RBP differentiation contributes to selective transmissibility of OPT3 mRNAs

Abstract

Long-distance mobile mRNAs play key roles in gene regulatory networks that control plant development and stress tolerance. However, the mechanisms underlying species-specific delivery of mRNA still need to be elucidated. Here, the use of grafts involving highly heterozygous apple (Malus) genotypes allowed us to demonstrate that apple (Malus domestica) oligopeptide transporter3 (MdOPT3) mRNA can be transported over a long distance, from the leaf to the root, to regulate iron uptake; however, the mRNA of Arabidopsis (Arabidopsis thaliana) oligopeptide transporter 3 (AtOPT3), the MdOPT3 homolog from A. thaliana, does not move from shoot to root. Reciprocal heterologous expression of the two types of mRNAs showed that the immobile AtOPT3 became mobile and moved from the shoot to the root in two woody species, Malus and Populus, while the mobile MdOPT3 became immobile in two herbaceous species, A. thaliana and tomato (Solanum lycopersicum). Furthermore, we demonstrated that the different transmissibility of OPT3 in A. thaliana and Malus might be caused by divergence in RNA-binding proteins between herbaceous and woody plants. This study provides insights into mechanisms underlying differences in mRNA mobility and validates the important physiological functions associated with this process.

The long-distance movement of OPT3 is selective between herbaceous and woody plants as shown using Malus and Arabidopsis thaliana plants.

Introduction

Plant grafting is a vegetative propagation technique for horticultural trait improvement, such as stress tolerance, development, and flowering characteristics (Taller et al., 1998; Edelstein et al., 2005; Venema et al., 2008). Experiments with various horticulturally important plant species have shown that abiotic stress can be alleviated by grafting scions to stress-tolerant rootstocks (Rivero et al., 2003; Papadakis et al., 2004a, 2004b; Sanchez-Rodriguez et al., 2012; Wang et al., 2017). Such studies have also revealed that physiological and morphological features can be altered by rootstock–scion interactions (Warschefsky et al., 2015). Grafting-induced phenotypic changes have been proposed to be associated with the actions of mobile plant hormones, RNAs, and proteins (Haywood et al., 2005; Molnar et al., 2010; Chen et al., 2016), but the underlying mechanisms are not well understood.

Previous studies have shown that grafts involved in two different species or a transgenic plant and its corresponding wild-type (WT) can be used to study long-distance transmission of specific signals (Xia et al., 2018). Grafting experiments have confirmed that endogenous RNA molecules exist in phloem sap (Ham and Lucas 2017) and mRNAs move in both the stock-to-scion and scion-to-stock directions. The long-distance transport of RNAs is facilitated by specific proteins that prevent RNAs from being degraded during transport (Kehr and Kragler 2018). It is known that pumpkin (Cucurbita maxima) endogenous proteins PHLOEM PROTEIN 16 (CmPP16) binds to its own RNA to form a ribonucleoprotein (RNP) complex in pumpkin (C. maxima). This complex interacts with the plasmodesmata protein, noncell autonomous phloem protein 1, thereby increasing the size exclusion limit of the plasmodesmata that connect companion cells (CCs) and sieve tubes and facilitating entry into the phloem for long-distance transport (Xoconostle-Cazares et al., 1999; Taoka et al., 2007). Proteomic analyses of phloem sap have shown that >10% of total phloem sap proteins are characterized as RNA-binding proteins (RBPs), and it is believed that the binding of phloem-residing RNAs to RBPs may play important roles in loading, unloading, and long-distance transport of RNA in the phloem sieve system (Lin et al., 2009; Rodriguez-Medina et al., 2011; Hu et al., 2016). Cucurbita maxima RBP 50 (CmRBP50) from pumpkin has been shown to specifically bind to RNAs harboring the polymer-pyrimidine CUCU domain to form the core of the RNP complex, which then assists long-distance RNA transport in the phloem by binding to additional proteins (Ham et al., 2009). Solanum tuberosum polypyrimidine tract-binding protein 1 (StPTB1) and StPTB6, the RBP50 homologs in potato (S. tuberosum), have been shown to be able to bind to the 3′-untranslated region (UTR) region of S. tuberosum BEL1-like transcription factor (StBEL5) mRNA and facilitate long-distance phloem transport of the transcripts to improve tuber yield (Cho et al., 2015).

Enormous amounts of mRNAs have been discovered to be mobile during nutrient stress. For example, a total of 3,546 distinct mRNAs were identified to be transported from a cucumber (Cucumis sativus) scion to a watermelon (Citrullus lanatus) stock in response to phosphorous deficiency (Zhang et al., 2016b). In another heterografting experiment using two Arabidopsis thaliana ecotypes (Columbia-0 [Col-0] and Pedriza-0), although 2,006 different mRNAs were discovered to be mobile in the shoot–root system under normal conditions, only 90 and 91 transcripts were found to be mobile in response to phosphorus (P) and nitrogen (N) deficiency, respectively (Thieme et al., 2015). In addition, Xia and Zhang (2020) established a Nicotiana benthamiana/tomato heterograft system to identify shoot-to-root mobile mRNAs that are produced in response to low N, P, or iron (Fe). Although the substantial amounts of mobile mRNAs identified from these studies imply the potential roles of these transcripts in adapting plants to low minerals, a definite biological function of these mRNAs has not been determined.

Fe is an essential micronutrient for plant growth (Brear et al., 2013) and insufficient availability often limits plant development. Previous studies, including physiological (Kobayashi and Nishizawa 2012), transcriptomic (Enomoto et al., 2007), metabolic (Hansch and Mendel 2009), and signal transmission (Mai et al., 2016) analyses, have improved our understanding of mechanisms involved in Fe homeostasis, and substantial evidence exists for shoot-to-root signaling in response to varying Fe conditions (Grusak and Pezeshgi 1996; Enomoto et al., 2007; Wu et al., 2012; Garcia et al., 2013; Grillet et al., 2018). AtOPT3, one of the most studied genes related to Fe-deficiency responses, is mainly expressed in vascular tissues and might regulate the long-distance transport and distribution of Fe in A. thaliana (Lubkowitz 2011). In the opt3-2 mutant, the expression of ferric reduction oxidase 2 (FRO2) and Fe/Zn/Mn uptake transporter Fe-regulated transporter 1 (IRT1) in roots was upregulated and the accumulation of Fe in the roots and leaves increased in comparison with WT, indicating that an OPT3-mediated long-distance mobile signal produced in the aboveground parts of the plant negatively regulates the uptake of Fe in roots (Stacey et al., 2008; Zhai et al., 2014). However, the identity of the signal remains elusive and whether the long-distance movement of AtOPT3 mRNA participates in this regulatory process is unknown.

