The morphological response of wheat to low Fe stress
The typical symptom of low Fe in plants is the thin green veins on the pale green leaves. As the symptoms worsen, the leaves become thinner and white (or pale yellow), and brown spots and bad tissue appear on the leaves. After the leaves mature, the boundary between the green veins and the light green or light yellow mesophyll becomes more obvious. Our results showed that under low Fe stress, the growth and development of the root system of wheat were inhibited, and the root hair density became higher (Fig. 1A, C). Leaves were pale yellow or whitish, young leaves were chlorosis, and there was obvious visible inter-vein yellowing on the leaves (Fig. 1A, B).
The biomass of wheat was measured and found that under low Fe stress, the fresh weight and dry weight of the shoots were reduced by about 1.2 times, and the fresh weight and dry weight of the roots were reduced by about 1.3 times and 1 time, respectively (Fig. 2A, B). However, the relative water content of shoots showed no significant difference under different treatments (Figure. 2C). Determination of photosynthetic pigment content in wheat found that under low Fe stress, the concentrations of total chlorophylls (including chlorophyll a and b) and carotenoids (including xanthophyll and carotene) were significantly reduced (Fig. 2D, F). The ratio of the concentration of chlorophyll a to chlorophyll b was increased significantly under low Fe stress, which was also a sign of plant adaptation to adversity stress (Fig. 2E). In addition, low Fe stress reduced the ratio of total chlorophyll concentration to carotenoid concentration (Fig. 2E). Then we measured the ion leakage of the root system and found that there was no difference in ion leakage rate under different treatment conditions (Fig. 2G).
The damage of organelles was observed by transmission electron microscope, and the intracellular ultrastructure of the morphological difference between the control and the Fe-deficiency stress was studied (Fig. 3A). Our results showed that under normal Fe supply condition, chloroplast was well developed, complete in structure, convex lens-shaped, rich in content, many thylakoids, clear and complete basal lamella, and chloroplast membrane, and multiple basal lamellas, neatly arranged. However, under the low Fe condition, the chloroplast deformed and became smaller and the chloroplast membrane was blurred; the lamella system could not be stacked to form basal particles, and most of the basal lamellas disappeared and became blurred.
Root growth is determined by the balance between cell division and cell elongation . To study the role of Fe in the growth and development of the root system, we observed the cell size of the meristematic zone and elongation zone in the root system grown under normal or low Fe stress (Fig. 3B). The meristem size was calculated as the area where isodiametric cells extend from the quiescent center (QC) to the cell, which was twice the length of the immediately preceding cell . The boundary of the transition zone is different in each cell type, so in all the analyses performed here, the cortical cell file was used to define the boundary . Our results showed that compared with the control, the number of cells in the meristematic zone of the root system under Fe-deficient condition was significantly reduced, and the length of the cells in the elongation zone was significantly shorter (Fig. 3B-D).
Under the low Fe treatment, the root-to-shoot ratio was reduced but the difference was not significant. It could be inferred that the low Fe stress inhibited the growth of the shoots higher than that of the roots (Fig. 4A). Subsequently, the specific effects of low Fe stress on the growth of the wheat root system were analyzed. Low Fe stress was significantly reduced root-related parameters, including total root length, main root length, root surface area, root volume, number of root tips, average root diameter, and lateral root length (Fig. 4B-H). Then we measured the root activity of wheat and found that the root activity of wheat seedlings decreased significantly under low Fe stress, indicating that low Fe stress reduced the ability of roots to absorb Fe (Fig. 4I). These results indicated that the growth and development of wheat seedlings under low Fe stress were severely inhibited.
Physiological response of wheat to low Fe stress
To further understand the physiological response of wheat to low Fe stress, the contents of some osmotic adjustment substances that may participate in the regulation of low Fe stress resistance were tested. Under low Fe stress, the Pro concentration in the shoots and roots was increased significantly, but the concentration of MAD was only increased significantly in the shoots (Fig. 5A, D). The content of MDA is a sign of plasma membrane peroxidation. Our results showed that compared with the control, there was no significant difference in the concentration of MAD in roots under the low Fe stress, which indicated that the low Fe stress did not cause peroxidative damage to the plasma membrane (Fig. 5D). Under the stress of low Fe, the concentrations of OFR and H2O2 in the shoots and roots were significantly higher than those of the control condition (Fig. 5B, C). The results showed that low Fe stress accumulated more ROS.
