Heat sensitivity in a bentgrass variant. Failure to accumulate a chloroplast heat shock protein isoform implicated in heat tolerance

Abstract

Two variants of creeping bentgrass (Agrostis stolonifera cv palustris), developed using tissue culture, have been used to determine the roles of chloroplast-localized small heat shock proteins (CP-sHSPs) in heat tolerance. Results from previous research indicate that the heat-tolerant variant expressed two additional CP-sHSP isoforms not expressed in the heat-sensitive variant, that accumulation of the additional CP-sHSP isoforms was genetically linked to thermotolerance, and that the presence of the additional isoforms in the heat-tolerant variant provided greater protection to photosystem II during heat stress. To determine the basis of the differential expression, we isolated the genes encoding the CP-sHSPs from both variants and characterized their structure and expression. Two genes, ApHsp26.2 and ApHsp26.7a, were isolated from the heat-tolerant variant, and three genes, ApHsp26.2m, ApHsp26.8, and ApHsp26.7b, were isolated from the heat-sensitive variant. The sequence of ApHsp26.2m from the heat-sensitive variant was identical to ApHsp26.2, except for a point mutation that generated a premature stop codon. Therefore, the protein product of ApHsp26.2m did not accumulate in the heat-sensitive line. Mass spectrometry analysis confirmed that ApHsp26.2 encoded for the CP-sHSP isoforms unique to the heat-tolerant variant. An identical mutation was detected in one of the three parental lines used to develop the creeping bentgrass variants. This suggests that ApHsp26.2m was inherited from this parent and did not arise from a mutation that occurred during tissue culture. The presence of two isoforms encoded by the same gene might be due to differential processing of the N-terminal amino acids during or after import into the chloroplast.

Figure 1.

Deduced amino acid sequence alignment of ApHsp26.2, ApHsp26.2m, ApHsp26.8, ApHsp26.7a, and ApHsp26.7b. Sequences that differ from the consensus are shaded with gray. Consensus region I (I), consensus region I (II), Met-rich region III (III), and chloroplast transit peptide IV (IV) are double underlined. The arrow marks the N-terminal amino acid for the lower spot in regions 1 and 3 in Figure 5.

Figure 2.

Analysis of mRNA level of ApHsp26.2, ApHsp26.8, ApHsp26.7a/b, ApHsp16, and actin by RT-PCR. The RNA was isolated from SB heat shocked for 4 h at 40°C (1), SB control (2), NSB heat shocked for 4 h at 40°C (3), and NSB control (4).

Figure 3.

Genomic PCR analysis of SB, NSB, and the three Penncross parental lines [11(38)4, 9(38)5, and 10(37)4]. The primer sets used to amplify ApHsp26.2 or ApHsp26.2m (1), gene ApHsp26.8 (2), and gene ApHsp26.7a or ApHsp26.7b (3) were the same as those used for RT-PCR analysis.

Figure 4.

Immunoblot analysis of CP-sHSPs present in the three parental lines of Penncross. Total proteins were extracted from leaves of the parental lines 11(38)4, 9(38)5, and 10(37)4 that were heat shocked at 40°C for 4 h. Proteins were separated by two-dimensional gel electrophoresis, blotted, and probed with an antibody to the Met-rich region of the CP-sHSPs. The white box indicates the position of CP-sHSP that is absent in 9(38)5.

Figure 5.

Two-dimensional gel electrophoresis of total protein isolated from heat-shocked (at 37°C for 12 h) SB (SBH), heat-shocked NSB (NSBH), control SB (SBC), and control NSB (NSBC) leaf tissues. Proteins were stained with Coomassie Brilliant Blue. Six protein spots from three regions (1-3) were excised from the gel for protein identification. Insets, Magnified sections of the gels. Black arrows, CP-sHSPs isoforms present in both SB and NSB. White arrows, CP-sHSP isoforms present only in SB.

Figure 6.

The MALDI-TOF MS spectra of CP-sHSP isoforms from region 1 (A), region 2 (B), and region 3 of the two-dimensional gels shown in Figure 5. Asterisks indicate peaks matching the peptide finger prints of ApHSP265.