One of the prerequisites for the identification of mobile mRNAs using the heterografting method is to have a certain degree of genetic difference between the scion and the rootstock (Xia and Zhang 2020). Self-incompatible apple (Malus) species have particularly high levels of genetic variation and the use of heterografts of Malus species allowed us to investigate the long-distance movement of Malus domestica (Md) oligopeptide transporter3 (MdOPT3) mRNA from the leaf to the root to regulate Fe uptake, and to contrast this with the movement of AtOPT3 mRNA in A. thaliana. These observations were then associated with differences in RBP profiles between Malus and Arabidopsis. Our extended studies in tomato and poplar trees suggest that the observed differential in transmissibility of OPT3 mRNAs may exist between herbaceous and woody plants.

Results

MdOPT3 mRNA moves from shoots to regulate Fe uptake in roots of Malus plants

To gain insight into long-distance mRNA signal transduction in apple plants in response to Fe stress, we established a heterologous apple grafting system (Figure 1, A and B). Long-distance movement of RNAs in the shoot–root system was determined using a scion of “Golden Delicious” (Md) and two rootstocks: Malus xiaojinensis (Mx), which is Fe uptake efficient, and Malus baccata (Mb), which has a much lower Fe uptake efficiency (Zhang et al., 2017). To obtain a profile of the mobile transcripts, we analyzed our previous RNA sequencing (RNA-Seq) data on the hetero-grafted samples (Sun et al., 2020; Figure 1, C and D; Supplemental Table S1). A total of 1,421 distinct mobile mRNAs were identified, of which 14 participated in bi-directional movement. We next searched for transmissible mRNAs that were specifically associated with the Fe-deficiency treatments and identified 193 transcripts, in which 150 moving from leaves to roots and 43 in the opposite direction (Supplemental Table S1). Gene ontology (GO) enrichment analysis showed that the mRNAs moving from leaves to roots were enriched in the “transport” (GO: 0006810), “response to stress” (GO: 0006810), and “biosynthetic process” (GO: 0009058) pathways (Supplemental Figure S1).

Figure 1.

Detection of transmissible mRNAs associated with responses to Fe stress in Malus. A, Model of Fe stress signals transported from root to leaf (black arrows) and leaf to root (blue arrows) The brown plant represents the Mx. The bright green shading represents the leaves of Malus plants. The terms 24 h and 3 d in (A) means the grafted Md/Mx were treated under +Fe and −Fe nutrient solution for 24 h and 3 d (B) Samples collected from the grafted rootstocks and scions at 0 h, 24 h and 3 d ± Fe treatment (0 h ± Fe treatment means the grafted Malus plants were treated under +Fe and −Fe nutrient solution for 0 h. The terms 24 h and 3 d in (B) means the grafted Md/Mx were treated under +Fe and −Fe nutrient solution for 24 h and 3 d. The nongrafted control samples were collected from Mx/Mb roots and Md leaves. The green plant represents the Mx. C, Design of the micro-graft experiments and experimental strategy. Modular bioinformatics schematic using RNA-seq data in Malus. The yellow module represents the alignment flow between reads and the reference genome in the root and leaf of the grafted sample, and the grey module represents the transfer mRNA identification flow. First, the clean data from stock or scion of the four samples were compared with the reference genomic GDDH13V1.1 to obtain the modified genome sequence (I\II\III\IV) of each sample. Second, mapped reads were turned into Merged seq transcripts by using the method of BLASTN. The GATK was used to annotate the SNP changed reads of each sample. When determining leaf-to-root mobile genes, the genes that retain the SNP (heterozygosity of rootstock loci) consistent with scions were screened. The accuracy of a SNP is judged according to the sequencing depth and the number of sample repeats. Mn stands for Mb or Mx, r: root, L: Leaf. HISAT2: graph-based alignment of next generation sequencing reads to a population of genomes (D) Specific screening methodology. SNPs between different samples were used to identify transmissible mRNAs. The different colors indicate total SNPs after comparison with reference genome.

Using the population of the specifically transmissible mRNAs, we performed a weighted gene co-expression network analysis and identified 14 distinct modules (Supplemental Figure S2). One of the 14 modules is closely related to the Fe-deficiency response and was of particular interest for this study (purple module). Further exploration of the Fe-associated module showed that OPT3 has high connectivity to other members in the network, indicating the possibility of this transcript in a wide range of Fe regulatory pathways (Figure 2A;Supplemental Table S2). The abundances of MdOPT3 transcripts were found to be more significantly accumulated in roots of Md/Mx and Md/Mb grafts subjected to Fe-deficiency treatment than those in control condition. Interestingly, this overaccumulation of MdOPT3 transcripts in roots coincided with the reduced accumulation of the same transcripts in leaves (Figure 2B). We next performed RNA fluorescence in situ hybridization (FISH) and localized the MdOPT3 mRNA to be in the phloem in stem cross-sections, whereas in root, MdOPT3 mRNA signal appeared to be unloaded and diffused to all vascular bundle cells (Figure 2C). Since OPT3 has been linked to phloem-specific Fe transport in A. thaliana (Stacey et al., 2002, 2008; Zhai et al., 2014), we then tested whether it is involved in Fe accumulation in Malus. We found that MdOPT3 transient suppressed Malus plant grown under normal Fe or Fe-deficient conditions showed weaker Fe³⁺ staining and a significant decrease of Fe contents compared with the control plants (Figure 2, D and E). This suggested that the MdOPT3 contributes to the coordination of Fe accumulation in Malus plant.

Figure 2.

The potentially mobile RNA OPT3 response to Fe deficiency in Malus plants. A, Weighted correlation network analysis (WGCNA) the mobile RNA response to Fe deficiency. B, Relative expression level of MdOPT3 (MD00G1163400) in Md/(Mx and Md/Mb grafted seedlings under +Fe and −Fe treatments. C, RNA FISH of MdOPT3 in Mb stem and root. The sections of stems and leaves stained with nuclear dye DAPI appear blue. The green fluorescence indicates the expression site of the target gene MdOPT3. The length of the ruler in the field of view of DAPI, FAM, and Merge is 200 μm. The length of the ruler in the field of view of Magnification is 20 μm. SE, sieve elements. The white box indicates the enlarged part (D) Perls’ staining of leaves and roots from transient transgenic Md seedlings. Analysis of Fe distribution was performed using Perls’ staining. Scale bars: 1 cm. E, Fe concentration in roots and leaves of MdOPT3-suppressed Malus plants. B and E, Values are means ± sd (n = 3), *Statistically significant difference (P < 0.05), **extremely statistically significant significant difference (P < 0.01), as determined by a Student’s t test.