Fe deprivation caused ROS accumulation
When plants are stressed by Fe deficiency, they will produce a large amount of ROS, which will cause oxidative damage. To analyze the effect of low Fe on the accumulation of ROS in wheat plants, DAB and NBT staining were performed on the shoots and roots of wheat after different treatments to observe the accumulation of H2O2 and O2¯ (Fig. 6A, B). The results showed that compared with normal Fe treatment, the DAB staining and NBT staining colors of wheat plant shoots and roots under low Fe stress were dark brown or dark blue, respectively, indicating that the shoots under low Fe stress, the concentrations of H2O2 and O2¯ in the roots and roots were significantly higher than those of the control (Fig. 6A, B). These research results showed that wheat plants produced more ROS after low Fe treatment. This result was consistent with the conclusion of Fig. 5B and C, that was, low Fe stress increased the accumulation of ROS.
Effect of low Fe stress on mineral nutrient elements in wheat
Inductively coupled plasma mass spectrometry (ICP-MS) is used to determine the ion expression profile of control and low Fe stress. ICP-MS quantitative analysis showed that the Fe+ concentration in the shoots and roots of wheat was significantly reduced under the low Fe stress treatment (Fig. 7B). Subsequently, we tested the concentrations of other metal cations, including Cu2+, Mn2+, Zn2+, Mg2+, Na+, Ca2+, Cd2+, and K+ (Fig. 7). In general, the response of low Fe stress to Cu2+, Mg2+, Na+, Cd2+, and K+ was similar. Under low Fe stress, the concentrations of Cu2+, Mg2+, Na+, Cd2+, and K+ in the shoots did not change significantly; on the contrary, the concentrations of these cations increased significantly in the roots (Fig. 7A, E, F, H, I). Under low Fe stress, the Mn2+ concentration was increased significantly in the shoots and roots (Fig. 7C). Unlike the above cations, the concentrations of Zn2+ and Ca2+ did not change significantly in the shoots and roots (Fig. 7D, G).
Transcriptional response of wheat to low Fe stress
To understand the molecular basis of Fe tolerance in wheat seedlings under low Fe stress, an RNA-seq library was established using the shoots and roots of low Fe seedlings as materials. The correlation between gene expression levels in samples is an important indicator for testing the reliability of experiments. In this study, under the same treatment, the Pearson correlation coefficient between each pair of biological replications was mostly higher than 0.90, indicating that the similarity between samples in the transcriptome was very high (Figure S1). RNA-Seq generated more than 1,145,14 million Raw reads (Table 1). Among these reads, the GC content of libraries was about 54.00%. After quality control, 1,134,550,000 Clean reads were obtained, the average error rate of sequencing bases was less than 0.0249%, the Q20 value was more than 98.03%, and the Q30 value was more than 94.06%. Among them, 7,592-10.23 million Clean reads were located in the wheat genome (Table 1). Principal component analysis showed that there were significant differences in the expression patterns between different treatments and different wheat tissues (Fig. 8A). 2,861 genes were identified as differentially expressed (FDR <0.05, fold change ≥ 2). There were a total of 137 differentially expressed genes (DEGs) in the shoots and roots (Fig. 8B). The number of DEGs in the shoots was relatively small (378), under Fe-deficient stress, 313 genes were up-regulated and 65 genes were down-regulated (Fig. 8B, C). There were a large number of DEGs in the roots (2,619), under low Fe stress, there were 1,589 up-regulated genes and 1,030 down-regulated genes (Fig. 8B, D).
To obtain the functional information of DEGs, GO annotation analysis was performed on DEGs, and DEGs were classified into three types: biological process (BP), cell component (CC), and molecular function (MF). GO entries with p-value < 0.001 were considered to be significantly enriched. In the shoots and roots under the stress of low Fe, BP was mainly enriched in the metabolic process and cellular process (Fig. 8E); CC was mainly enriched in the cell part and membrane part (Fig. 8E); in the MF, binding and catalytic activity were the two most abundant GO terms (Fig. 8E). The KEGG annotation divided the pathways that DEGs participate in into five categories: Cellular Processes, Environmental Information Processing, Genetic Information Processing, Metabolism, and Organismal Systems. Most DEGs in Cellular Processes were annotated to Endocytosis pathway, most DEGs in Environmental Information Processing were annotated to MAPK signaling pathway–plant pathway, most DEGs in Genetic Information Processing were annotated to Protein processing in endoplasmic reticulum pathway, most of Metabolism DEGs were annotated to the Phenylpropanoid biosynthesis pathway, and most DEGs in Organic Systems were annotated to the Plant-pathogen interaction pathway (Fig. 8F).
