Comparative studies of thermotolerance: different modes of heat acclimation between tolerant and intolerant aquatic plants of the genus Potamogeton

Abstract

Background and aims: Molecular-based studies of thermotolerance have rarely been performed on wild plants, although this trait is critical for summer survival. Here, we focused on thermotolerance and expression of heat shock transcription factor A2 (HSFA2) and its putative target gene (chloroplast-localized small heat shock protein, CP-sHSP) in two allied aquatic species of the genus Potamogeton (pondweeds) that differ in survival on land.

Methods: The degree of thermotolerance was examined using a chlorophyll bioassay to assess heat injury in plants cultivated under non- and heat-acclimation conditions. Potamogeton HSFA2 and CP-sHSP genes were identified and their heat-induction was quantified by real-time PCR.

Key results: The inhibition of chlorophyll accumulation after heat stress showed that Potamogeton malaianus had a higher basal thermotolerance and developed acquired thermotolerance, whereas Potamogeton perfoliatus was heat sensitive and unable to acquire thermotolerance. We found two duplicated HSFA2 and CP-sHSP genes in each species. These genes were induced by heat shock in P. malaianus, while one HSFA2a gene was not induced in P. perfoliatus. In non-heat-acclimated plants, transcript levels of HSFA2 and CP-sHSP were transiently elevated after heat shock. In heat-acclimated plants, transcripts were continuously induced during sublethal heat shock in P. malaianus, but not in P. perfoliatus. Instead, the minimum threshold temperature for heat induction of the CP-sHSP genes was elevated in P. perfoliatus.

Conclusions: Our comparative study of thermotolerance showed that heat acclimation leads to species-specific changes in heat response. The development of acquired thermotolerance is beneficial for survival at extreme temperatures. However, the loss of acquired thermotolerance and plasticity in the minimum threshold temperature of heat response may be favourable for plants growing in moderate habitats with limited daily and seasonal temperature fluctuations.

Fig. 1.

Thermotolerance of Potamogeton species examined by chlorophyll bioassay. The change in chlorophyll content was used as an index of thermotolerance, which is calculated as the chlorophyll content of submerged, heat-treated leaves (water at 30–45 °C, 24 h) minus that of non-heated leaves (at 25 °C, control). Heat-stress injury in plants is indicated by the reduction of chlorophyll content. (A) P. malaianus cultivated under non-acclimation (C25: constant 25 °C) conditions was injured by heat shock above 40 °C, while plants cultivated under heat-acclimation (A30: alternating 25/30 °C and C30: constant 30 °C) conditions survived 40 °C heat shock. (B) P. perfoliatus cultivated under both non-acclimation and heat-acclimation conditions was injured by heat shock above 35 °C. Bars indicate the means of five replicates ± s.d. Columns with the same upper-case and lower-case letters are not significantly different at P < 0·05 in temperature series comparisons (Fisher's PLSD).

Fig. 2.

Multiple alignment of deduced amino acid sequences of duplicated HSFA2 from P. malaianus (Pm-HSFA2a1 and Pm-HSFA2a2) and P. perfoliatus (Pp-HSFA2a1 and Pp-HSFA2a2). Conserved structures of a class A-HSF are indicated. DBD, DNA-binding domain; HR-A/B, oligomerization domains; NLS, nuclear localization signal; AHA1 and AHA2, functional motifs (aromatic, hydrophobic and acidic amino acids). Amino acids identical to Pm-HSFA2a1 are shown as dots (.); gaps are indicated by a dash (-).

Fig. 3.

Phylogenetic tree of HSFA genes based on the nucleotide sequence of the DNA-binding domain. Duplicated HSFA2 from P. malaianus (Pm-HSFA2a1 and Pm-HSFA2a2) and P. perfoliatus (Pp-HSFA2a1 and Pp-HSFA2a2) form a monophyletic clade with high bootstrap support based on 1000 replicates. At, Arabidopsis thaliana; Ah, Arachis hypogaea; Ll, Lilium longiflorum; Os, Oryza sativa subsp. japonica; Sp, Solanum peruvianum. The GenBank accession numbers for the genes are shown in Supplementary Data Table S3.

Fig. 4.

HSE module of duplicated HSFA2 in Potamogeton. Three HSEs (open box), a TATA box (grey box) and the transcriptional start site (+1) are presumed to be within the promoter regions of HSFA2a1 and HSFA2a2 in P. malaianus and P. perfoliatus. Core consensus sequence head (nGAAn, nGAnn or nGnAn) and tail (nTTCn, nnTCn or nTnCn) modules in the active HSE are highlighted on a green or blue background, respectively. Active HSE motifs (palindromic repeats of the head and tail modules) were identified according to Nover et al. (2001). In the Pp-HSFA2a2 promoter, there is no perfect match to the HSE consensus sequences, and a long insertion is located within the HSE II region (532 bp: highlighted on a grey background).

Fig. 5.

Expression of duplicated HSFA2 (HSFA2a1, HSFA2a2) and CP-sHSP (HSP21a, HSP21b) in Potamogeton cultivated under non-acclimation conditions (C25: constant 25 °C). (A) RT-PCR analysis of submerged leaves during heat-shock treatment (water temperature at 35 °C for 0, 1, 2, 4 or 8 h). Active transcripts of the examined genes were induced by the treatment, except for HSFA2a2 in P. perfoliatus. For HSFA2a2, a 624-bp transcript was found to be the active transcript, while the 882- and 775-bp transcripts were spliced isoforms. Actin served as a loading control. (B) Structures of alternatively spliced HSFA2a2 isoforms (Type 1 and Type 2). Active HSFA2 transcripts have properly spliced mRNA sequences. Both isoforms retain an intron that includes a premature stop codon (arrowhead). The Type 2 isoform has the same structure as the Type 1, except for a 107-bp deletion in the middle of exon 2. Arrows indicate the positions of the primers used for gene-specific amplification (477C and 1320 in Supplementary Data Table S1). Numbers show the length (bp) of specific regions.

Fig. 6.

Heat-stress response of the HSFA2 and CP-sHSP genes in Potamogeton. Plants cultivated under non-acclimation (C25: constant 25 °C) and heat-acclimation (A30: alternating 25/30 °C) conditions were subjected to heat shock (0, 1, 2, 4 or 8 h). The expression levels of transcripts were determined using real-time PCR, and normalized to that of Actin (= 1). Significant differences between non-heated and heat-acclimated plants (asterisks) are observed at 40 °C in heat-tolerant P. malaianus (A) and at 30 °C in heat-sensitive P. perfoliatus (B) (P < 0·05, t-test for each comparison). Data are expressed as the mean values of three individual experiments (n = 3). Error bars indicate s.d.