To further validate MdOPT3 mRNA mobility in Malus grafts, we analyzed the OPT3 gene sequences from cv “Golden Delicious,” M. xiaojinensis, and Mb. A single-nucleotide polymorphism (SNP) (T/C) was identified at +995-bp downstream from the ATG start site, comprising a MdOPT3^T allele present in “Golden Delicious,” a MbOPT3^C allele in Mb, and an Mx oligopeptide transporter 3 (MxOPT3^T/C) allele in M. xiaojinensis (Figure 3A;Supplemental Figure 3A). A combination of OPT3 homozygous genotypes of “Golden delicious” (MdOPT3^T) and Mx (MbOPT3^C) allowed us to distinguish the origin of OPT3 in Md/Mb grafts accurately. In this heterograft, the MdOPT3^T/C was identified in roots under normal growth conditions (Figure 3B). However, after Fe-deficiency treatment of grafted seedlings, MdOPT3^T was the only form of transcript detected in the roots of Md/Mb grafts (Figure 3B). This showed that, under Fe-deficient growth condition, the expression of the root-derived OPT3 was prohibited and the detected OPT3 in the root originated from the scion via the leaf-to-root long-distance movement.

Figure 3.

OPT3 is a mobile mRNA in Malus Plants, but not in Arabidopsis. A, A stable SNP (+995 bp) site was found through the OPT3 sequence alignment between Md and Mb. The SNP site in the root and leaf of Md/Mb grafted seedlings changed after grafting under +Fe and −Fe treatments. B, The stable SNP site was used to identify the mobility of OPT3 from shoot to root in the Md/Mb graft complexes under +Fe and −Fe treatments. Circles of different shapes and colors represent different SNP sites in different OPT3 (MdOPT3^T,MbOPT3^C). The arrow indicates that the MdOPT3 in the leaf can move to the root. C, Effect of girdling on the expression of OPT3 in roots of Md/Mb grafted seedlings under +Fe and −Fe treatments. WOX13 was used as the positive control for this experiment. Two schematic diagrams, respectively, show un-girdled and girdled apple grafted seedlings. (D) The relative expression of AtOPT3 in Arabidopsis graft complex (WT/WT, opt3-2/opt3-2, WT/opt3-2, and opt3-2/WT) shoots and roots under +Fe and −Fe treatments. In the diagram, the blue plants represent WT A. thaliana, and the green plants represent the opt3-2 mutant. Different colors of Arabidopsis leaves and roots represent different grafting combinations. C and D, Values are means ± sd (n = 3), *Statistically significant difference (P < 0.05), **extremely statistically significant difference (P < 0.01) as determined by a Student’s t test.

The movement of nutrients and signaling molecules from the leaf-to-root normally take place in phloem (Aloni et al., 2010; Aoki et al., 2005; Kehr and Buhtz 2008). To further verify whether MdOPT3^T is mobile from the leaf-to-root in the Malus grafts, phloem girdling, a technique that disrupts the phloem transport, was employed. The expression of MdOPT3 in the girdled plant was barely detected. In addition to MdOPT3, we also analyzed the abundance of MdWOX13 (homologous gene of PbWOX1) in the girdled plant. PbWOX1 was previously reported to be mobile in pear (Duan et al., 2015). Its homologous gene MdWOX13 has also been detected to be mobile in our study. The significantly reduced abundance of WUSCHEL-related homeobox 13 (WOX13) mRNA in the roots of the girdled Md/Mb heterografts indicated that our observation on MdOPT3 was not a coincidence (Figure 3C). This result indicated that the MdOPT3^T mRNAs were indeed originated from the leaf and transported from the Md scion to the Mb rootstocks via the phloem.

AtOPT3 mRNA does not move from shoots to roots in A. thaliana

AtOPT3 was shown to promote the movement of Fe from xylem to phloem in A. thaliana and regulate the redistribution of Fe signal and associated signaling in developing and mature tissues (Mendoza-Cozatl et al., 2014). The discovery of the movement of the Malus OPT3 inspired us to investigate whether the long-distance movement of the OPT3 mRNA is a common phenomenon in plants. We reciprocally grafted shoots of the AtOPT3 knockdown mutant (opt3-2) onto WT rootstocks (opt3-2/WT) and WT shoot scions onto opt3-2 rootstocks (WT/opt3-2), and determined AtOPT3 expression in the two graft combinations. Compared to the WT/WT control, the expression of AtOPT3 was hardly detected in the roots of WT/opt3-2 (Figure 3D), indicating that the AtOPT3 mRNAs in WT leaves were not transported to the opt3-2 roots in Arabidopsis.

The mechanistic basis of the differences in OPT3 mRNA mobility between herbaceous and woody plant species

The above results showed that OPT3 mRNAs from A. thaliana and Malus plants differ in their mobility. The alignment analysis between MdOPT3 and AtOPT3 showed that the two sequences share 73% identity at the cDNA level. To determine whether the mRNA sequence differences affect their mobility, transient expressions of MdOPT3 in A. thaliana rosette leaves or AtOPT3 in apple leaves were conducted (Figure 4). PCR primers that can distinguish the two transcripts were used to measure the expression levels of OPT3 in the roots of control and transient transgenic plants (Supplemental Table S3). We found that AtOPT3 mRNAs could be detected in the roots of the transient transgenic apple seedlings but not in the control, although AtOPT3 mRNA levels in apple roots were lower than those in the shoot (Figure 4). In contrast to the results from the transient transgenic apples, the MdOPT3 transcripts could not be detected in the Arabidopsis roots although the transcripts could be detected in high abundance in leaves with a previously described mobile mRNA A. thaliana translationally controlled tumor protein 1 (AtTCTP1; Yang et al., 2019) as a positive reporter (Figure 4). To confirm the efficiency of the mobile mRNA detection in Arabidopsis using transient expression, we also conducted the transient expression of the known mobile mRNA AtTCTP1 fused to eGFP in Arabidopsis rosette leaves. We found that eGFP-AtTCTP1 mRNAs could be detected in the roots of the transient transgenic Arabidopsis seedlings but not in the control (Supplemental Figure S4). Taken together, these results indicated that factors other than the sequence specialty of OPT3 conferred the differential shoot-to-root mobility in the two species.

Figure 4.

Using transient transformation of heterologous OPT3 expression in Malus plant and Arabidopsis to identify the mobility difference of OPT3 between Malus plant and Arabidopsis. The expression of heterologous OPT3 was detected by RT-qPCR in the roots of transient transgenic plants, and the untransformed plants were used as controls. AtTCTP1 specifically expressed in the phloem of A. thaliana was used as the positive control of this experiment. TG, transgenic plant. The dashed-dotted line divides the schematic plant into left and right parts. The left side represents the Md, and the right side represents the A. thaliana. The green portion of the plant represents the shoot, and the yellow portion of the plant represents the root. The blue arrows mean that AtOPT3 can move from the transiently transgenic apple leaf to the root. Large RNA indicates mobile mRNA which transport from leaves, while small RNA indicates the local mRNA which express in roots.