To further deepen our understanding of the functions of these DEGs, we conducted GO enrichment analysis (Fig. 8G). Among them, GO terms with p-value < 0.001 were considered to be significantly enriched. We found that most DEGs were related to plant metabolism and biosynthesis processes, such astricarboxylic acid biosynthetic process, nicotianamine synthase activity, nicotianamine metabolic process, nicotianamine biosynthetic process, nicotianamine aminotransferase activity, and L−methionine biosynthetic process, etc. These results indicated that the synthesis of Fe chelator and Fe transport was regulated to maintain Fe homeostasis. Significantly enriched GO terms related to ion transport and ion homeostasis included iron ion transmembrane transport, intracellular sequestering of iron ion, sequestering of metal ion and sequestering of iron ion, etc. The above results indicated that the transporter played an important role during the low Fe period. In addition, the GO terms related to photosynthesis, such as photosynthesis, light harvesting in photosystem I, were also significantly enriched, indicating that low Fe affected the photosynthesis of wheat, which was closely related to the decrease of chlorophyll content in the shoots (Fig. 2D, E, F). These results emphasized the importance of membrane and/or membrane-localized metal ion transporters and regulatory and metabolic proteins in the stress of low Fe.
The KEGG database was used to further determine the pathways involved in the response of wheat to low Fe stress (Fig. 8H). Studies have shown that in plants exposed to abiotic stress, the accumulation of amino acids was believed to have a beneficial effect in the process of plant stress adaptation [17-19]. Most of the DEGs were involved in amino acid metabolism pathways, such as Alanine, aspartate and glutamate metabolism, Tyrosine metabolism, Cysteine, and methionine metabolism and Phenylalanine metabolism, and other KEGG pathways, which indicated that amino acid metabolism and protein synthesis played an important role in the response of wheat to Fendeficiency stress. DEGs were also involved in pathways related to photosynthesis, such as Photosynthesis − antenna proteins and Carotenoid biosynthesis, which indicated that low Fe had a significant impact on the photosynthesis of wheat. In addition, Phenylpropanoid biosynthesis, MAPK signaling pathway-plant, and other KEGG pathways were also significantly enriched (Fig. 8H).
Transcriptional response of Fe transport-related genes under low Fe stress
GO enrichment indicated that the synthesis of Fe chelator and Fe transport played an important role during low Fe stress. Among many DEGs, genes related to Fe ion homeostasis are the key genes for wheat tolerance to low Fe. Therefore, we analyzed the expression of genes related to Fe uptake and transport under Fe-deficiency stress. Figure S2 showed a molecular model of genes involved in Fe absorption and transport in plant roots (Figure S2A), chloroplasts, mitochondria, and vacuoles (Figure S2B). Transcription results analysis showed that most of the key genes involved in strategy I uptake of Fe, such as Fe3+ chelate reductase (FRO), iron-regulated transporter (IRT), natural resistance-associated macrophage protein (NRAPM), and Yellow stripe-like (YSL), were significantly up-regulated under Fe-deficiency stress. Among them, TaFRO2-2A (Figure S3A) and TaIRT1a-4A (Figure S3E), TaNRAMP2-4A (Figure S3B), and TaYSL15a-6D (Figure S3C) were up-regulated by 3 times, 6 times, 5 times, and 70 times, respectively, which might play a decisive role in the process of Fe uptake by plants.
