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SUPPLEMENTARY INFORMATIONDOI: 10.1038/NPLANTS.2016.33

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Plant vasculature-mediated signaling involved in early phosphate stress response

Zhaoliang Zhang, Yi Zheng, Byung-Kook Ham, Akiko Yoshida, Leon. V. Kochian, Zhangjun Fei, William J. Lucas

Supplementary methods

Soluble phosphate measurement. Leaf soluble phosphate content was measured, as previously described1, with modifications. Briefly, leaf discs (~ 0.1 g) were cut using an 8 mm cork borer and then ground for soluble phosphate extraction with 1 ml de-ionized and glass-distilled water (ddH2O). After centrifugation at 12,000 g for 30 min, the supernatant containing soluble phosphate was analyzed by the P-molybdate blue method2. A 200 µl aliquot of the supernatant was mixed with 100 µl fresh 10% ascorbic acid and 4.7 ml ddH2O was added to yield a final volume of 5 ml. After 30 s, 200 µl of color reagent was added and incubated, at room temperature, for 15 min before measuring absorbance at 700 nm on a Beckman DU 640 spectrophotometer. Color reagent was prepared by dissolving 10 g (NH4)6Mo7O24.4H2O in 450 ml ddH2O, followed by mixing with 153 ml concentrated H2SO4, 100 ml 5 g/L K(SbO)C4H4O6, and ddH2O to a final volume of 1 liter.

Grafting procedures. Grafting experiments were performed, as previously described22, with some modifications. Hetero-grafted watermelon (Wm)/cucumber (Cu) (scion/stock, G1), and control homo-grafted Cu/Cu (G2) experiments were performed in order to assay for mobile mRNAs delivered to the developing leaves (DL) and shoot apex (SA). Hetero-grafted Cu/Wm (scion/stock, G3) and control homo-grafted Cu/Cu (G4) experiments were performed to assay for shoot-to-root mobile mRNAs. Homo-grafted Cu/Cu (G5) plants on which leaves were not removed served as an additional control. Plant growth conditions were as described above. Twenty two-day-old cucumber and watermelon plants were used for hetero- or homo-grafting experiments. Stock plants were prepared by excising the stem just above the fifth leaf. Scions were excised 8 cm back from the plant apex and the scion stem was inserted into an incision made in the stem of stock plants. The graft union was then sealed with Parafilm and the scion covered with a clear plastic bag. Bags were removed 5 days after grafting (DAG) (G1, G2, G4 and G5) or 7 DAG (G3). To enhance phloem translocation across the graft union, source scion leaves of G1 or G2 grafted plants were excised 9 DAG; stock leaves of G3 and G4 grafted plants were excised 16 and 11 DAG, respectively. Given that the compatibilities between grafting combinations were different, grafting times were adjusted to ensure that plants were of a similar height when tissue samples were harvested. Grafts G1 and G2 were +Pi or -Pi treated 13 DAG; grafts G3 and G4 were +Pi or -Pi treated 23 and 18 DAG, respectively; grafts G5 were +Pi or -Pi treated 12 DAG. Twenty six-day-old control non-grafted cucumber plants were given +Pi or -Pi treatments. All plants were + Pi treated for 24 h and then sink tissues (DL, SA and RT [terminal ~3 cm root tip]) were

Vascular-mediated signalling involved in early phosphate stress response in plants

http://dx.doi.org/10.1038/nplants.2016.33

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SUPPLEMENTARY INFORMATION DOI: 10.1038/NPLANTS.2016.33

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sampled. DL, SA and RT samples from four +Pi treated and four -Pi treated watermelon plants were also used for RNA-Seq to prepare a reference watermelon transcriptome. Three independent biological replicates were performed, with 4 plants being used for each treatment. Messenger RNA was extracted from these samples and 78 cDNA libraries were prepared and sequenced using the Illumina HiSeq 2000 platform. Details relating to RNA-Seq data processing is provided in Supplementary methods.