In plants, RBPs play a key role in mRNA transport (Singh et al., 2015), and the 3′-UTR of mobile mRNAs was demonstrated to be sufficient for the long-distance movement of various mRNAs. To identify putative MdOPT3 RPBs, we used a 3′-UTR MdOPT3 sequence as a probe (Figure 5A), and performed RNA-pull down coupled with mass spectrometry. A total of 911 proteins from Md were identified to be able to interact with MdOPT3 mRNA (Supplemental Table S4). The candidate proteins were functionally annotated and a GO enrichment analysis revealed that the most enriched category belongs to “response to stimulus” (Figure 5B). These 911 proteins included five RBPs: MD04G1208800, MD12G1223200, MD05G1230700, MD07G1160000, and MD14G1031800 (Figure 5, C and D), of which MD04G1208800 and MD12G1223200 expression were upregulated in roots of both Md/Mx and Md/Mb grafted seedlings subjected to Fe-deficiency treatment (Figure 5C).

Figure 5.

Analysis of proteins that interact with MdOPT3 3′-UTR. A, Analysis of the secondary structure of MdOPT3 3′-UTR sequence. Different numbers indicate different base sequences of MdOPT3 3′-UTR. The blue circle represents the sequence that can form a tRNA-like structure, and the black circle represents the rest of sequence. B, GO enrichment analysis of proteins interacting with MdOPT3 3′-UTR sequence. The X-axis represents the number of genes enriched in GO. C, Candidate RBPs relative expression levels in roots and leaves of Md/Mx and Md/Mb graft complexes under +Fe and −Fe treatments. Color bar in the heat map indicate the level of gene expression (D) Candidate RBP domain analysis. RRM: RNA recognize motif. The thick black line in the figure indicates the amino acid sequence that cannot form a specific domain. E, RRM domain analysis of two RBPs in herbaceous and woody plants. Gray and pink strips indicate two RBP proteins with different amino acid sequence in woody plants. The numbers 76–77 in the figure represent the 76–77th amino acids of the RBP protein. AA, AE, and EE, respectively, indicate that the amino acids at positions 76–77 are alanine–alanine, alanine–glutamate, and glutamate–glutamate.

To quantify the binding affinities of Malus and A. thaliana RBPs with their associated mRNAs, we used surface plasmon resonance (SPR) assays with immobilized oligonucleotides. We expressed five Malus RBPs and the corresponding A. thaliana proteins in Escherichia coli, each as a fusion with glutathione S-transferase (GST), and purified the recombinant fusion proteins (Figure 6A). The two Malus RBPs (MD04G1208800 and MD12G1223200) were found to be bound to the 3′-UTR of the MdOPT3 and AtOPT3 probes with high affinity, whereas the corresponding A. thaliana RBP (AT3G26420.1) had a much lower binding value than the Malus RBPs, suggesting the low affinity of the A. thaliana RBP with the 3′-UTR of the MdOPT3 and AtOPT3 probes (Figure 6, B–D). To investigate whether RBPs contribute to MdOPT3 mRNA mobility, we studied the role of RBPs with a transient expression system in which tobacco rattle virus (TRV) was employed to silence RBP expression in the apple seedlings. We then grafted shoots of the MdRBP transiently suppressed Malus onto WT rootstocks, and determined MdOPT3 expression in the roots. Our analysis showed that MdOPT3 transcripts were barely detected in the apple roots (Supplemental Figure S5). This result showed that the mobility of MdOPT3 may be associated with the characteristics of the MdRBP in apple. To verify whether the mobility of the AtOPT3 can be increased if the MdRBPs are present in Arabidopsis, we overexpressed MdRBP in the Arabidopsis CCs and grafted this transient transgenic plant on the opt3-2 mutant rootstock (AtSUC2::MdRBP/opt3-2). Compared to the WT/opt3-2 control, the expression of AtOPT3 was detected at much higher abundance in the roots of AtSUC2::MdRBP/opt3-2 (Supplemental Figure S6). This result not only validated the relationship between the mobility of MdOPT3 and the presence of MdRBPs in apple, it also indicated that the lack of mobility of AtOPT3 in Arabidopsis may be due to the absence of a compatible AtRBP.

Figure 6.

SPR analysis of the binding of candidate RBPs to MdOPT3, MxOPT3, and AtOPT3 3′-UTR. A, The binding of candidate RBPs with the MdOPT3, MxOPT3, and AtOPT3 3′-UTR sequences was tested and the response curves of three RBPs, (B) MD12G1223200, (C) MD04G1208800, and (D) AT3G26420.1 to the MdOPT3, MxOPT3, and AtOPT3 3′-UTR sequences are shown. The different colored squares in the heat map and the black table in the figure parts (B–D) indicate the level of binding ability of the RNA probe to the RBP protein, expressed in terms of Absolute Response RU. The black box in the figure indicates the proteins that can obviously interact with the RNA probe.

In order to find out whether the above findings also apply to other plant species, we conducted transient expressions of MdOPT3 in Solanum lycopersicum leaves and AtOPT3 in Populus tremula leaves (Supplemental Figure S3B). Similar to our discovery in the Malus transient expression study, we found that AtOPT3 mRNAs could be detected in the roots of the transient transgenic P. tremula (Supplemental Figure S3C). On the contrary, the MdOPT3 transcripts could not be detected in roots of transient transgenic S. lycopersicum although the transcripts were in high abundance in leaves (Supplemental Figure S3C). These results led us to speculate that the differences between A. thaliana and apple in the transmissibility of OPT3 mRNAs may also be associated with the different functions of the RBP proteins present in other herbaceous plants and woody plants.

To validate whether special sequence motifs associated with their RBPs exist in herbaceous and woody plants, we performed a multiple sequence alignment of RNA binding domain (RRM) of RBPs (MD12G1223200 and MD04G1208800) from multiple herbaceous and woody plant species. Our analysis discovered that the RRM of the RBPs formed separate clusters between herbaceous and woody plants and showed the presence of the conserved “AA” at the 76–77th amino acid site in most herbaceous species, whereas these two amino acids were replaced by “EE” or “EA” in most woody plants (Figure 5E;Supplemental Figure S7). Whether the difference of the two amino acids in the RBPs is associated with OPT3 mobility is worth of future exploration.

