*, *Solyc01g094890.3((FRO2)* and *Solyc01g094910.3(FRO),* which potentially improved the Fe uptake and transport capacity of the root system under low-iron stress.

**Figure 6.** Heat map of differentially expressed genes related to plant hormone signal transduction and sucrose metabolism. Low Fe + Spd\_Root sample vs. Low Fe\_Root sample.

**Figure 7.** Diagram of plant sucrose metabolism pathway.

Finally, expression trends of six selected differentially-expressed genes related to iron transport or sucrose metabolism in the root were validated by qRT-PCR. The trends for the gene expression in qRT-PCR (Supplementary Figure S1) were approximately the same as the transcriptome sequencing results, indicating that the results were credible.