RNA isolation and qRT-PCR. Total RNA was isolated from tissues and phloem sap (PS) using TRIzol reagent and TRIzol LS reagent (Invitrogen), respectively, following the manufacturer's instructions. Total RNA was treated with DNase I to remove any contaminating DNA and cDNA was synthesized using the SuperScript III first-strand synthesis system (Invitrogen). qRT-PCR was carried out using iQTM SYBR® Green Supermix (Bio-rad) on an iQTM 5 optical system. Gene specific primers are listed in Supplementary Table 4. For miRNA detection, stem-loop qRT-PCR was performed using a previously described protocol3. Briefly, cDNA was synthesized using stem-loop reverse transcription primer and qRT-PCR was performed using a miRNA specific primer and a universal reverse primer. The PCR was performed using iQTM SYBR® Green Supermix with the following conditions: 95℃ 5 min, followed by 40 cycles of 95°C for 5 s, 60°C for 5 s, and 72°C for 8 s. Fluorescence signals were recorded when the temperature increased from 65°C to 90°C at 20°C per second. The stem-loop RT primers, miRNA-specific primers and universal reverse primer are listed in Supplementary Table 5.

RNA-Seq library preparation. Strand-specific RNA sequencing libraries were prepared as previously described4. Briefly, the procedure was as follows: (i) mRNA was first enriched, DNase-digested and fragmented; (ii) mRNA was used for reverse transcription with dNTP to synthesize the first strand and RNA/cDNA hybridization was purified; (iii) second strand was synthesized with dNTP (dATP, dGTP, dCTP and dUTP); (iv) double-stranded DNA was end-repaired, dA-tail added and ligated with adapter; (v) size selection of library; (iv) dUTP containing second strand was digested; (vii) PCR enrichment for library production. Three independent replicates were prepared and sequenced on an Illumina HiSeq 2000 system.

Small RNA (sRNA) library preparation was performed as follow: (i) Small RNA was purified by cutting the sRNA bands from PAGE gels; (ii) 3’ adapter was linked to the purified sRNA using T4 RNA ligase 2; (iii) following 3’ adapter ligation the resultant product was run on a PAGE gel and the sRNA recovered; (iv) 5’ adapter was linked using T4 RNA ligase 1; (v) cDNA was synthesized using the SuperScript III first-strand synthesis system; and (vi) PCR amplification and purification of the library was carried out. Three independent replicates were prepared and sequenced on an Illumina HiSeq 2000 platform.

RNA-Seq data processing. Raw RNA-Seq reads were first processed to trim the adaptor and low quality sequences using Trimmomatic5, and the trimmed reads




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shorter than 40 bp were discarded. The remaining high quality reads were aligned to a ribosomal RNA database6 using Bowtie7, allowing up to three mismatches. These ribosomal RNA mapped reads were discarded in subsequent analyses.

Differential gene expression. For both cucumber and watermelon RNA-Seq reads, the cleaned read pairs were mapped to the corresponding genomes using Tophat28. Following alignments, raw counts for each gene model were derived and then normalized using the FPKM (fragments per kilobase of exon per million fragments mapped) method. Raw counts were then fed to edgeR9 to identify differentially expressed genes with a cutoff of FPKM > 2 in at least one treatment, the p-value less than 0.05 and fold-change greater than two.

Small RNA sequence processing and analysis. Raw small RNA (sRNA) reads were processed to trim adaptor and low quality bases using an in-house Perl script. Trimmed reads shorter than 15 bp were discarded. The remaining high quality reads were then aligned to the ribosomal RNA database6 using Bowtie7, allowing up to one mismatch, and the mapped reads were excluded in the downstream analyses. The final cleaned sRNA reads were then processed by collapsing identical reads into unique reads, and raw counts for each unique sRNA were derived and normalized into TPM (transcripts per million).