In strategy II, the expression abundance of genes involved in plant Fe vector synthesis and Fe uptake and transport-related genes was significantly up-regulated. The process of synthesis and secretion of plant Fe siderophores is very complicated and requires the participation of 13 enzymes, but there are mainly four key enzymes: methionine synthetase (SAM), Nicotinamide synthase (NAS), Nicotinamide aminotransferase (NAAT), and Deoxyergate synthase (DMAS). Transcription analysis showed that these four key enzyme genes were significantly induced under low Fe stress. Among them, TaSAM3-6B (Figure S3D), TaNAS1c-6D (Figure S3I), TaNAAT1b-1B (Figure S3G), and TaDMAS1-4A (Figure S3F) had the most differential expression fold changes, which were up-regulated by 3 times, 3105 times, and 2718, and 24 times, respectively. The GO enrichment results also found that the GO terms related to nicotinamide (NA) synthesis were significantly enriched under low Fe stress (Fig. 8G). It might be related to the up-regulation of the expression abundance of the four key enzyme genes mentioned above, SAM, NAS, NAAT, and DMAS. And the synthesis of plant Fe carrier is related to the methionine salvage pathway because SAM is the key precursor for the synthesis of malic acid substances (MAs), which are produced by methionine. Transcriptome KEGG enrichment analysis also showed that methionine metabolism, L-methionine biosynthetic process, and L-methionine salvage from the S-adenosylmethionine pathway were significantly enriched (Fig. 8H). The above results indicated that amino acid metabolism played an important role in wheat response to low Fe. In strategy II, genes involved in Fe3+ transport such as transporter of mugnetic acid (TOM) (Figure S3J) and multidrug and toxin efflux family (MATE) (Figure S3K) were significantly up-regulated under low Fe stress. Among them, the differential expression of TaTOM-2B and TaMATE-4A changed the most, up-regulated by 2 times and 20 times, respectively. It was speculated that it might play a key role in the transport of iron from the xylem to neighboring cells. Oligopeptide transporter (OPT) is a polypeptide transporter located on the plasma membrane of plant epidermal cells. Transcriptome analysis showed that it was significantly induced by low Fe, and the differential expression of TaOPT3-5B was significantly up-regulated by low Fe by 44 times (Figure S3M), which might play a key role in the process of transmembrane transport of Fe3+-PS complexes near epidermal cells to plants.
The process of vacuolar iron transporter ginseng (VIT) for vacuolar membrane positioning and vacuole loading of Fe into seed embryos not only promotes the development of seeds but is also crucial for seed germination. NRAMP is located on the vacuole membrane, mediates the transport of Fe from the vacuole of the seed embryo to the cytoplasm, and participates in the supply of Fe during seed germination. Transcriptome results showed that the expression abundance of most VIT genes was significantly down-regulated under low Fe stress, especially TaVIT2-5B was down-regulated by 3 times (Figure S3R), which might play a key role in promoting seed development. NRAMP3/4 was significantly induced by low Fe stress, and TaNRAMP3-7D was up-regulated and expressed 3 times, which might play an important role in the transport of Fe from the vacuole of the seed embryo to the cytoplasm (Figure S3B). Mitochondrial m-type thioredoxin in chloroplast (ATM) is mainly involved in the process of exporting Fe-S to mitochondria, thereby maintaining the balance of Fe ion in mitochondria. Transcriptome analysis found that TraesCS5A02G110300 had the highest expression abundance in ATM, which might play an important role in maintaining Fe ion balance in mitochondria (Figure S3H). Permease in chioroplasts (PIC) is the first protein identified to participate in chloroplast Fe transport. Its expression is not regulated by Fe and is a constitutively expressed protein. Transcriptome analysis showed that PIC was significantly induced by low Fe in the shoots, and TraesCS1A02G165700 had the highest expression abundance (Figure S3L). Iron efflux transporter ferroportin (FPN) located in the chloroplast can regulate the intracellular Fe content by participating in the transport of NA or Fe-NA complex into the chloroplast. Transcriptome results showed that FPN was significantly induced by low Fe in roots, and TaFPN1-7A was significantly up-regulated by 60 times (Figure S3P), which might play a key role in maintaining Fe homeostasis in chloroplasts under low Fe stress. Nonintrinsic ABC protein (NAP) is located on the inner chloroplast membrane. It encodes a non-plasma membrane-localized nucleotide-binding domain subunit of the ABC transporter. Transcriptome analysis showed that NAP was significantly induced by low Fe in the shoots, and TraesCS3A02G047200 was significantly up-regulated. This result indicated that it might be part of the chloroplast Fe-ABC transport complex and played an important role in regulating the dynamic balance of Fe in the chloroplast (Figure S3Q). Another contributor to promoting the transfer of Fe ion into the chloroplast is mitoferrin-like, which contains chloroplast transit peptides and is mainly located in rosette leaves. Transcriptome results showed that the expression abundance of most mitoferrin or mitoferrin-like under low Fe stress decreased significantly, and the expression levels of TraesCS6D02G228200 and TraesCSU02G183100 were significantly down-regulated. It was speculated that they might play an important role in maintaining Fe content in chloroplasts (Figure S3N, O). The above results emphasized the importance of Fe ion uptake and transport proteins and regulatory and metabolic proteins in the stress of low Fe.
