Phloem small RNAs, nutrient stress responses, and systemic mobility

Abstract

Background: Nutrient availabilities and needs have to be tightly coordinated between organs to ensure a balance between uptake and consumption for metabolism, growth, and defense reactions. Since plants often have to grow in environments with sub-optimal nutrient availability, a fine tuning is vital. To achieve this, information has to flow cell-to-cell and over long-distance via xylem and phloem. Recently, specific miRNAs emerged as a new type of regulating molecules during stress and nutrient deficiency responses, and miR399 was suggested to be a phloem-mobile long-distance signal involved in the phosphate starvation response.

Results: We used miRNA microarrays containing all known plant miRNAs and a set of unknown small (s) RNAs earlier cloned from Brassica phloem sap 1, to comprehensively analyze the phloem response to nutrient deficiency by removing sulfate, copper or iron, respectively, from the growth medium. We show that phloem sap contains a specific set of sRNAs that is distinct from leaves and roots, and that the phloem also responds specifically to stress. Upon S and Cu deficiencies phloem sap reacts with an increase of the same miRNAs that were earlier characterized in other tissues, while no clear positive response to -Fe was observed. However, -Fe led to a reduction of Cu- and P-responsive miRNAs. We further demonstrate that under nutrient starvation miR399 and miR395 can be translocated through graft unions from wild type scions to rootstocks of the miRNA processing hen1-1 mutant. In contrast, miR171 was not transported. Translocation of miR395 led to a down-regulation of one of its targets in rootstocks, suggesting that this transport is of functional relevance, and that miR395, in addition to the well characterized miR399, could potentially act as a long-distance information transmitter.

Conclusions: Phloem sap contains a specific set of sRNAs, of which some specifically accumulate in response to nutrient deprivation. From the observation that miR395 and miR399 are phloem-mobile in grafting experiments we conclude that translocatable miRNAs might be candidates for information-transmitting molecules, but that grafting experiments alone are not sufficient to convincingly assign a signaling function.

Figure 1

List of miRNAs that were enriched in phloem, leaves or roots, respectively, in plants grown under full nutrition. Only families where at least one member showed a statistically significant differential accumulation in one organ are shown (p < 0.05, n = 3). Values are log2s between P/L: phloem vs. leaves, P/R: phloem vs. roots and L/R: leaves vs. roots. Markedly (log2 values >1 or <-1, indicating a two-fold difference) phloem-enriched miRNAs are marked in blue, leaf-enriched in green, and root-enriched in red. The statistical significance is indicated as: * p < 0.05; ** p < 0.01; *** p < 0.001.

Figure 2

List of unknown sRNAs that were organ-enriched grown under full nutrition. List of unknown sRNAs, sequenced from Brassica phloem sap [1], that showed statistically significant differences between phloem sap, leaves and roots, respectively (p < 0.05, n = 3). Values are log2s between P/L: phloem vs. leaves, P/R: phloem vs. roots and L/R: leaves vs. roots. Markedly (log2 values >1 or <-1, indicating a two-fold difference) phloem-enriched miRNAs are marked in blue, leaf-enriched in green, and root-enriched in red. The statistical significance is indicated as: * p < 0.05; ** p < 0.01; *** p < 0.001.

Figure 3

List of nutrient-responsive sRNAs. List of sRNAs that showed a strong positive reaction to S, Cu or Fe deprivation, respectively, shown as log2 values of stressed vs. FN samples. Only sRNAs that fulfilled the criteria described in the Methods section (positive response, log2 >2 in one of the stress treatments, signal value >100 in FN or deprived sample) in at least one of the comparisons are listed. The insets show results obtained by miRNA sqRT-PCR (after 25 cycles) from an independent experiment. To allow a better overview, values for known nutrient starvation-responsive miRNAs (398 and 857 for -Cu and 2111 for -P) were included, although they only showed a negative response or were not detectable. Arrows indicate directions of changes obtained in a second, independent -Cu experiment. n.d.: not detectable (both, FN and stress, signal values <100). X: not on chip.

Figure 4

Effect of copper and iron deficiency on known nutrient-responsive miRNAs. Graphic summary of the opposite effect of copper and iron deficiency on the known -Cu responsive miRNAs 397, 398, 408 and the -P responsive miRNAs 399 and 2111. Phloem responses are compared to data obtained from leaves and roots. All data were obtained from miRNA array hybridization experiments. Differences between stress and control plants are shown as log2 values, only Arabidopsis miRNAs are depicted. n.d.: not detectable.

Figure 5

WT/hen1-1 grafting experiments. Analysis of mature miR395, miR399 and miR171 by RNA gel blot analysis in scions and rootstocks of reciprocal hen1-1/WT and WT/hen1-1 grafts under sulfate and phosphate deficiency. A: miRNAs 395 and 399 were translocated from WT scions to hen1-1 rootstocks but not in the opposite direction, miR171 was immobile. One representative result is shown for WT, and three replications for hen1-1 roots and shoots. The hen1-1 graft parts kept their growth retardation phenotype, indicating that not all necessary miRNAs could be transferred. The 5.8 ribosomal RNA band served as a loading control. B: Control of miR395 expression in WT and hen1-1 mutant plants. In WT plants miR395 was induced by sulfate deficiency in shoots and roots, while no signal was detected in hen1-1 mutants under both conditions.

Figure 6

Analysis of the targets of miR395 in roots. Analysis of the mRNA levels of the miR395 targets SULTR2;1, APS1 and APS4 by semi-quantitative RT-PCR. A: PCR results from root tissue of hydroponically grown Arabidopsis hen1-1 mutants and WT/hen1-1 rootstocks (35 cycles, UBC10, At5g53300 served as a control). B: Changes of target mRNAs in B. napus roots under -S compared to full nutrition (FN) (35 cycles, UBP1B, At1g17370 served as a control).