Micro-RNA identification and differential expression analysis. The unique sRNAs with expression > 5 TPM in at least one of the analyzed tissues and lengths of 20-24 nucleotides were aligned to the cucumber genome10 using Bowtie7 with perfect matches. Only sRNAs with no more than 20 hits in the genome were kept and their flanking sequences on the genome (200 bp on each side) were extracted and then folded in silico using the RNAfold program11. Resulting folded structures were checked with miRcheck12 with default parameters. Candidate miRNAs whose precursors passed miRcheck were then aligned to miRBase13 using Bowtie7 allowing up to two mismatches. Those miRNAs sharing homology to known miRNAs were identified as conserved miRNA candidates and those not sharing homology were regarded as novel miRNA candidates.

Differentially expressed miRNAs were determined using the following criterion: fold-change for the mean of three independent biological replicates > 2; fold change in each of the three independent biological replicate > 1.8; and mean of TPM for the three replicates > 2 in at least one treatment.

In situ RT-PCR analysis

In situ RT-PCR analyses were performed as described previously14-16. In brief, cucumber petiole sections were pretreated with DNase I (Fermentas; Thermo Fisher Scientific) for 15 min to remove DNA, and then subjected to in situ RT-PCR by using Titanium One-Step RT-PCR Kit (Clontech, TAKARA) as the manufacturer's instructions. Fluorescence analysis was performed on a Leica TCS-SP2 confocal laser




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scanning microscope (Leica Microsystems) using an Ar/ArKr laser. Primers used in In situ RT-PCR analyses were listed in Supplementary Table 6.




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Supplementary references

1. Qin, L. et al. The high-affinity phosphate transporter GmPT5 regulates phosphate transport to nodules and nodulation in soybean. Plant Physiol. 159, 1634-1643 (2012).

2. Murphy, J. & Riley, J. P. A modified single solution method for the determination of phosphate in natural waters. Anal. Chim. Acta 27, 31-36 (1962).

3. Varkonyi-Gasic, E., Wu, R., Wood, M., Walton, E. F. & Hellens, R. P. Protocol: a highly sensitive RT-PCR method for detection and quantification of microRNAs. Plant Methods 3, 12 (2007).

4. Zhong, S. et al. High-throughput illumina strand-specific RNA sequencing library preparation. Cold Spring Harb. Protoc. 2011, 940-949 (2011).

5. Bolger, A. M., Lohse, M. & Usadel, B. Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics 30, 2114-2120 (2014).

6. Quast, C. et al. The SILVA ribosomal RNA gene database project: improved data processing and web-based tools. Nucleic Acids Res. 41, D590-596 (2013).

7. Langmead, B., Trapnell, C., Pop, M. & Salzberg, S. L. Ultrafast and memory-efficient alignment of short DNA sequences to the human genome. Genome Biol. 10, R25 (2009).

8. Kim, D. et al. TopHat2: accurate alignment of transcriptomes in the presence of insertions, deletions and gene fusions. Genome Biol. 14, R36 (2013).

9. Robinson, M. D., McCarthy, D. J. & Smyth, G. K. edgeR: a Bioconductor package for differential expression analysis of digital gene expression data. Bioinformatics 26, 139-140 (2010).

10. Huang, S. et al. The genome of the cucumber, Cucumis sativus L. Nature Genet. 41, 1275-1281 (2009).

11. Hofacker, I. L. Vienna RNA secondary structure server. Nucleic Acids Res. 31, 3429-3431 (2003).

12. Rajagopalan, R., Vaucheret, H., Trejo, J. & Bartel, D. P. A diverse and evolutionarily fluid set of microRNAs in Arabidopsis thaliana. Genes Dev. 20, 3407-3425 (2006).

13. Kozomara, A. & Griffiths-Jones, S. miRBase: annotating high confidence microRNAs using deep sequencing data. Nucleic Acids Res. 42, D68–D73 (2013).

14. Ruiz-Medrano, R., Xoconostle-Cazares, B. & Lucas, W. J. Phloem long-distance transport of CmNACP mRNA: implications for supracellular regulation in plants. Development 126, 4405-4419 (1999).

15. Haywood, V., Yu, T. S., Huang, N. C. & Lucas, W. J. Phloem long-distance trafficking of GIBBERELLIC ACID-INSENSITIVE RNA regulates leaf development. Plant J. 42, 49-68, (2005).