Involved in the transcriptional response of photosynthesis-related genes under low Fe stress
The GO enrichment analysis showed that GO terms related to photosynthesis, such as photosynthesis, light harvesting in photosystem I, were significantly enriched (Fig. 8G). And the KEGG enrichment analysis also showed that Photosynthesis − antenna proteins and carotenoid biosynthesis KEGG pathways were significantly enriched under low Fe stress (Fig. 8H), which indicated that low Fe significantly affected the photosynthesis of wheat plants. Many genes related to photosynthesis, such as photosynthetic antenna protein and key genes in the biosynthesis of carotenoids were identified (Figure S4). Through KEGG pathway analysis, a total of 33 genes encoding photosynthesis-antenna proteins (Figure S4A) and 39 genes encoding carotenoid biosynthesis (Figure S4B) were screened, and their expression levels were shown in Figure S4. The results showed that the expression levels of most genes involved in the photosynthetic antenna protein pathway and carotenoid biosynthetic pathway were significantly induced by low Fe (Figure S4A, B). The ratio of the concentration of chlorophyll a to chlorophyll b under low Fe stress was significantly higher than that under the control condition (Fig. 2E), which was a characteristic of plants adapting to adversity stress. Among them, chlorophyll an oxygenase (CAO), chlorophyll (ide) b reductase (CBR), chlorophyll synthase (CS), and chlorophyllase play an important role in the conversion between chlorophyll a and chlorophyll b (Figure S4C). Among the DEGs involved in photosynthesis, we found that the genes encoding these enzymes showed an increased expression pattern under low Fe stress (Figure S4C). There are two synthetic pathways for plant carotenoid substances: MEP pathway and MVA pathway. These two pathways generate geranylgeranyl diphosphate (GGPP), the direct precursor substance for carotenoid synthesis. GGPP generates the first carotenoid substance phytoene. After dehydrogenation, cyclization, hydroxylation, epoxidation, etc., it is transformed into other carotenoids. Transcriptome analysis showed that the expression of key enzyme genes in the process of carotenoid syntheses such as phytoene synthase (PSY), β-carotene hydroxylase (β-OHase), zeaxanthin epoxidase (ZEP), 9-cis-epoxycarotenoid dioxygenase (NCED), LUT5, and violaxanthin de-epoxidase (VDE) was significantly inhibited under low Fe stress (Figure S4D). And we found that Chlorophyll a/b binding proteins (Figure S4E), Photosynthesis II reaction proteins (Figure S4F), RuBisCo subunit binding proteins (Figure S2G), Mg2+ chelatase (Figure S4H), and Ribulose bisphosphate carboxylase/oxygenase activase (Figure S4I) were significantly down-regulated under low Fe stress. These results all indicated that the severe degradation of wheat chlorophyll and photosynthesis was inhibited under low Fe stress.
