Ethylene induces antifreeze activity in winter rye leaves

Abstract

Antifreeze activity is induced by cold temperatures in winter rye (Secale cereale) leaves. The activity arises from six antifreeze proteins that accumulate in the apoplast of winter rye leaves during cold acclimation. The individual antifreeze proteins are similar to pathogenesis-related proteins, including glucanases, chitinases, and thaumatin-like proteins. The objective of this study was to study the regulation of antifreeze activity in response to ethylene and salicyclic acid, which are known regulators of pathogenesis-related proteins induced by pathogens. Nonacclimated plants treated with salicylic acid accumulated apoplastic proteins with no antifreeze activity. In contrast, when nonacclimated plants were exposed to ethylene, both antifreeze activity and the concentration of apoplastic protein increased in rye leaves. Immunoblotting revealed that six of the seven accumulated apoplastic proteins consisted of two glucanases, two chitinases, and two thaumatin-like proteins. The ethylene-releasing agent ethephon and the ethylene precursor 1-aminocyclopropane-1-carboxylate also induced high levels of antifreeze activity at 20 degrees C, and this effect could be blocked by the ethylene inhibitor AgNO(3). When intact rye plants were exposed to 5 degrees C, endogenous ethylene production and antifreeze activity were detected within 12 and 48 h of exposure to cold, respectively. Rye plants exposed to drought produced both ethylene and antifreeze activity within 24 h. We conclude that ethylene is involved in regulating antifreeze activity in winter rye in response to cold and drought.

Figure 1

Antifreeze activity of rye leaves treated to manipulate ethylene levels. Antifreeze activity was determined by observing the morphology of ice crystals grown in crude leaf apoplastic extracts of plants treated with 1 μL L⁻¹ ethylene for 120 h (Ethylene+) and its control (Ethylene−); 10 mm ethephon for 168 h (Ethephon+) with 2 mm HCl and 2 mm H₂PO₃ used as its control (Ethephon−); 10 mm ACC for 168 h (ACC+) and its control (ACC−); 200 μm salicylic acid (SA+) and its control (SA−) at 192 h; and 200 μm AgNO₃ (Ag+) and its control (Ag−) at 168 h. Ice crystals grown in extracts from plants that were grown under cold-acclimating conditions (CA) for 7 weeks or nonacclimating conditions (NA) for 3 weeks are shown for comparison. A representative crystal obtained from one of three independent experiments is shown. For all circular ice crystals, the basal plane is parallel to the plane of the page. For all hexagonal ice crystals, the basal plane is perpendicular to the plane of the page. Bar = 10 μm.

Figure 2

Time course of accumulation of apoplastic proteins in rye leaves treated to manipulate ethylene levels. Apoplastic proteins were extracted at 24-h intervals from the leaves of NA rye plants treated with 1 μL L⁻¹ ethylene (▪) or air (□) as a control inside a closed chamber (A), sprayed with 10 mm ethephon and 0.005% (v/v) Tween 20 (▪) or with 2 mm HCl, 2 mm H₂PO₃, and 0.005% (v/v) Tween 20 (□) as a control (B), sprayed with 10 mm ethephon and 0.005% (v/v) Tween 20 then watered with Hoagland solution containing 200 μm AgNO₃ (▪) or sprayed with 2 mm HCl, 2 mm H₂PO₃, and 0.005% (v/v) Tween 20 (□) then watered with Hoagland solution as a control (C), or sprayed with 10 mm ACC and 0.005% (v/v) Tween 20 (▪) or with 0.005% (v/v) Tween 20 as a control (□) (D). Total apoplastic proteins were measured using the Bio-Rad method with bovine serum albumin as the standard protein and are presented as the means ± se (n = 3). An asterisk indicates that the total apoplastic protein content of treated winter rye leaves was significantly different (P = 0.05) from the control leaves at that time point.

Figure 3

Examination of polypeptides present in apoplastic extracts of ethephon-treated winter rye leaves by SDS-PAGE and immunoblotting. A, For SDS-PAGE, apoplastic proteins were extracted at 24-h intervals from NA rye leaves sprayed with 10 mm ethephon (+), from NA leaves sprayed with 2 mm HCl/H₂PO₃ (−) as a negative control, and from CA leaves (CA) as positive control and were denatured, separated on 15% (w/v) polyacrylamide gels, and stained with Coomassie Brilliant Blue R-250 (Bio-Rad, Mississauga, Canada). An equal volume of each apoplastic extract per gram leaf fresh weight (30 μL) was loaded on each lane. For immunoblotting, SDS-polyacrylamide gels loaded with 10 μL per lane of each apoplastic extract were blotted and probed with antisera produced against the cold-induced winter rye 32-kD glucanase (B), the cold-induced winter rye 35-kD chitinase (C), and the cold-induced winter rye 25-kD TLP (D). Low-range prestained SDS-PAGE standards (M) were used in both SDS-PAGE and immunoblotting analysis to determine the molecular mass (kD). The molecular mass of each immunodetected polypeptide is indicated on the right.

Figure 4

Time course of ethylene produced endogenously by plants transferred to low temperature. Ethylene content was measured by gas chromatography with analyzed ethylene standards and is presented as the means ± se (n = 3). Antifreeze activity was also determined in leaf apoplastic extracts at various time points after the plants were transferred to cold temperature. Representative ice crystals are shown for each time point. The two crystals on the left are shown with the basal plane parallel to the plane of the page. The two crystals on the right are shown with the basal plane normal to the plane of the page. Bars = 5 μm.

Figure 5

Time course of ethylene produced endogenously by plants exposed to drought. Ethylene content was measured by gas chromatography with analyzed ethylene standards and is presented as the means ± se (n = 3). Antifreeze activity was also measured in leaf apoplastic extracts at various time points after the plants were subjected to water stress. Representative ice crystals are shown for each time point. The three crystals on the left are shown with the basal plane parallel to the plane of the page. The three crystals on the right are shown with the basal plane normal to the plane of the page. Bars = 5 μm.