Mobile MdOPT3 mRNAs are required for the Fe-deficiency response in Malus plants

It was previously reported that Arabidopsis mutant opt3-2 exhibited a constitutive Fe-deficiency response in the root (Zhai et al., 2014). Consistent with this early finding, our results showed that the expressions of the IRT1 and FIT in roots of the opt3-2 mutant were constitutively upregulated, indicative of an Fe-deficiency response (Supplemental Figure S8, B and D). To confirm MdOPT3 is the functional homolog of the Arabidopsis AtOPT3. We evaluated the phenotypes of the transient ProAtOPT3::MdOPT3-expressing lines. Since the Arabidopsis mutant opt3-2 shows a constitutive Fe-deficiency response in roots including the upregulation of FCR, A. thaliana Fe-regulated transporter 1 (AtIRT1) and A. thaliana FRO2 (AtFRO2) AtFRO2, we tested whether expression of MdOPT3 was able to complement both phenotypes. We found that in FCR activity, AtIRT1 and AtFRO2 expressions were greatly reduced in the roots of ProAtOPT3::MdOPT3 expressing plants compared to the opt3-2 mutant (Supplemental Figure S9). These results show that MdOPT3 is sufficient for proper regulation of Fe homeostasis and apple MdOPT3 is indeed the homolog of the Arabidopsis AtOPT3. To explore whether MdOPT3 is involved in Fe-deficiency responses in apple plants, we measured the expression levels of the two Fe-deficiency marker genes, MdIRT1 and MdFIT, in transient MdOPT3 suppressed apple plants. To our surprise, both IRT1 and FIT were downregulated in the suppressed plants when compared with the control plants (Supplemental Figure S8, F and G). The contrast response to the suppressed expression of OPT3 in leaf between Malus and Arabidopsis inspired us to hypothesize that OPT3 mobility could play a role in this physiological process.

It is known that OPT3 is located on the vascular tissues of both roots and shoots (Zhai et al., 2014). To distinguish the importance of leaf and root AtOPT3 in response to Fe deficiency, a few graft combinations, that is, opt3-2/WT, opt3-2/opt3-2, and WT/opt3-2, were created and the expressions of IRT1, FIT, and FRO2 in roots were measured. Higher expression levels of the marker genes were found in both opt3-2/WT and opt3-2/opt3-2, whereas WT/opt3-2 grafts had lower expressions of these genes (Figure 7, A–C). These results indicated that the Fe-deficiency responses in Arabidopsis roots were stimulated by a signal from the shoot mediated by OPT3 but OPT3 transcript is not the mobile signal. To investigate whether the MdOPT3 mRNA could be the mobile signal in apple plants under Fe-deficiency, it is important to create some Malus plants without the mobile OPT3. We analyzed the 3′-UTR of OPT3 from Mx, Mb, and Md, and identified a deletion of 15 bp specific to MxOPT3 in Mx (Supplemental Figure S3A). We observed that OPT3 mRNAs in Mx leaves were immobile, whereas OPT3 mRNAs in Mb leaves were mobile (Supplemental Figure S10). The variation in transmissibility of OPT3 mRNAs between Mx and Mb was used to investigate whether OPT3 mRNAs mobility contribute to Fe-deficiency response. We created two graft systems, taking Md as the rootstock, and Mx or Mb as the scion (Figure 7, D–G). The lack of the Mx derived OPT3^C mRNA in the rootstock Md indicated the mRNA in the Mx leaves could not be transferred to the roots, whereas the detection of the Mb derived OPT3^C mRNA in the rootstock Md indicated that the mRNA in the Mb leaves could be transferred. We found the expressions of Fe uptake-related gene (IRT1, FRO2) were much higher in the Mb/Md grafts in which OPT3 was mobile in comparison with the expression in the Mx/Md, in which the OPT3 transcript was not mobile. Interestingly, we did not observe significant differences in the expression of FIT in both Mb/Md and Mx/Md, indicating that the MdOPT3 mobility is not involved in the transcriptional regulation of Fe uptake. Taken together, our results showed that the long-distance movement of OPT3 mRNA from shoots-to-roots involves in Fe uptake in roots of Malus plants.

Figure 7.

OPT3 moves from shoots to regulate Fe-deficiency response in roots of Malus plants. The relative expression of (A) AtIRT1, (B) AtFRO2, and (C) AtFIT1 in Arabidopsis graft complex (blue color plant: WT/WT, green color plant: opt3-2/opt3-2, blue/green plant: WT/opt3-2, green/blue plant: opt3-2/WT) shoot and root under +Fe and −Fe treatments. The relative expression of (D) OPT3, (E) IRT1, (F) FRO2 and (G) FIT in reverse graft complex (green color plant: Mx/Md, brown color plant: Mb/Md) leaf and root under +Fe and −Fe treatments. A–G, Values are means ± sd (n = 3). *Statistically significant difference (P < 0.05), **extremely statistically significant difference (P < 0.01), as determined by a Student’s t test.

Discussion

Based on the data presented in this study, we propose a model regarding the mobility of OPT3 mRNA in Malus and Arabidopsis plants (Figure 8). MdOPT3 moves long distance from shoots to regulate Fe uptake in roots of Malus plants while this is not the case in Arabidopsis. We further provided evidence to demonstrate that the differential transmissibility of OPT3 in Arabidopsis and Malus might represent a generic phenomenon in herbaceous and woody plants although further experiments are needed before these conclusions can be generalized.

Figure 8.

Schematic overview of OPT3 mobility from shoot to root in Malus plants (left) and A. thaliana (right). OPT3 mRNA mobility leads to differences in Fe redistribution between Malus plants and A. thaliana roots. A, OPT3 mRNAs in Malus shoots are transported to roots by binding to RBPs. B, OPT3 mRNAs detected in Malus roots include sequences endogenous to the root, as well as OPT3 mRNAs transferred from the shoot. The OPT3 from shoot and OPT3 in root jointly participate in the Fe redistribution within the root (from xylem to phloem). C and D, A. thaliana OPT3 mRNAs are not transported from the shoot to the root. Blue cells indicate phloem and xylem, green cells indicate CC. N means nucleus. The green part represents the shoot, and the yellow represents the root.

At the genome level, various graft-transmissible mRNA putative orthologs were found to be shared among different plant species; however, each plant has a unique phloem mRNA population. For example, 2,006, 3,333, and 3,546 graft-transmissible mRNAs were identified in A. thaliana, grape (Vitis vinefera), and cucumber phloem, respectively (Thieme et al., 2015; Yang et al., 2015; Zhang et al., 2016b), but only 38% and 33% of the mobile mRNA putative orthologs from A. thaliana and grape, respectively, were also detected in cucumber (Zhang et al., 2016b). The core sieve tube system (STS) mRNAs may play roles that are essential for phloem function and therefore are conserved during plant vascular tissue evolution. Conversely, the species-specific STS-mRNAs may reflect differences in growing conditions, or evolutionary adaptations, such as those exhibited by rosette plants (A. thaliana), annual plants and perennial vines (grape; Ham and Lucas 2017). The difference in the transmissibility of OPT3 between apples and A. thaliana may be related to differences in the development of vascular bundles, and specifically phloem, between herbaceous and woody plants. In addition to Malus and Arabidopsis, we further selected two other herbaceous and woody plants which demonstrated that the originally immobile AtOPT3 can be transported from the shoot to the root in P. tremula, while the originally mobile MdOPT3 does not show such movement in S. lycopersicum. Multiple factors, for example, expression site, association with RBPs, and so on have been suggested to be associated with the mobility of mRNAs (LeBlanc et al., 2013; Kim et al., 2014; Morris 2018). The noncell-autonomous phloem RBP CmPP16 has been shown to bind to mRNAs in a nonspecific manner, increase the plasmodesmata size exclusion limit, and transmit as an RNP complex between cells (Xoconostle-Cazares et al., 1999). Phloem sap proteomic analysis showed that >10% of phloem sap consists of RBP proteins, which may play an important role in the long-distance transport of RNA in the phloem (Lin et al., 2009; Rodriguez-Medina et al., 2011; Hu et al., 2016). Thus, it is legitimate to assume that the variation in RBP orthologs between apple and A. thaliana may contribute to differences in phloem transcript profiles between the two species.

