Heat shock factor HSFB2a involved in gametophyte development of Arabidopsis thaliana and its expression is controlled by a heat-inducible long non-coding antisense RNA

Abstract

Heat stress transcription factors (HSFs) are central regulators of the heat stress response. Plant HSFs of subgroup B lack a conserved sequence motif present in the transcriptional activation domain of class A-HSFs. Arabidopsis members were found to be involved in non-heat shock functions. In the present analysis we investigated the expression, regulation and function of HSFB2a. HSFB2a expression was counteracted by a natural long non-coding antisense RNA, asHSFB2a. In leaves, the antisense RNA gene is only expressed after heat stress and dependent on the activity of HSFA1a/HSFA1b. HSFB2a and asHSFB2a RNAs were also present in the absence of heat stress in the female gametophyte. Transgenic overexpression of HSFB2a resulted in a complete knock down of the asHSFB2a expression. Conversely, asHSFB2a overexpression leads to the absence of HSFB2a RNA. The knockdown of HSFB2a by asHSFB2a correlated with an improved, knockdown of asHSFB2a by HSFB2a overexpression with an impaired biomass production early in vegetative development. In both cases the development of female gametophytes was impaired. A T-DNA knock-out line did not segregate homozygous mutant plants, only heterozygots hsfB2a-tt1/+ were viable. Approximately 50% of the female gametophytes were arrested in early development, before mitosis 3, resulting in 45% of sterile ovules. Our analysis indicates that the "Yin-Yang" regulation of gene expression at the HSFB2a locus influences vegetative and gametophytic development in Arabidopsis.

Fig. 1

A Position of the T-DNA insertions in the HSFB2a genomic region. Grey boxes depict the coding sequence, the open box the intron, hatched boxes the 5′-UTR and 3′-UTR, and arrowheads the direction of transcription. Black upright boxes mark perfect, open boxes imperfect HSEs. Crosswise black boxes show the position of in situ hybridization probes. Arrows denote primer positions, numbers refer to nucleotide positions relative to the transcription start and lines indicate the insertion site of the respective T-DNA. The expression levels of HSFB2a (B) and asHSFB2a (C) mRNA in wild-type and the respective overexpressing and knockout (SALK_012418) plants at different temperatures are indicated by the numbers above the columns. All values were normalized with respect to Actin2 mRNA (=100 %)

Fig. 2

a Sterile ovules in siliques of the heterozygous mutant, indicated by arrow heads. In situ hybridization of carpel sections detecting HSFB2a mRNA with DIG labelled sense control (b) and antisense (c) probe

Fig. 3

GUS expression in HSFB2a-Promoter:GUS plants. In roots (a) of 8 day old and nodes (b) of 15 day old seedlings, leaves (d) of 7 weeks old plants, anthers of flowers (e) of stage 13 (Smyth et al. 1990) and embryos (c), seed (f) and the junction of the petiole of ripe siliques (g)

Fig. 4

Proportions of mature and immature female gametophytes in (A) wt and five HSFB2a overexpressing (35S:HSFB2a, 1–5) and (B) wt and five asHSFB2a (35S:asHSFB2a, 1–5) overexpressing lines. Open boxes represent the proportion of mature female gametophytes, grey boxes the proportion of immature female gametophytes, numbers in and above boxes the respective average percentage of five plants per line. Prevalent aberrant phenotypes of HSFB2a and asHSFB2a overexpressing lines. C Embryo sac with one nucleus. D Embryo sac with four nuclei. Arrow heads depict the position of visible nuclei in the female gametophytes. Text below the pictures indicates the percentage of gametophytes showing the above phenotype in the respective lines