Recovery of heat shock-triggered released apoplastic Ca2+ accompanied by pectin methylesterase activity is required for thermotolerance in soybean seedlings

Abstract

Synthesis of heat shock proteins (HSPs) in response to heat shock (HS) is essential for thermotolerance. The effect of a Ca(2+) chelator, EGTA, was investigated before a lethal HS treatment in soybean (Glycine max) seedlings with acquired thermotolerance induced by preheating. Such seedlings became non-thermotolerant with EGTA treatment. The addition of Ca(2+), Sr(2+) or Ba(2+) to the EGTA-treated samples rescued the seedlings from death by preventing the increased cellular leakage of electrolytes, amino acids, and sugars caused by EGTA. It was confirmed that EGTA did not affect HSP accumulation and physiological functions but interfered with the recovery of HS-released Ca(2+) concentration which was required for thermotolerance. Pectin methylesterase (PME, EC 3.1.1.11), a cell wall remodelling enzyme, was activated in response to HS, and its elevated activity caused an increased level of demethylesterified pectin which was related to the recovery of the HS-released Ca(2+) concentration. Thus, the recovery of HS-released Ca(2+) in Ca(2+)-pectate reconstitution through PME activity is required for cell wall remodelling during HS in soybean which, in turn, retains plasma membrane integrity and co-ordinates with HSPs to confer thermotolerance.

Fig. 1.

Thermotolerance is abolished by EGTA treatment during the recovery after HS but restored by Ca²⁺. Two-day-old etiolated soybean seedlings with an embryonic axis of ∼1.5 cm were tested. (A) HS regimen is shown on the top and inside the panel. Treatments 1–5 and 12 were used as references. Thermotolerance was lost with treatment at 40HS→28C+EGTA→45HS (treatment 6) and restored by adding Ca²⁺, Sr²⁺ or Ba²⁺ (treatments 7–9, respectively); Mg²⁺ was less effective (treatment 10), and K⁺ was not effective (treatment 11). Seedlings were grown at 28 °C in a dark growth chamber for an additional 72 h after treatment, and the length was measured. Data are means ±SD of three independent replicates, with 30 seedlings for each treatment. a and b represent significantly different values (P <0.05). (B) Typical seedlings were photographed at 72 h after treatment as indicated in (A). An asterisk indicates that the treatment is lethal.

Fig. 2.

EGTA treatment increases cellular leakage of electrolytes, amino acids, and sugars and is counteracted by treatment with Ca²⁺. Treatments are indicated inside the panel. Data are means ±SD of three independent replicates, with 30 seedlings for each treatment. An asterisk indicates that the treatment is lethal.

Fig. 3.

Atomic absorption spectrometry of Ca²⁺ and K⁺ leakage in response to HS and EGTA treatment. Treatments are indicated inside the panel. Data are means ±SD of 3–5 independent replicates, with 25 seedlings for each treatment. a, b, and c represent significantly different values (P <0.05).

Fig. 4.

HS-triggered release of Ca²⁺ and its recovery is required for the development of thermotolerance. Treatments are indicated inside the panel. The EGTA incubation medium was replaced with Milli-Q-purified water every 30 min during the 2 h 28 °C recovery period after 40HS (treatment 5, indicated by an asterisk). After treatment, seedlings were grown at 28 °C in a dark growth chamber for an additional 48 h, and the length of seedlings was measured. Data are means ±SD of three independent replicates, with 30 seedlings for each treatment. a and b represent significantly different values (P <0.05).

Fig. 5.

Effect of EGTA on class-I sHSP accumulation, and organelle association and dissociation in response to HS. Treatments are indicated inside the panel. Proteins were extracted by (A) SDS-extraction buffer or (B) were collected from the post-ribosomal supernatant (PRS) after treatment. Extracted proteins were separated by SDS-PAGE, then analysed for class-I sHSP content. 40HS treatment, as an internal standard, contains 0.98 μg class-I sHSP 100 μg^-1 of total proteins is sufficient to confer thermoprotection. Data are means ±SD of three independent replicates.

Fig. 6.

Thermostabilization of [³⁵S]-labelled proteins by addition of a class-I sHSP-enriched fraction from different treatments. Treatments are indicated inside the panel. The thermodenatured proteins were quantitated as described in the Materials and methods. The values relative to the 28C control (100%) were presented. Data are means ±SD of three independent replicates.

Fig. 7.

Pectin methylesterase (PME) and polygalacturonase (PG) activities are affected by HS and EGTA treatment. Treatments are indicated inside the panel. (A) PME activity was analysed by acidic continuous native-PAGE. One typical stained gel is shown (top panel), and the relative activity of PME to 28C control treatment was quantified (bottom panel). (B) Enzymatic analysis of PG was by colorimetric assay. Data are means ±SD of three independent replicates.

Fig. 8.

Ruthenium red staining characterizes pectic substances in hypocotyl parenchymal cells under HS and EGTA treatment. Treatments were (A) 28C, (B) 40HS, (C) 40HS→28C, (D) 40HS→28C+EGTA, (E) 40HS→28C→45HS, (F) 40HS→28C+EGTA→45HS, and (G) 40HS→28C+EGTA+Ca²⁺→45HS. (H) 28C control treated with 1 N NaOH before staining. (I) 28C+EGTA treatment. Scale bars, 100 μm.

Fig. 9.

Immunolocalization of the egg-box structures in hypocotyl parenchymal cells under HS and EGTA treatment. Treatments were (A) 28C, (B) 40HS, (C) 40HS→28C, (D) 40HS→28C+EGTA, (E) 40HS→28C→45HS, (F) 40HS→28C+EGTA→45HS, and (G) 40HS→28C+EGTA+Ca²⁺→45HS. (H) 28C without prior NaOH treatment, (I) 28C+EGTA, and (J) 28C without the primary antibody were controls. Except for specimens in (H), all specimens were incubated with 50 mM NaOH before immunolocalization. The egg-box structures are indicted by arrows. Scale bars, 50 μm.