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. 2009 Dec;151(4):2120-32.
doi: 10.1104/pp.109.147280. Epub 2009 Oct 23.

Uncovering small RNA-mediated responses to phosphate deficiency in Arabidopsis by deep sequencing

Li-Ching Hsieh  1 , Shu-I Lin, Arthur Chun-Chieh Shih, June-Wei Chen, Wei-Yi Lin, Ching-Ying Tseng, Wen-Hsiung Li, Tzyy-Jen Chiou
Affiliations

Uncovering small RNA-mediated responses to phosphate deficiency in Arabidopsis by deep sequencing

Li-Ching Hsieh et al. Plant Physiol. 2009 Dec.

Abstract

Recent studies have demonstrated the important role of plant microRNAs (miRNAs) under nutrient deficiencies. In this study, deep sequencing of Arabidopsis (Arabidopsis thaliana) small RNAs was conducted to reveal miRNAs and other small RNAs that were differentially expressed in response to phosphate (Pi) deficiency. About 3.5 million sequence reads corresponding to 0.6 to 1.2 million unique sequence tags from each Pi-sufficient or Pi-deficient root or shoot sample were mapped to the Arabidopsis genome. We showed that upon Pi deprivation, the expression of miR156, miR399, miR778, miR827, and miR2111 was induced, whereas the expression of miR169, miR395, and miR398 was repressed. We found cross talk coordinated by these miRNAs under different nutrient deficiencies. In addition to miRNAs, we identified one Pi starvation-induced DICER-LIKE1-dependent small RNA derived from the long terminal repeat of a retrotransposon and a group of 19-nucleotide small RNAs corresponding to the 5' end of tRNA and expressed at a high level in Pi-starved roots. Importantly, we observed an increased abundance of TAS4-derived trans-acting small interfering RNAs (ta-siRNAs) in Pi-deficient shoots and uncovered an autoregulatory mechanism of PAP1/MYB75 via miR828 and TAS4-siR81(-) that regulates the biosynthesis of anthocyanin. This finding sheds light on the regulatory network between miRNA/ta-siRNA and its target gene. Of note, a substantial amount of miR399* accumulated under Pi deficiency. Like miR399, miR399* can move across the graft junction, implying a potential biological role for miR399*. This study represents a comprehensive expression profiling of Pi-responsive small RNAs and advances our understanding of the regulation of Pi homeostasis mediated by small RNAs.

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Figures

Figure 1.
Figure 1.
Global profiling of small RNAs. Correlations between the small RNA length and the proportion of total sequence reads (A) or unique sequence signatures (B) are shown. nt, Nucleotides.
Figure 2.
Figure 2.
Overrepresentation of tRNA-derived small RNAs in the Pi-starved roots. A, Small RNA gel analyses of tRNA-derived small RNAs in +Pi or −Pi roots or shoots. B, The tRNA regions corresponding to the 5′ and 3′ end probes are designated by the black line, and the anticodon probe is designated by the gray line. The black arrowheads in A and B indicate the 5′ cleaved products and corresponding cleavage site in tRNA, respectively. The vertical gray lines in A indicate the products of tRNA halves from the cleavage at the anticodon loop (gray arrowhead in B). nt, Nucleotides.
Figure 3.
Figure 3.
Differentially expressed MIRNA genes in response to Pi deficiency in roots (A) and shoots (B). The significantly differentially expressed MIRNA genes with greater than 1.5-fold relative change are shown. The miRNAs with a relative change ratio greater than 3 are highlighted in boldface.
Figure 4.
Figure 4.
Validation of differentially expressed miRNAs under different nutrient deficiencies. Small RNA gel analyses of Pi starvation-up-regulated miRNAs (A) and Pi starvation-down-regulated miRNAs (B) are shown. miR778 was detected using the probe corresponding to miR778-3p.1 and miR778-3p.2.
Figure 5.
Figure 5.
Characterization of miR2111 in response to Pi deficiency. A and B, Small RNA gel analyses of miR2111-5p and miR2111-3p under different nutrient-deficient conditions (A) and in small RNA biogenesis mutants (B). Col, Ecotype Columbia; Ler, ecotype Landsberg erecta; wt, wild type. C, Hairpin secondary structures of miR2111a and miR2111b precursors. D, RLM 5′-RACE analysis of miR2111-5p cleavage on At3g27150 mRNA. E, Quantitative RT-PCR analysis of At3g27150 mRNA in response to −Pi. One of two biological replicates is presented, and the error bars indicate the sd of two technical replicates.
Figure 6.
Figure 6.
Pi starvation-induced small RNAs derived from LTR of retrotransposon. A and B, Small RNA gel analysis of smRPi1LTR under different nutrient-deficient conditions (A) and in small RNA biogenesis mutants (B). Col, Ecotype Columbia; hetero, heterozygous plants; wt, wild type. C, Arrangement of Copia95 LTR in the intergenic region of At5g27990 and At5g28000 and the potential hairpin structure and location of smRPi1LTR.
Figure 7.
Figure 7.
Autoregulatory mechanism of PAP1/MYB75 via miR828 and TAS4-siR81(−). A, Small RNA gel analysis of TAS4-siRNAs under different nutrient-deficient conditions. B, Small RNA gel analysis of TAS4-siR81(−) and miR828 in the shoots of wild-type (wt) plants and tas4, mir828, and PAP1/MYB75 activation (pap1-D) mutants. C, Expression of PAP1/MYB75, PAP2/MYB90, and MYB113 by quantitative RT-PCR analysis in tas4 and mir828 mutants. One of two biological replicates is presented, and the error bars indicate the sd of two technical replicates. D, Alteration of anthocyanin accumulation in tas4, mir828, and pap1-D mutants. Error bars represent the sd (n = 5). FW, Fresh weight; OD, optical density. E, A proposed model of the autoregulatory pathway of PAP1/MYB75 via miR828 and TAS4-siR81(−).
Figure 8.
Figure 8.
Expression and long-distance movement of miR399*. A, Small RNA gel analysis of miR399*. B, Quantitative RT-PCR analysis of miR399f and miR399f* in the rootstocks of grafted plants. Combination of grafts is indicated as scion/rootstock. 399f, miR399f-overexpressing plants; wt, wild-type plants. The error bars represent sd (n = 4–5 independent grafted plants).
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