16. Ham, B. K. et al. A polypyrimidine tract binding protein, pumpkin RBP50, forms the basis of a phloem-mobile ribonucleoprotein complex. Plant Cell 21, 197-215, (2009).

17. Guo, S. et al. The draft genome of watermelon (Citrullus lanatus) and resequencing of 20 diverse accessions. Nature Genet. 45, 51-58 (2013).




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Supplementary Figures

Supplementary Fig. 1: Cucumber – a model plant for vascular biology and Pi stress studies. a, Cucumber plants undergoing a Pi-stress treatment; indicated lower (3rd), middle (5th, 6th, or 7th , as indicated) and upper (7th, 8th , 9th or 12th, as indicated) leaves were excised and used to measure soluble Pi content. b, Soluble Pi content measured in lower, middle and upper leaves of plants shown in (a), at the indicated time points. Data points are mean (n = 3) + SD. c, Development of Pi deficiency symptoms in leaves of -Pi treated cucumber. Images were taken 15 d after the onset of the -Pi treatment. d, Presence of primary (pri)-CsmiR399 in leaf and CsmiR399 in PS of +Pi and -Pi treated plants. Data points are mean (n = 3) + SD. ACTIN and CsmiR164 were used as internal controls for pri-CsmiR399 and CsmiR399, respectively. e-i, Tissues from which samples were collected in this study. e, Phloem sap (PS) exuding from cucumber stem; f, Internodal stem segment (left image) and related transverse section showing the location of internode vascular bundles (IVB) (right image) and a peeled IVB; petiole segment (left image) and a peeled petiole vascular bundle (PVB) (right image), scale bar, 1 cm; g, Major veins (MV) and lamina; h, Cucumber (left) and watermelon (right) developing leaf (DL) and shoot apex (SA); i, Cucumber root tips (RT); scale bar, 1 cm. j, Hetero-grafting between cucumber (Cu) and watermelon (Wm). GU, graft union.




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Supplementary Fig. 2: Response of pri-CsmiR399 and CsmiR399 levels to Pi deficiency in cucumber. a,b, Detection of pri-CsmiR399 (a) and CsmiR399 (b) levels in various tissues and phloem sap (PS) 0, 6, 12 h after -Pi treatment determined by qRT-PCR. Note that pri-CsmiR399 was not detected in PS. ACTIN and CsmiR164 were used as internal controls for pri-CsmiR399 and CsmiR399, respectively. Data points are mean (n = 3) + SD.




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Supplementary Fig. 3: Validation of RNA-Seq results (12 h +/-Pi) by qRT-PCR. a-g, Validation of differentially expressed genes (DEGs) in lamina (a), major veins (MV) (b), petiole vascular bundles (PVB) (c), internode vascular bundles (IVB) (d), root tips (RT) (e), shoot apex (SA) (f) and differentially detected transcripts (DDTs) in phloem sap (PS) (g), and their gene annotations. ACTIN was used as the internal control for DEGs in tissues and CsPP1 (Cucumber PHLOEM PROTEIN 1) was used as the internal control for DDTs in the PS. Data points are mean (n = 3) + SD.




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Supplementary Fig. 4: In situ RT-PCR performed to detect induction of Pi stress -responsive genes in cucumber vascular tissues. Cross-section of cucumber PVB analyzed by in situ RT-PCR using gene-specific primers for Pri-CsmiR399a (a to d) and UDP-glucose glucosyltransferase (CsUGT, Csa3M038620) (e to h), respectively. b, d, f and h are the bright fields of a, c, e and g, respectively. PVB structure is indicated in (b); CA, cambium; EP, external phloem; IP, internal phloem; Xy, xylem. Cucumber plants were supplied with phosphate (control, +Pi) or given a 12 h Pi-stress (-Pi) treatment. Positive signal (green) originated from cellular PCR products incorporating Alexa 488–labeled nucleotides, red signal represents tissue autofluorescence. Bars, 200 µm.




