Warm temperatures induce transgenerational epigenetic release of RNA silencing by inhibiting siRNA biogenesis in Arabidopsis

Abstract

Owing to their sessile nature, plants have evolved sophisticated genetic and epigenetic regulatory systems to respond quickly and reversibly to daily and seasonal temperature changes. However, our knowledge of how plants sense and respond to warming ambient temperatures is rather limited. Here we show that an increase in growth temperature from 22 °C to 30 °C effectively inhibited transgene-induced posttranscriptional gene silencing (PTGS) in Arabidopsis. Interestingly, warmth-induced PTGS release exhibited transgenerational epigenetic inheritance. We discovered that the warmth-induced PTGS release occurred during a critical step that leads to the formation of double-stranded RNA (dsRNA) for producing small interfering RNAs (siRNAs). Deep sequencing of small RNAs and RNA blot analysis indicated that the 22-30 °C increase resulted in a significant reduction in the abundance of many trans-acting siRNAs that require dsRNA for biogenesis. We discovered that the temperature increase reduced the protein abundance of SUPPRESSOR OF GENE SILENCING 3, as a consequence, attenuating the formation of stable dsRNAs required for siRNA biogenesis. Importantly, SUPPRESSOR OF GENE SILENCING 3 overexpression released the warmth-triggered inhibition of siRNA biogenesis and reduced the transgenerational epigenetic memory. Thus, our study reveals a previously undescribed association between warming temperatures, an epigenetic system, and siRNA biogenesis.

Fig. 1.

Dwarf phenotype of NRG1(d) is caused by PTGS of the endogenous BRI1 gene. (A) Schematic diagram of NRG1 and its mutant variants, NRG1mL and NRG1mK (20). LRR, leucine-rich repeat; TM, transmembrane domain; JM, juxtamembrane domain; NOS-ter, termination sequence of the nopaline synthase gene. (B) Transgenic plants grown at 22 °C expressing NRG1 or its mutant variants. (Scale bar, 1 cm.) (C) RNA blot analysis of BRI1 and NRG1 transcripts and BRI1-specific siRNAs. Reprobing the filters with a ³²P-labeled 18S rRNA cDNA or ³²P-labeled U6 was used to control equal loading. (D) Four-week-old Col (WT), strong (d), and semidwarfed (sd) segregates of a semidwarf NRG1 line and WT-like (wt) NRG1 seedlings. (Scale bar, 1 cm.) (E) RNA blot analysis of BRI1 and NRG1 transcripts in Col-0, three segregates of the semidwarf NRG1 line, and a WT-like NRG1 line (wt). The filter was rehybridized with an 18S rRNA probe to control equal loading. (F) Immunoblot analysis of BRI1 abundance in 4-wk-old plants. Coomassie blue staining of the protein gel (*) served as a loading control. (G) Four-week-old rdr6/NRG1(d), sgs3/NRG1(d), and hyponastic leaves (hyl1)/NRG1(d) plants, 6-wk-old ago1/NRG1(d) and dcl4/NRG1(d) homozygous filial generation 3 (F3) plants, and 4-wk-old control NRG1(d) plants grown in soil.

Fig. 2.

Release of S-PTGS by warming ambient growth temperatures. (A) Four-week-old Col-0 and NRG1(d) plants grown at 22 and 30 °C. (Scale bar, 1 cm.) (B) RNA blot analysis of BRI1 and NRG1 transcripts and BRI1-specific siRNAs. (C and D) Percentage of four different types of NRG1(d) plants at different growth temperatures. About 100 plants at each temperature were analyzed. (Scale bar, 1 cm.) The average (± SD) values from 3 repeats of the analysis are shown (D). (E) Histochemical staining of GUS activity in 4-wk-old L1 seedlings grown at 22 and 30 °C. (F) RNA blot analysis of GUS mRNA and GUS-specific siRNAs in 22/30 °C-grown 4-wk-old L1 plants. (G and H) Fluorescent microscopic examination of the GFP signal in the apical root meristem (G) and maturation zone (H) of 4-wk-old GxA seedlings grown at 22 and 30 °C. (I) RNA blot analysis of GFP mRNA and GFP-specific siRNAs. Rehybridization with a ³²P-labeled 18S rRNA cDNA (B), a ³²P-labeled U6 probe (B, F, and I), or ethidium bromide staining of the rRNAs (F and I) served as loading controls.

