The wheat peptidyl prolyl cis-trans-isomerase FKBP77 is heat induced and developmentally regulated

Abstract

We isolated a cDNA encoding a 568-amino acid, heat-stress-induced peptidyl prolyl isomerase belonging to the FK506-binding-protein (FKBP) family. The open reading frame encodes for a peptidyl prolyl isomerase that possesses three FKBP-12-like domains, a putative tetratricopeptide motif, and a calmodulin-binding domain. Specific antibodies showed that the open reading frame encodes a heat-induced 77-kD protein, the wheat FKBP77 (wFKBP77), which exhibits 84% identity with the wFKBP73 and 42% identity with the human FKBP59. Because of the high similarity in sequence to wFKBP73, wFKBP77 was designated as the heat-induced isoform. The wFKBP77 mRNA steady-state level was 14-fold higher at 37 degreesC than at 25 degreesC. The wFKBP77 transcript abundance was the highest in mature embryos that had imbibed and 2-d-old green shoots exposed to 37 degreesC, and decreased to 6% in 6-d-old green shoots. The transcript level returned to the level detected at 25 degreesC after recovery of the embryos for 90 min at 25 degreesC. We compared wFKBP73 and wFKBP77 with the heat-shock proteins having cognate and heat-stress-induced counterparts.

Figure 1

Alignment of the deduced amino acid sequences of wFKBP73 (Blecher et al., 1996) and ROF1 (Vucich and Gasser, 1996) with that of the heat-induced wFKBP77 according to FASTA analysis. Single-letter codons are used for amino acid residues, black or gray boxes indicate identical or similar residues, respectively, and dots indicate gaps introduced to allow optimal alignment of the sequence.

Figure 2

Temperature-dependent accumulation of wFKBP77 RNA in embryos. Slot-blot analysis of RNA extracted from embryos that were exposed for 2 h to temperatures from 25°C to 42°C. The blots were hybridized with the FKBP77 and the 26S rRNA probes. Scale represents 100% for the wFKBP77 mRNA extracted from embryos that were exposed for 2 h at 37°C.

Figure 3

Northern-blot analysis of poly(A⁺) RNA extracted from embryos that were exposed to 25°C or 37°C for 2 h. One microgram of mRNA from FKBP77 (25°C) was hybridized with the FKBP77 probe, and 4 μg with the FKBP73 probe.

Figure 4

Accumulation of wFKBP77 in mature embryos exposed to 37°C. Northern blot of RNA extracted from embryos that were exposed to 37°C or 25°C after 2 h at 37°C and hybridized with the cDNA probes from the common region of the wFKBP73 (FKBP73/77) and the specific probes FKBP77, FKBP73, wHSP82 (HSP82), and the wheat 26S rRNA as a control for the amount of RNA that was loaded.

Figure 5

Disappearance of wFKBP77 after heat stress in embryos that had imbibed. Slot-blot analysis of RNA extracted from embryos that were exposed to 25°C after 2 h at 37°C. The blots were hybridized with the FKBP77 and the 26S rRNA probes. Scale represents 100% for the wFKBP77 mRNA extracted from embryos that were exposed to 37°C for 2 h.

Figure 6

Expression of wFKBP77 and wFKBP73 in tissues exposed to heat stress. Northern-blot analysis of RNA extracted from 2- to 6-d-old etiolated and green shoots (A), 2- to 6-d-old roots (B), anthers (Ant), and pistils (Pis) (results of an independent experiment; see Methods) (C). RNA was extracted only from the basal first 1 cm of the shoots and the distal first 1 cm of the roots. Blots were hybridized with the FKBP77, FKBP73, and 26S rRNA probes. Scale of green shoots (D) and roots (E) represents 100% for the transcripts abundance of 2-d-old green shoots exposed for 2 h at 37°C. Solid lines, wFKBP77; dashed lines, wFKBP73.

Figure 7

Western blot of proteins (20 μg/lane) extracted from embryos that were exposed at 25°C for 2 h (lane 1) or at 37°C for 15 min (lane 2), 30 min (lane 3), 60 min (lane 4), or 120 min (lane 5). For recovery the embryos that were exposed for 2 h at 37°C were incubated at 25°C for an additional 4 h (lane 6), 6 h (lane 7), 8 h (lane 8), or 24 h (lane 9). The blot was immunodecorated with the polyclonal antibodies raised against recombinant wFKBP73 (anti 73).

Figure 8

Western blot of proteins (20 μg/lane) extracted from embryos exposed to 37°C for 2 h and immunodecorated with the polyclonal anti 73 or with polyclonal antibodies produced against a synthetic peptide of 24 amino acids from the C terminus of wFKBP77 (anti HS24C).

Figure 9

Expression of wFKBP77 and wFKBP73 in various tissues. Western blot of proteins (20 μg/lane) extracted from pistils (Pis), anthers (Ant), embryos that had been allowed to imbibe for 24 h (Emb), shoots (S), or roots (R) that had been allowed to imbibe for 2 d, and leaves (L) or roots (R) that had been allowed to imbibe for 6 d exposed to 25°C or 37°C. The antibodies anti 73 and anti HS24C were used to immunodecorate the blots.

Figure 10

Expression of wFKBP77 in wheat immature embryos exposed to heat stress. Western blot of proteins (80 μg/lane) extracted from immature embryos (18–45 d after anthesis) exposed to 37°C for 2 h and immunodecorated with anti HS24C or anti HVA1 antibodies.