Potato StBEL5 mRNA was shown to move via the phloem in the veins to the tip of the subsurface stolon (Banerjee et al., 2006; Hannapel 2010), and increasing the long-distance transmission was able to improve tuber yield (Banerjee et al., 2006). The UTR of StBEL5 was found to be related to its long-distance transmission and other studies also have shown the importance of the 3′-UTR in binding to RBPs for long distance RNA movement (Ferrandon et al., 1994). We analyzed the 3′-UTR of OPT3 from Mx, Mb, and Md, and identified a deletion of 15-bp specific to MxOPT3 (Supplemental Figure S3A). We observed that OPT3 mRNAs in Mx leaves were mobile whereas OPT3 mRNAs in Mb leaves were immobile. The variation in transmissibility of OPT3 mRNAs between Md, Mx, and Mb may reflect variation in the 3′-UTR sequences. It was demonstrated that a potential secondary structure motif resembling tRNA in the 3′-UTR is sufficient for long-distance mRNA movement (Zhang et al., 2016a). The deletion of 15 bp in the 3′-UTR in Mx may affect the tRNA structure of MxOPT3. Moreover, our results show that variation in the 3′-UTR sequences may affect the mobility of the mRNA, which might represent a general tool for precisely regulating communication between scion and rootstock.

The plant OPT3 gene was first described in A. thaliana (Stacey et al., 2002), and in subsequent studies of its function the focus was mainly on the tissue localization of expression changes as a consequence of Fe deficiency, as well as the identification of opt3-2 mutants (Stacey et al., 2006). It was also found that AtOPT3 is a key component of the Fe-signaling network connecting shoots to roots (Zhai et al., 2014). Through phloem localization and radioactive Fe tracer tests, OPT3 were shown to mediate the transport of Fe or Fe ligand complexes from the xylem to the CCs and then to the phloem for long-distance transport (Zhai et al., 2014). Even though phloem RNAs are known to play important developmental roles (Haywood et al., 2005; Rodriguez-Medina et al., 2011; Zhang et al., 2016b), and AtOPT3 is mainly transcribed and expressed in the phloem (Stacey et al., 2006; Mendoza-Cozatl et al., 2014; Zhai et al., 2014), through an interactive grafting test involving opt3-2 and WT, we found no evidence that the OPT3 mRNA can move from the shoot to the root in A. thaliana and conclude that the factor mediating the Fe signal transduction from shoot to root in Arabidopsis is less likely the mRNA form of OPT3.

To show the overlap of differential regulation in both opt3-2 and TRV-MdOPT3 lines, we compared RNA-seq profiling between opt3-2 and TRV-MdOPT3 (Supplemental Table S5). About 43 (root) and 47 (leaf) of the GO pathways were shared in both opt3-2 and TRV-MdOPT3 lines. A large proportion of the common enriched terms are related to “response to iron,” “iron chelate transport,” “response to hormone” category, and so on (Supplemental Figure S11). Different from Arabidopsis, mobile MdOPT3 mRNA is required for the Fe-deficiency response in Malus plants. Our results showed MdOPT3 mRNA acts as a Fe signal transduced from shoot to root. It is unknown why the shoot-to-root movement of OPT mRNAs is needed and how the root-arriving OPT3 participates in the Fe-deficiency responses. Small peptides have diverse functions in cellular signaling in many organisms (Murphy et al., 2012; Endo et al., 2014; Luo et al., 2019). In addition to Fe, OPT proteins can transport diverse substrates which are peptide-containing compounds or peptide derivatives. Also, we speculated that scion-originated OPT3 may deliver into the specific terminal regions of the root which is different from the localization of the root-originated OPT3. Future experiments are needed to verify whether the oligopeptide transport function plays a role in regulating Fe-deficiency response in Malus plants.

Materials and methods

Plant materials

Malus xiaojinensis and Mx as rootstocks, and Md cv “Golden Delicious” as scion were propagated on Murashige and Skoog (MS) medium (Gao et al., 2011) containing 0.5 mg L⁻¹ 6-benzylaminopurine and 0.5 mg L⁻¹ indole-3-butytric acid for 1 month (growth temperature 25 ± 2°C day/21 ± 2°C night, 250 μmol m⁻² s⁻¹ light intensity, 16-/8-h light/dark photoperiod). The seedlings were transferred to MS medium with 1.0 mg L⁻¹ indole-3-butytricacid for rooting (Han et al., 1994). Seedlings were grown for 3 weeks prior to experimental treatments. Fe sufficient (+Fe) or Fe-deficient (−Fe, 0 μM FeNaEDTA) treatments were carried out for 3 d.

Arabidopsis thaliana lines included WT (Col-0) and OPT3 knockdown mutants (opt3-2) (Stacey et al., 2008). The seeds were surface-sterilized, stratified at 4°C for 48 h in the dark, and seeded on solid half MS medium (pH 5.8) with an aseptic pipette suction head under sterile conditions. The plates were then placed at room temperature (16-/8-h light/dark cycle, temperature 22°C) and cultured vertically for 6 d. +Fe and −Fe media were prepared as previously described (Khan et al., 2018).

Plant grafting

Before grafting, Malus plants were grown in MS medium for 1 month in a controlled environment as described above. Rootstocks and scions with similar stem diameters were selected for cleft grafting (Melnyk 2017), and the grafted materials were transferred to rooting medium (0.5 mg L⁻¹ indole butyric acid). After one and a half months, the grafted plants were used for Fe-deficiency treatment in hydroponic culture as described above. Arabidopsis thaliana grafting was performed using a flat surface collar-free method as previously described (Marsch et al., 2013). Grafting combinations were as follows: WT Col-0/WT Col-0, opt3-2/opt3-2, WT Col-0/opt3-2, and opt3-2/WT Col-0.