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Supplementary Fig. 5: GO enrichment analysis of DEGs (12 h, -Pi) in lamina, MV, PVB, IVB, SA and RT tissues. P-value cutoff for significant represented GO terms was < 0.001. GO terms enriched in these DEGs were identified with a tool in the cucurbit genomics databases website (http://www.icugi.org/cgi-bin/ICuGI/tool/GO_enrich.cgi).




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Supplementary Fig. 6: Hetero-grafting systems between cucumber and watermelon employed to identify phloem-mobile graft-transmissible mRNAs. a, Grafting combinations used between cucumber (Cu) and watermelon (Wm): G1, Wm scion/Cu stock with scion mature leaves removed; G2, Cu homo-grafted with scion mature leaves removed; G3, Cu scion/Wm stock with stock leaves removed; G4 and G5, Cu homo-grafted with stock leaves removed (G4), or no leaves removed (G5); NgCu, non-grafted cucumber. GU, graft union. Developing leaf (DL), shoot apex (SA) and RT (terminal ~3 cm of the root tip) samples were collected 24 h after +Pi or -Pi treatment. Scale bar, 15 cm. b, Homo-grafting and leaf removal had negligible influence on gene expression in cucumber sink tissues. Number of differentially expressed genes (DEGs) detected in DL and SA of G2 (upper leaves removed) and G5 (no leaves removed) plants. Number of DEGs detected in the RT of G4 (lower leaves removed) and G5 (no leaves removed) plants. Number of DEGs detected in DL, SA and RT of G5 and NgCu plants. Note that the higher level of up-regulated DEGs in RT of G5 compared with NgCu plants was due, in large part, to inhibited scion growth during the period in which the graft union was being established; under these conditions, root growth was promoted in the G5 grafted plants. c, Number of DEGs detected in DL, SA and RT of hetero-, homo- and non-grafted plants after a 24 h -Pi treatment.




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Supplementary Fig. 7: Tissue-specific graft-transmissible mRNAs and their response to -Pi treatment. a, Venn diagram of the total number of graft-transmissible (GT) mRNAs transported to the DL, SA and RT identified by the indicated criterion, under +Pi and -Pi conditions. b, Venn diagrams illustrating the tissue-specific distribution of GT mRNAs under +Pi or -Pi conditions, using the indicated filtering criteria. c, Major changes in the population of GT mRNAs occurred in watermelon DL, SA and RT tissues under Pi stress conditions (based on filtering criteria used in b). Green and red circles, mRNAs present only under +Pi and -Pi conditions, respectively; brown overlapping sectors, mRNAs unresponsive to Pi stress. d, Venn diagram of overlapping mRNAs between PS DDTs (12 h, -Pi) and GT mRNAs (based on filtering criteria used in b) showing 14 DDTs in PS (12 h, -Pi) are GT mRNAs.




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Supplementary Fig. 8: Relationship between phloem sap mRNA levels and their graft transmissibility. a, Phloem sap (PS) mRNAs were separated into six groups based on their transcript levels (FPKM>1000, 500-1000, 100-500, 10-100, 1-10, <1). Blue and red columns show the number of PS mRNAs and GT mRNAs in each group, respectively. b, Percentage of GT mRNAs in each PS mRNA group.




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Supplementary Fig. 9: DL GT mRNA expression in cucumber source PVB and sink DL. a, Expression in source PVB, of DL GT Cu mRNAs. b, Expression in sink DL, of DL GT Cu mRNAs that were highly expressed in source PVB (PVB FPKM > 100). c, Expression in sink DL, of DL GT mRNAs that had low expression in source PVB (PVB FPKM < 100). DL GT mRNAs means cucumber GT mRNAs detected in DL.




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Supplementary Fig. 10: SA GT mRNA expression in cucumber source PVB and sink SA. a, Expression in source PVB, of SA GT Cu mRNAs. b, Expression in sink SA, of SA GT Cu mRNAs that were highly expressed in source PVB (PVB FPKM > 100). c, Expression in sink SA, of SA GT mRNAs that had low expression in source PVB (PVB FPKM < 100). SA GT mRNAs means cucumber GT mRNAs detected in SA.