Fig. 3.

Transgenerational inheritance of warmth-induced release of PTGS. (A) Each vertical bar represents one line with ∼100 individual plants, and each color bar indicates the percentage of plants within a given NRG1(d) population exhibiting the four color-coded morphological phenotypes shown (Left). Seeds from 22 °C-grown NRG1(d) dwarf plants were germinated and grown at 30 °C, producing all phenotypically WT-looking plants (first generation). Seeds from a randomly chosen WT-like NRG1 plant were germinated and grown at 22 °C to produce the second generation. The offspring of three individual second-generation WT-like NRG1 plants and three individual third-generation plants of each phenotypic group grown at 22 °C were independently analyzed. (B) Transgenerational inheritance of warmth-triggered PTGS in the L1 line. More than 100 offspring of two individual second-generation WT-like plants and two individual third-generation plants of each of the three phenotypic groups were analyzed.

Fig. 4.

Growth at 30 °C likely inhibits dsRNA formation. (A) Four-week-old plants of WT (Left) and a BRI1-RNAi line (Right) grown at 22 and 30 °C. Two independent stable lines with similar phenotype were analyzed. (Scale bars, 1 cm.) (B) A qPCR analysis of relative BRI1 mRNA levels. (C) RNA blot analysis of three tasiRNAs in 22/30 °C-grown NRG1(d) and WT seedlings. (D) Schematic representation of the assay for sense and antisense transcripts of BRI1 (see Materials and Methods for details). (E and F) qPCR analysis of sense (E) and antisense (F) BRI1 transcripts in 22/30 °C-grown seedlings of each of the following lines: 1, 22 °C-grown Col-0; 2, 30 °C-grown Col-0; 3, 22 °C-grown NRG1(d); 4, 30 °C-grown NRG1(d); 5, 22 °C-grown second-generation type 4 plants; 6, 22 °C-grown third-generation type 1 plants; 7, 22 °C-grown third-generation type 4 plants; 8, 22 °C-grown NRG1(d)/rdr6; 9, 22 °C-grown NRG1(d)/sgs3; and 10, 22 °C-grown NRG1(d)/ago1. (G) Four-week-old WT and BRI1 antisense plants grown at 22 or 30 °C. (Scale bar, 1 cm.) Eight independent stable lines with similar phenotype were analyzed. (H) RNA blot analysis of BRI1-derived siRNAs in WT and BRI1 antisense plants grown at 22 or 30 °C. For RNA blot analysis, reprobing with a U6 probe (C and H) was used to serve as a loading control, whereas qPCR assays used the ACTIN2 signal for normalization. The average (±SD) values from three biological repeats are shown (B, E, and F).

Fig. 5.

SGS3 overexpression releases warmth-triggered inhibition of siRNA biogenesis and reduces transgenerational epigenetic memory. (A) Immunoblotting of SGS3-GFP in a pUbi::SGS3-GFP sgs3-12 transgenic line grown at different temperatures. The same amounts of total proteins extracted from 4-wk-old plants were separated by 10% SDS/PAGE and analyzed by immunoblotting with a commercial anti-GFP antibody, and the same blots were reprobed with an anti-ACTIN antibody to control equal loading. (B) qPCR analysis of SGS3 transcript levels that were normalized to the ACTIN2 signals. The average values (±SD) from three replicates of the qPCR experiments are shown. (C) Immunoblot analysis of SGS3-GFP abundance in the p35S::SGS3-GFP line. (D) RNA blot analysis of siR255 in 22/30 °C-grown plants of sgs3-11, sgs3-12, a p35S::SGS3-GFP transgenic line, and the WT. A U6 blot was used as a loading control. (E) Histochemical staining of the GUS activity of 4-wk-old 22/30 °C-grown seedlings. (Scale bars, 1 cm.) (F) Percentages of WT-looking plants in offspring of 22 °C-grown NRG1(d) and pUbi::SGS3 NRG1(d) lines. Approximately 100 independent T1 lines (equivalent of the second generation in Fig. 3) and 30 T2 plants (equivalent of the third generation in Fig. 3) of six independent pUbi::SGS3 NRG1(d) lines [three being SGS3 overexpressors (OE) and the other with low SGS3 transcript levels (not OE)] were analyzed.