Phloem girdling

The stems of hydroponically rooted Md/Mb grafted seedlings were used for the girdling experiments. The bark 2 mm above the graft joint was removed. The girdling part was wrapped with plastic wrap to prohibit water loss. To prevent the healing of the girdling incision, the seedlings after girdling were immediately treated with +Fe and −Fe.

Mobile mRNA bioinformatic analysis

Total RNA was extracted from grafted tissues using an RNAprep Pure Plant Plus Kit (TIANGEN, DP441). Library preparation was as described in our previous study (Sun et al., 2020).

The mobile mRNA transcripts were derived from grafted (and un-grafted) scion or rootstock samples and compared with the reference genome (ftp://ftp.bioinfo.wsu.edu/ species/Malus_x_domestica/Malus_x_domestica-genome_GDDH13_version 1.1/assembly) using the HISAT2 software (Gao et al., 2011; https://ccb.jhu.edu/software/hisat2/index.shtml). Before comparing to the reference genome, we determined four groups of sequences (nongrafted, Md/Mn 0 h, +Fe Md/Mn, and −Fe Md/Mn) using the rootstocks and scion raw data, referred to as reference I, II, III, and IV, respectively. These sequences were first compared to the reference genome, and unmapped reads were removed. To identify leaf to root mobile transcripts, we compared Mn (r) versus Md/Mn (r) (Mn: Mx or Mb, r: root, L: leaf), and searched for SNPs and InDels (insertion–deletions) using Mn (r) as control. SNP/InDels detected in Md/Mn (r) but not in Mn (r) were considered as candidates for transmission from the leaf to the root in Md/Mn. Similar criteria were used to identify candidates for transmission from the root to the leaf. To identify mobile transcripts in response to Fe deficiency, we compared the SNP/indels in sequences from Md/Mn (r) grown under normal Fe conditions with those from sequences grown under Fe-deficiency conditions. SNPs and InDels were identified and mapped to the apple genome using Genome Analysis Toolkit (GATK;http://www.broadinstitute.org/gatk/). The GATK identification criteria used were: (1) No more than three single base mismatches in the range of 35 bp; (2) Compared with the reference genome, all unique loci in the grafted sample had a sequencing depth >2.0 and two replicates were retained for further analysis. The snpEff software was used for annotation, and only homozygous variants were kept for further analysis. An overview of the methods used in our study is showed in Figure 1, C and D.

Reverse transcription quantitative PCR

First-strand cDNA was synthesized using HiScript^® II Q RT SuperMix for qPCR (Vazyme, Nanjing, China; R232-01). RT-qPCR was performed using an ABI QuantStudio™ 6 Flex system (ThermoFisher Scientific, Waltham, MA, USA) with a SensiFAST™ SYBR Lo-ROX Kit (Bioline, London, UK; BIO-94005) For reverse transcription quantitative PCR (RT-qPCR), the thermocycler protocol was as follows: predenaturation, 1 min at 95°C, 40 cycles of 95°C for 15 s, 56°C for 15 s, and 72°C for 45 s. ACTIN (Md) was used as reference gene, and the relative expression levels were calculated using the 2^−ΔΔCT method (Livak and Schmittgen 2001). Primers used are listed in Supplemental Table S3.

RNA FISH of OPT3

The roots and stems of hydroponically grown Md seedlings were used as RNA FISH experimental materials. The roots and stems of ∼1-cm long were fixed as described previously (Yang et al., 2020). The samples were then dehydrated and buried in paraffin (Yang et al., 2016). A slicer was used to cross-cut the tissue into 8-μm slices. The treatment on these slices, that is, dewaxing, rehydration, and dehydration, followed a previous study (Yang et al., 2016). The probe labeled with 5-Carboxyfluorescein (FAM) was synthesized by Gefan Biological Co., Ltd. (Shanghai, China). The sequence of the probe is shown in Supplemental Table S3. Hybridization is the same as described previously (Yang et al., 2016). During hybridization, FAM-labeled probe was used to react with the sample in the dark at 65°C for 48 h. The nuclei were stained with 4',6-diamidino-2-phenylindole staining solution for 5 min, and then the sections were processed and sealed. The image was taken with OLYMPUS FV3000 confocal microscope.

Perls’ staining

K₄[Fe(CN)₆]:HCl = 1:1 (Perls’) was used to visualize Fe³⁺ localization as previously described (Meguro et al., 2007). The roots and leaves of transient transgenic Md were incubated tin the Perls’ solution for 1 h then washed with water. A methanol solution containing 10 mM sodium azide and 0.3% (v/v) H₂O₂ was used to clear the roots and leaves. The samples were then washed in 0.1 M phosphate-buffered saline solution (composed of NaH₂PO₄ and Na₂HPO₄, pH 7.4).

Cloning and sequencing of OPT3

A 5′ and 3′ Rapid Amplification of cDNA Ends procedure was used to confirm the full-length mRNA sequence of MdOPT3, MxOPT3, and MbOPT3 as previously described (Scotto-Lavino et al., 2006a, 2006b). In order to confirm the stable SNPs, the full-length coding sequence (CDS) of MdOPT3, MxOPT3, and MbOPT3 were cloned from cDNA reverse transcribed from total mRNA using the pTOPO-Blunt Cloning vector (AidLab, CV15). All primers are listed in Supplemental Table S3.

SNP typing

To study the mRNA mobility of MdOPT3, MxOPT3, and MbOPT3, we detected the stable SNP of OPT3 in the roots and leaves of Mx/Md, Mb/Md, Md/Mb, and Md/Mx grafted seedlings under +Fe and −Fe treatments. SNP typing was completed by Sangon Biotec Co., Ltd (Shanghai, China).

Transient expression

To study the OPT3 mobility in Malus and Arabidopsis, the mRNA sequence of MdOPT3 and AtOPT3 were separately cloned into the BamH1 site of the pRI101-AN vector. To completement the Atopt3 by the MdOPT3, the promoter sequence of AtOPT3 and mRNA sequence of MdOPT3 were cloned into the BamH1 site of the pCAMBIA1391 vector. To increase the abundance of MdRBP in leaf CCs, the promoter sequence of AtSUC2 and CDS of MdRBP were cloned into pCAMBIA1391 in tandem. To suppress MdOPT3 expression in “Golden Delicious” using virus-induced gene silencing, the MdOPT3 fragment (Supplemental Table S3) was cloned into the pTRV2 vector (Ramegowda et al., 2014).

All constructs were transformed into GV3101 Agrobacterium tumefaciens cells (Weidi Biotechnology, Nanjing, China; AC1001). Positive clones were mixed to give equal concentrations of pTRV1 (Liu et al., 2002) and pTRV2, or its derivatives, and introduced into Md and A. thaliana seedlings using vacuum infiltration and injection, respectively (Sun et al., 2020). The transformation procedure used a pressure of 0.8 MPa, which was applied twice for 5-min duration. Distilled water was used to wash away the bacteria on the surface of the seedlings.

