Detecting freeze injury and seasonal cold-hardening of cells and tissues in the gall fly larvae, Eurosta solidaginis (Diptera: Tephritidae) using fluorescent vital dyes

This study identified a hierarchy in levels of cold tolerance for diverse tissues from larvae of Eurosta solidaginis. Following freezing at -80 degrees C, larval survival and the viability of specific tissues were assessed using membrane-permeant DNA stain (SYBY-14) and propidium iodide.Integumentary muscle, hemocytes, tracheae, and the crystal-containing portion of the Malpighian tubules were most susceptible to freezing injury. A second group consisting of fat body, salivary glands, and the proximal region of the Malpighian tubules were intermediate in their susceptibility, while the foregut, midgut, and hindgut were the most resistant to freezing injury. Seasonal increases in larval cold tolerance were closely matched by changes in the cold tolerance of individual tissues. Compared to larvae collected in September, the survival rates for each of the six tissues tested from October-collected larvae increased by 20-30%. The survival rate in all tissues was notably higher than that of whole animals, indicating that larval death could not be explained by the mortality in any of the tissues we tested. This method will be useful for assessing the nature of chilling/freezing injury, the role cryoprotectants, and cellular changes promoting cold tolerance.     

Acquisition of freezing tolerance in early autumn and seasonal changes in gall water content influence inoculative freezing of gall fly larvae,Eurosta solidaginis (Diptera,Tephritidae)

We examined seasonal changes in freeze tolerance and the susceptibility of larvae of the gall fly, Eurosta solidaginis to inoculative freezing within the goldenrod gall (Solidago sp.). In late September, when the water content of the galls was high (approximately 55%), more than half of the larvae froze within their galls when held at -2.5 degrees C for 24 h, and nearly all larvae froze at -4 or -6 degrees C. At this time, most larvae survived freezing at > or = -4 degrees C. By October plants had senesced, and their water content had decreased to 33%. Correspondingly, the number of larvae that froze by inoculation at -4 and -6 degrees C also decreased, however the proportion of larvae that survived freezing increased markedly. Gall water content reached its lowest value (10%) in November, when few larvae froze during exposure to subzero temperatures > or = -6 degrees C. In winter, rain and melting snow transiently increased gall water content to values as high as 64% causing many larvae to freeze when exposed to temperatures as high as -4 degrees C. However, in the absence of precipitation, gall tissues dried and, as before, larvae were not likely to freeze by inoculation. Consequently, in nature larvae freeze earlier in the autumn and/or at higher temperatures than would be predicted based on the temperature of crystallization (T(c)) of isolated larvae. However, even in early September when environmental temperatures are relatively high, larvae exhibited limited levels of freezing tolerance sufficient to protect them if they did freeze.     

Effect of Cold Hardening on SOD and Glutathion Reductase Activities and on the Contents of the Reduced Form of Glutathion and Ascorbic Acid in Rice and Cucumber Seedlings

Rice and cucumber seedlings were employed in the study on changes of superoxide dismatase (SOD) and glutathion reductase (GR) activities, the contents of the reduced form of glutathion (GSH) and ascorbic acid (ASA) in leaves and the chilling resistance as well as the level of lipid peroxidation products after cold hardening and chilling stress under light. The seedlings hardened under a day/night temperature variation of 15 ℃/10 ℃ and photon flux density (PFD) of 250μmol · m-2 · s-1 for 12 h/d indicated an increase of the activities of SOD and GR, and the contents of GSH and AsA. The resistance of the seedlings to chilling and light stress was enhanced by cold hardening. Under the stress condition, the stabilities of SOD and GR activities, and contents of GSH and AsA in hardened seedlings were higher than those in the unhardened seedlings; the lipid peroxidation was also less than that in the latter. It was thus concluded that cold-hardening under appropriate light leads on to the enhancement of function of membrane protective system and increase of cell membrane stability which is an important part of chilling-resistance mechanism in the plant.     

