Identification of phloem-associated translatome alterations during leaf development in Prunus domestica L

Abstract

Phloem plays a fundamental role in plants by transporting hormones, nutrients, proteins, RNAs, and carbohydrates essential for plant growth and development. However, the identity of the underlying phloem genes and pathways remain enigmatic especially in agriculturally important perennial crops, in part, due to the technical difficulty of phloem sampling. Here, we used two phloem-specific promoters and a translating ribosome affinity purification (TRAP) strategy to characterize the phloem translatome during leaf development at 2, 4, and 6 weeks post vernalization in plum (Prunus domestica L.). Results provide insight into the changing phloem processes that occur during leaf development. These processes included the early activation of DNA replication genes that are likely involved in phloem cell division during leaf expansion, as well as the upregulation of phloem genes associated with sink to source conversion, induction of defense processes, and signaling for reproduction. Combined these results reveal the dynamics of phloem gene expression during leaf development and establish the TRAP system as a powerful tool for studying phloem-specific functions and responses in trees.

Fig. 1. Prunus domestica L. promoter:HF-RPL18 transgenic plants.

a Histochemical analysis of Arabidopsis pSUC2 and pSULTR2;2 promoters in transgenic plums visualized by GUS staining in mid-vein cross sections. Phloroglucinol was used to stain xylem red. x xylem, p phloem. b Relative HF-RPL18 transgene expression in leaves. Quantitative RT-PCR analysis was performed with a primer set specific to HF-RPL18 and 18S rRNA was used as the internal control. Bars represent the mean of three biological replicates ± standard error. c Representative photographs of leaves collected at 2, 4, and 6 weeks post vernalization

Fig. 2. Isolation of translatome RNA.

a Representative Agilent 2100 bioanalyzer gel-like image showing RNA isolated after incubation with Anti-FLAG magnetic beads taken from the unbound (blue labels) or bound (orange labels) fraction. High-quality RNA was successfully recovered in the bound fraction for all translatome HF-RPL18 plant lines, but not from non-transgenic controls. b Quantification of RNA recovered from the bound fraction. RNA was measured on a NanoDrop 2000 and is shown in nanograms. Bars represent the mean of four biological replicates ± standard error. c Relative HF-RPL18 transcript accumulation detected in total RNA (white bars) and translating mRNA samples (orange bars). Bars represent the mean of three biological replicates ± standard error. All samples are normalized to p35S total RNA

Fig. 3. Phloem-enriched genes.

a Total number of genes with > 2-fold expression that are unique or shared by pSUC2 or pSULTR2;2 phloem translatomes compared with p35S translatome at 2, 4, and 6 weeks post vernalization. b Number of unique and shared phloem-enriched genes across time points. Phloem-enriched genes were defined as > 2-fold expression in both pSUC2 and pSULTR2;2 phloem translatomes compared with p35S translatome

Fig. 4. Phloem-enriched gene categories altered over development.

A subset of gene ontology (GO) biological process terms overrepresented in phloem-enriched genes that are altered over the 2- to 6-week sampled time frame. Bars represent the percentage of genes in each category that are downregulated (blue) or upregulated (red). All significantly overrepresented GO biological process terms are listed in Table S7

Fig. 5. Heatmaps of phloem-enriched gene expression over development.

Heatmaps showing changes to genes involved in a DNA replication, b systemic acquired resistance, c response to nutrients, and d reproduction in Prunus domestica L. leaves 2, 4, and 6 weeks post vernalization. Mean FPKM values from three biological replicates are shown on a log2 scale with z-scaling by row. Gene names from A. thaliana best BLAST matches are shown to the right of all heatmaps