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Supplementary Fig. 11: RT GT mRNA expression in cucumber source PVB and sink RT. a, Expression in source PVB, of RT GT Cu mRNAs. b, Expression in sink RT, of RT GT Cu mRNAs that were highly expressed in source PVB (PVB FPKM > 100). c, Expression in sink RT, of RT GT mRNAs that had low expression in source PVB (PVB FPKM < 100). RT GT mRNAs means cucumber GT mRNAs detected in RT.




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Supplementary Fig. 12: Gene ontology (GO) enrichment analysis of biological pathways of Group I, II, III and IV GT mRNAs. P-value cutoff for significant represented GO terms was < 0.001. GO terms were identified with a tool in the cucurbit genomics databases website (http://www.icugi.org/cgi bin/ICuGI/tool/GO_enrich.cgi).




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Supplementary Fig. 13: Comparison of 380 Pi stress-responsive Group IV GT mRNAs detected in DL and 380 randomly selected Group I DL GT mRNAs for their association with either Pi stress or general cellular processes.




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Supplementary Fig. 14: Comparison of graft-transmissible mRNAs in cucumber, Arabidopsis and grape. a, Venn diagram of overlapping and species-specific cucumber (Cu) and Arabidopsis (At) graft-transmissible (GT) mRNAs. b, Venn diagram of overlapping and species-specific cucumber and grape (Gr) GT mRNAs. c, Numbers of GT mRNAs identified under +Pi and -Pi (3-week treatment) conditions in reference 20. d, -Pi-repressed, -unresponsive and -activated cucumber GT mRNAs and the response of their Arabidopsis graft-transmissible orthologs to Pi stress.




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Supplementary Datasets and Tables

Supplementary Dataset 1 (separate file)

Dataset S1. Differentially expressed genes in cucumber lamina, MV, PVB, IVB, RT, SA and differentially detected transcripts in cucumber PS after a 12 h -Pi treatment.


Dataset S2. Differentially expressed or detected miRNAs in cucumber lamina, MV, PVB, PS, IVB, RT and SA after a 12 h -Pi treatment.


Dataset S3. Graft-transmissible mRNAs detected in DL, SA and RT under +Pi or -Pi conditions. (Detected at least twice and total reads ≥ 2 in three replicates).


Dataset S4. Graft-transmissible mRNAs detected in DL, SA and RT under +Pi or -Pi conditions. (Detected at least twice and total reads ≥ 5 in three replicates).


Dataset S5. Tissue-specific and common graft-transmissible mRNAs shown in Figure 4b and Supplementary Figure 7a.


Dataset S6. Common or condition-specific graft-transmissible mRNAs as shown in Figure 4c and Supplementary Figure 7c.


Dataset S7. Differentially expressed genes in hetero-, homo- and non-grafted plants 24 h after a -Pi treatment.


Dataset S8. Overlapping transcripts between 417 PS DDTs (12 h -Pi treatment) and GT mRNAs as shown in Figure 4d and Supplementary Figure 7d.


Dataset S9. Classification of GT mRNAs delivered to DL as shown in Figure 5a.


Dataset S10. Classification of GT mRNAs delivered to SA as shown in Figure 5a.


Dataset S11. Classification of GT mRNAs delivered to RT as shown in Figure 5a.


Dataset S12. Tissue-specific and common Group IV GT mRNAs as shown in Figure 5 b,c.




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Dataset S13. Representative Group IV GT mRNAs responsive to Pi stress.

Supplementary Table 1 (separate file)

Table S1. Representative differentially detected transcripts in cucumber phloem sap after a 12 h -Pi treatment.


Table S2. Identified miRNAs and their levels in cucumber lamina, MV, PVB, PS, IVB, RT and SA.


Table S3. Predicted sequence conservation between the identified cucumber miRNAs and the watermelon genome.


Table S4. Induction of RNA-binding protein coding genes in PS and PVB following a 12 h -Pi treatment.


Table S5. Primers used in this study.


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