A small RNA mediated regulation of a stress-activated retrotransposon and the tissue specific transposition during the reproductive period in Arabidopsis

Abstract

Transposable elements (TEs) are key elements that facilitate genome evolution of the host organism. A number of studies have assessed the functions of TEs, which change gene expression in the host genome. Activation of TEs is controlled by epigenetic modifications such as DNA methylation and histone modifications. Several recent studies have reported that TEs can also be activated by biotic or abiotic stress in some plants. We focused on a Ty1/copia retrotransposon, ONSEN, that is activated by heat stress (HS) in Arabidopsis. We found that transcriptional activation of ONSEN was regulated by a small interfering RNA (siRNA)-related pathway, and the activation could also be induced by oxidative stress. Mutants deficient in siRNA biogenesis that were exposed to HS at the initial stages of vegetative growth showed transgenerational transposition. The transposition was also detected in the progeny, which originated from tissue that had differentiated after exposure to the HS. The results indicated that in some undifferentiated cells, transpositional activity could be maintained quite long after exposure to the HS.

Keywords: Arabidopsis thaliana; ONSEN; environmental stress; small RNA; transposon.

FIGURE 1

Structure of the gene construct used to produce transgenic plants, and the insertion position of the transgene in the genome. (A) The structure of the DNA sequence between the left border (LB) and the right border (RB) of the T-DNA. Intact long terminal repeat (LTR) of ONSEN (AT5G13205) was fused with the green fluorescent protein (GFP) gene. (B) The transgene was inserted in the intergenic region between At1g26850 and At1g26860, in the same direction. Numerals indicate the nucleotide number according to the AGI map.

FIGURE 2

Expression analysis of GFP driven by the ONSEN promoter. (A) Scheme of the heat stress (HS) experiment. (B) Photographs of bright field and GFP fluorescence images. Wild-type plants (WT) and nrpd1 plants (nrpd1) were observed immediately after HS and after the recovery phase (Recovery). (C) Relative transcription level of GFP. WT and nrpd1 plants (nrpd1) were analyzed immediately after HS and after the recovery phase (R3). (D) Relative transcription level of endogenous ONSEN. The same samples as those used in c were used. NS, non-stress control samples. Error bars represent the mean ± SEM, n = 3; values are relative to heat-stressed wild-type (WT HS).

FIGURE 3

Activation of ONSEN by high light stress and oxidative stress. Plants before stress treatment, after continuous stress treatment for 1 h and after continuous stress treatment for 6 h were analyzed. (A) Relative transcription level of ONSEN immediately after high light stress (HL). (B) Relative transcription level of ONSEN immediately after Paraquat treatment (PQ). Error bars represent SDs (n = 3).

FIGURE 4

The quantitative analyses of transcripts, extrachromosomal DNAs, and newly transposed copies in progeny. WT and nrpd1 plants were exposed to HS for 6 h and for 24 h at 7 days after germination. NS, non-stressed samples. (A) Relative transcription level of ONSEN. Error bar represents the mean ± SEM, n = 3, values relative to 24 h heat-stressed WT. (B) Relative number of copies of extrachromosomal DNA of ONSEN. Error bar represents the mean ± SEM, n = 3; values are relative to the NS WT seedlings. (C) Southern blot analysis of ONSEN in progeny plants.

FIGURE 5

Transgenerational transposition of ONSEN in young seedling. (A) Scheme of the HS experiment. On each day after germination, seedlings were exposed to HS for 24 h. (B,C) Southern blot analysis of ONSEN using the progeny of WT (B) and nrpd1 (C) plants that were exposed to HS, as indicated in (A). Durations of HS are indicated over the lanes. NS plants were used as controls. Arrowheads indicate the transposed copies of ONSEN.

FIGURE 6

The effect of daylight conditions on flowering time. The average of the day on which bolting occurred was measured in 3 to 8 plants that grew under long-day (LD) or short-day (SD) conditions. Plants were exposed to HS for 24 h at 7, 10, 14, and 21 days after germination. The WT and nrpd1 plants grew under LD and SD were compared, respectively, using a t-test. An asterisk indicates significantly different, p < 0.05. NS control plants.

FIGURE 7

Transgenerational transpositions in the WT (A) and nrpd1(B) plants. Data for plants grown under long-day (LD) and short-day (SD) conditions are shown in the left panels and right panels, respectively. 7, 10, 14, and 21 denote plants that were exposed to HS for 24 h at 7, 10, 14, and 21 days after germination, respectively. NS, non-stressed plants.

FIGURE 8

Southern hybridization of ONSEN using the progeny of heat-stressed plants. (A) DNAs were extracted from the progeny of one nrpd1 plant. Numbers over lanes indicate the seed pods having seeds to grow progeny. Four progeny from the same seed pod were analyzed. The location of each seed pod on the parent plant is shown on the illustrated plant. Arrowheads indicate the new copies of ONSEN and the same color of arrowhead denotes that they transposed to the same loci. (B) DNAs were extracted from the progeny of another nrpd1 plant. Symbols are the same as those used in (A).

FIGURE 9

Transgenerational transposition in late-differentiated branches. (A) A scheme to obtain seeds for progeny. Parent nrpd1 plants were exposed to HS for 24 h at 7 days after germination, then the primary flower stalk was cut immediately after bolting, and seeds were harvested from secondary branches. (B) Southern hybridization of ONSEN. DNAs of progeny from two NS parents and five HS parents were analyzed. Numbers over the lanes indicate that DNAs were extracted from the progeny of different parents. Arrowheads indicate the new copies of ONSEN.

FIGURE 10

Tissue-specific expression of ONSEN. (A) A paraffin section of a young seedling. (B) A paraffin section of a young seedling after the shoot apex was isolated by laser capture microdissection. (C) Relative transcription level of ONSEN in young leaf tissue and the shoot apex immediately after being subjected to HS and 5 days after the heat-stressed (HS + R5). WT: hypocotyl tissue in WT plants. nrpd1: hypocotyl tissue in nrpd1 mutant plants. WT SA: the shoot apex in WT plants. nrpd1 SA: the shoot apex in nrpd1 mutant plants. Error bars represent the mean ± SEM, n = 3; values are relative to heat-stressed WT.