Calcium Effect on the Process of Cold-Hardening of Rice Seedlings

Rice seedlings for cold-hardening were germinated from seeds treated with or without immersion in 30 mmol/L of CaCl2 solution. Change of membrane system protectivity in leaves at various periods (viz: following cold-hardening, chilling stress and on the 3rd day of recovery); the ratio of seedling survival and the ability of recovery were investigated. The results showed that cold-hardening increased the contents of endogenous antioxidants (the reduced form of glutathione, GSH; ascorhic acid, ASA), SOD activity and the content of heat stable protein in soluble protein. Cold-hardening combined with the CaC12 treatment of seeds enhanced the above-mentioned effects of cold-hardening, and obviously increased the activities of catalase (CAT) and peroxidase (POD). Both cold-hardening with or without the CaCl2 treatment reduced the declining degree of GSH, AsA content, the SOD activity and the heat stable protein content caused by chilling stress, augmented the increase in the level of GSH, AsA, CAT, SOD, POD and heat stable protein in the recovery periods. The CaC12 pretreatment especially had more effect on the level of heat stable protein, augmenting the A protein to rise to the level in normal seedlings. Furthermore, cold-hardened seedlings from seeds pretreated with CaClz grew faster and better in the recovery period of chilling stress than those from seeds without CaClz pretreatment. This promotive effect of CaC12 treatment on seeds prior to cold-hardening of seedling could be associated with an aug- mentation of membrane protectivity induced by the combined treatment.     

Cold-hardening-induced resistance to photoinhibition of photosynthesis in winter rye is dependent upon an increased capacity for photosynthesis

Analyses of chlorophyll fluorescence and photosynthetic oxygen evolution were conducted to understand why cold-hardened winter rye (Secale cereale L.) is more resistant to photoinhibition of photosynthesis than is non-hardened winter rye. Under similar light and temperature conditions, leaves of cold-hardened rye were able to keep a larger fraction of the PS II reaction centres in an open configuration, i.e. a higher ratio of oxidized to reduced Q_A (the primary, stable quinone acceptor of PSII), than leaves of non-hardened rye. Three fold-higher photon fluence rates were required for cold-hardened leaves than for non-hardened leaves in order to establish the same proportion of oxidized to reduced Q_A. This ability of cold-hardened rye fully accounted for its higher resistance to photoinhibition; under similar redox states of q_a cold-hardened and non-hardened leaves of winter rye exhibited similar sensitivities to photoinhibition. Under given light and temperature conditions, it was the higher capacity for light-saturated photosynthesis in cold-hardened than in non-hardened leaves, which was responsible for maintaining a higher proportion of oxidized to reduced Q_A. This higher capacity for photosynthesis of cold-hardened leaves also explained the increased resistance of photosynthesis to photoinhibition upon cold-hardening.Abbreviations F_m and F'_m fluorescence when all PSII reaction centres are closed in dark- and light-acclimated leaves, respectively - F_o and F'_o fluorescence when all PSII reaction centres are open in darkness and steady-state light, respectively - F_v variable fluorescence (F'_m-F'_o) under steady-state light conditions - F_v/F_m the ratio of variable to maximum fluorescence as an expression of the maximum photochemical yield of PSII in dark-acclimated leaves - Q_A the primary, stable, quinone electron acceptor of PSII - q_N non-photochemical quenching of fluorescence due to high energy state (pH) - q_p photochemical quenching of fluorescence - RH cold-hardened rye - RNH non-hardened rye This work was supported by a Natural Sciences and Engineering Research Council of Canada (NSERCC) Operating Grant to N.P.A.H. G.Ö. was supported by an NSERCC International Exchange Award and by the Swedish Natural Science Research Council.     

Effect of Cold-Hardening on Thylakoid Membrane Lipids and Proteins of Spring Wheat and Winter Wheat

By comparison of thylakoid membrane lipids and their fatty acid composition, the supermolecular structure of light harvesting chlorophyll a/b-protein complex of Photosystem Ⅱ (LHC Ⅱ ) and the spectroscopic characteristics of thylakoids in winter wheat (Yanda 1817) with those in spring wheat (8901) before and after cold-hardening, it was found that after cold-hardening: (1)The trans-3-hexadeeenoic acid content of phosphatidyl alycerol (PG) in both cultivars decreased significantly, the ratio of monogalactosyl diglyceride (MGDG)/digalactosyl diglyceride (DGDG) in the thylakoid of Yanda 1817 decreased, but had no distinct change in 8901. (2)The lipid/chlorophyll ratio in thylakoids of Yanda 1817 increased significantly, but had no distinct change in 8901. (3) The LHC Ⅱ oligomer content decreased in thylakoids of both cultivars. (4) The A683/A652 ratio of the 4th derivative absorption spectra increased in both cultivars. (5)The F685/F738 ratio of low temperature (77K) fluorescence spectra of thylakoids in 8901 increased but was not affected in Yanda 1817. It was concluded that one of the major strategies of wheat to adapt low temperature was the increase of thylakoid membrane fluidity, and that the decrease of MGDG content may play an important role in stabilizing the bilayer structure of the thylakoid membrane at low temperature.