RNA–protein pull down and mass spectrometry

The MdOPT3 3′-UTR fragment was fused with T7 promoter sequence at both ends by PCR. About 2 μg of PCR products were transcribed in vitro using in vitro Transcription T7 Kit (TaKaRa, Shiga, Japan; 6140) kit. The integrity of transcripts was checked by agarose gel electrophoresis. Proteins were extracted from apple plant seedlings using the Plant Total Protein Extraction Kit (Sangon, Shanghai, China; C500053). The target RNAs were labeled using the Thermo Scientific Pierce RNA 3’Desthiobiotinylation Kit (Thermo Scientific, Waltham, MA, USA; 20163). The successive steps of RNA attachment to magnetic beads, incubation in the buffer, elution of RNA–protein complex, and final mass spectrometry detection all follow the Pierce Magnetic RNA–Protein Pull-Down Kit (Thermo Scientific; 20164). RNA–protein complex was detected by Q Exactive HF-X sequencer. The mass-charge ratio of peptides and peptides was collected according to the following method: Twelve fragment profiles (mass spectrometry 2 scan) were acquired after each full scan (mass spectrometry1 scan), MS1 scan in profile mode with a resolution of 70,000 and MS2 scan in profile mode with a resolution of 17,500. Collision energy: 27.0%, isolation window: 1.2 m/z. window: 1.2 m/z. The MaxQuant version 1.6.0.16 software was used to analyze the original data file (raw file). Maxquant’s algorithm was used to identify peptides and proteins, and gave the relative quantitative information (LFQ). After obtaining the proteins bound to the MdOPT3 3′-UTR, we used http://bioinformatics.cau.edu.cn/AppleMDO/ for functional annotation and GO enrichment analysis of target proteins.

SPR

The 3′-UTR RNA probes of MdOPT3, MxOPT3, and AtOPT3 were transcribed using the In vitro Transcription T7 Kit (Takara, Shiga, Japan; 6140). The GST-tagged RBPs were expressed using the pGEX4T-1 prokaryotic expression vector (cwbiotech, Beijing, China; CW2198) in E. coli BL21 (Transgen, Strasbourg, France; CD901-02). SPR measurements were performed using the GST Capture Kit on the Biacore X100 platform (GE Healthcare Chicago, IL, USA). RNA and RBP binding levels were tested using a Biacore Binding Analysis program (GE Healthcare) with purified RNA probes as the mobile phase, and GST-tagged RBPs as the stationary phase. Binding level is defined as the value of the binding number of the Reaction Unit given by the binding analysis program after the stable combination of the sample and the fixed phase.

Statistical analysis

For all multiple comparisons, GraphPad Prism software (https://www.graphpad.com) was used to analyze the significance of the differences between different groups through a one-way analysis of variance with a significance level of 0.01 and 0.05. * Represents P < 0.05, and ** represents P < 0.01.

Accession numbers

Sequence data from this article can be found in the GDR and TAIR data libraries under the following accession numbers: MdOPT3 (MD00G1163400), MdIRT1 (MD05G1255500), MdFRO2 (MD01G1068200), MdFIT (MD03G1129100), AtOPT3 (AT4G16370.1), AtIRT1 (AT4G19690.2), AtFRO2 (AT1G01580.1), and Fe-deficiency-induced transcription factor 1 (AtFIT; AT2G28160.1). RNA-seq data has been uploaded to the NCBI public database (SRP071838)

Supplemental data

The following materials are available in the online version of this article.

Supplemental Method S1. Plant materials.

Supplemental Method S2. SNPs between different samples were used to identify transmissible mRNAs.

Supplemental Method S3. Transient expression.

Supplemental Method S4. Ferric-chelate reduction activity.

Supplemental Figure S1. Spatiotemporal differential expression and GO enrichment analysis of mobile mRNAs in response to Fe stress.

Supplemental Figure S2. Weighted gene co-expression network analysis (WGCNA) of Malus mobile mRNAs associated with responses to Fe deficiency.

Supplemental Figure S3. AtOPT3 and MdOPT3 have different transmissibility in P. tremula and S. lycopersicum.

Supplemental Figure S4. A mobile mRNA AtTCTP1 in A. thaliana can move from shoot to root in Arabidopsis seedlings grafted WT/opt3-2.

Supplemental Figure S5. Inhibiting the expression of (Md) RBP (MdRBP) in leaves could block the movement of MdOPT3 mRNA from Malus leaves to roots.

Supplemental Figure S6. Transient overexpression of ProSUC2::MdRBP in the leaves of Arabidopsis seedlings grafted WT/opt3-2 could promote the mobility of AtOPT3 from shoot to root.

Supplemental Figure S7. The RRM domain of MdRBP in herbaceous and woody plants.

Supplemental Figure S8. OPT3 in both Malus and A. thaliana responds to Fe-deficiency stress.

Supplemental Figure S9. Transient overexpression of MdOPT3 in Arabidopsis opt3-2 mutant leaves can complement part of the phenotype of opt3-2.

Supplemental Figure S10. There are differences in mobility between MbOPT3 and MxOPT3.

Supplemental Figure S11. GO Analysis of WT versus opt3-2 in A. thaliana and TRV-MdOPT3 versus Control in Malus seedlings.

Supplemental Figure S12. The secondary structure of AtOPT3 3′-UTR sequence.

Supplemental Table S1. Total mobile mRNAs in response to Fe treatment.

Supplemental Table S2. List of genes associated with OPT3 in WGCNA.

Supplemental Table S3. Primers used in this study.

Supplemental Table S4. Protein annotation of MdOPT3 3′ UTR sequence interactors identified in an RNA–protein pull-down analysis.

Supplemental Table S5. DEG and GO analysis of TRV-MdOPT3 versus Control and WT versus opt3-2.

Funding

This work was supported by the National Natural Science Foundation of China (No. 31572097), the China Agricultural Research System (CARS-27), the Construction of Beijing Science and Technology Innovation and Service Capacity in Top Subjects (CEFF-PXM2019_014207_000032), the 111 Project (B17043), and the 2115 Talent Development Program of China Agricultural University.

Conflict of interest statement. The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

 

T.W. and Z.H. conceived and designed the research. Y.S., X.L., P.H., M.F., and Z.X conducted the experiments. J.T., Y.W., X.X., and X.Z. contributed reagents and analytical tools. T.W. and X.L. wrote the manuscript. C.Z gave advice and edited the manuscript. All authors read and approved the manuscript.

The authors responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (https://academic.oup.com/plphys/pages/General-Instructions) are: Ting Wu (wuting@cau.edu.cn) and Zhenhai Han (rschan@cau.edu.cn).