Transcriptional response of other ion transporters to low Fe stress
Among the numerous DEGs, genes related to ion homeostasis are the key genes for wheat resistance to Fe-deficiency stress. Fig. 9A showed a molecular model of the key genes responsible for regulating K+, Na+/Cl-, and Ca2+ transport. Transcription analysis showed that most of the K+ transporter genes, including the K+ efflux transporter gene KEA located on the chloroplast, the vacuolar K+ inflow transporter gene KCO, the plasma membrane-located K+ inflow transporter gene AKT/KAT and HKT, and the K+ efflux gene SKOR was up-regulated under low Fe stress (Fig. 9B). The expression of Na+/H+ antiporter gene NHX, especially NHX2 involved in the Na+ compartment in the vacuole, and SOS1/NHX7 regulated the outflow of Na+ in the cell, were significantly up-regulated. To avoided cell damage due to excessive accumulation of Na+ in the cell (Fig. 9C). NHD could regulate the efflux of Na+, and its expression level was significantly up-regulated under low Fe stress, especially the expression of TaNHD1-5B was significantly up-regulated by 2 times, which might help reduce chloroplast damage caused by excessive Na+ (Fig. 9C). The chloroplast-localized bile acid: Na+ cotransporter (BASS) could regulate Na+ influx, and its expression level was significantly inhibited by low Fe stress, especially the expression level of TaBASS1D-1D was significantly down-regulated by 3 times (Fig. 9C). In addition, low Fe stress also induced the expression of most ALMT genes, which were related to the transport of Cl- in vacuoles, especially the expression of TaALMT12-1B was significantly up-regulated by a factor of 2 under low Fe stress (Fig. 9C). Subsequently, the gene expression related to Na+/Ca2+ transport was studied, including CCX, CAX, ANXD, GLR, and CNGC. The expression levels of most genes changed significantly under low Fe stress. This result indicated that these genes might be involved in ion homeostasis under low Fe stress (Fig. 9D). For example, under low Fe stress, TaCCX expression was increased, TaCAX expression was decreased, and TaANXD expression was increased. This might be an important reason for reducing cytoplasmic Na+ and increasing cytoplasmic Ca2+ concentration (Fig. 9D). Most of the plasma membrane-localized NSCC, including GLR and CNGC, were up-regulated under low Fe stress (Fig. 9D). This might be an important way for Ca2+ and Na+ to enter the cytoplasm under low Fe stress (Fig. 9D). In addition, under low Fe stress, the expression levels of genes PHT2;1 (Fig. 9E) and COPT (Fig. 9F) involved in the uptake and transport of Pi and Cu2+ by roots were significantly down-regulated, while genes involved in Mg2+ uptake and transport were significantly induced by low Fe tress (Fig. 9G). The above results all indicated that the maintenance of ion homeostasis in cells was of great significance for plants to respond to low Fe tress.
Expression profile of cell cycle-related genes and ROS metabolism-related genes under low Fe stress
To study whether the inhibitory effect of Fe deficiency on root growth is related to the degree of cell division and differentiation, we combined the transcriptome to analyze the rich expression of cell cycle-related genes (Figure S5A). Cyclin and cyclin-dependent kinase belong to cell cycle control proteins, which play a key role in the process of mitosis .LRP (lateral root primordia) is specifically expressed in adventitious roots and lateral root primordia. It is a transcription factor that regulates root elongation and has strong tissue specificity . Transcriptome analysis showed that under low Fe tress, the expression abundance of these genes was significantly down-regulated (Figure S5A). It indicated that the shortening of roots under low Fe stress might be due to the decrease in the division frequency of cells in the meristematic zone and the shortened cell elongation in the elongation zone.
According to the results in Fig. 5 and Fig. 6, we combined the transcriptome to analyze the expression of key enzyme genes in the ROS metabolic pathway (Figure S5B, C). The respiratory burst oxidase homologous gene (RBOH) encodes NADPH oxidase and plays a key role in the synthesis of ROS . Under low Fe stress, 11 RBOH differentially expressed genes were significantly up-regulated in shoots or roots (Figure S5B). The expression of SOD, CAT, and PDH genes in most shoots and roots increased under low Fe stress, which might be necessary for ROS clearance under low Fe stress (Figure S5C).
Gene co-expression network analysis
In allohexaploid wheat, multiple copies of genes are ubiquitous. Therefore, the identification of core genes is an important prerequisite for understanding the molecular mechanisms of important agronomic traits. The systematic analysis of the transcriptional response of genes related to Fe uptake and transport under low Fe tress will help us to fully understand the adaptation mechanism of plants to Fe-deficiency stress. To identify the core members of genes related to Fe uptake and transport, we constructed a gene co-expression network (Figure S6A). The results showed that some Fe2+ absorption and transport related genes TaIRT1b-4A and plant siderophores related genes such as TaNAS2-6D, TaNAS1a-6A, TaNAS1-6B and TaNAAT1b-1D were identified as core target genes, which might play a key role in plant response to low Fe stress (Figure S6A).