Imbibitional Chilling Injury in Pollen

Chilling injury is sustained by dry pollen of Typha latifolia L. upon hydration in germination medium at 0°C. This injury is evidenced as poor germination, low vigor, and depressed respiration. Isolated mitochondria showed multiple sites of impaired electron transport. Besides losses of cytochrome (Cyt) c and NAD+, the activities of membrane-bound enzyme complexes such as Cyt oxidase, NADH-duroquinone oxidoreductase, succinate-duroquinone oxidoreductase, and malate-duroquinone oxidoreductase were severely affected. Similarly, as in isolated mitochondria, in situ tests of mitochondrial activity showed that Cyt c was partially lost from its site of action. Readdition of the lost Cyt c to the grains restored the N,N,N',N'-tetramethyl p-phenylenediamine dihydrochloride plus ascorbate-mediated electron transport from Cyt c to 02, but did not significantly accelerate the overall 02 uptake. Electron flow to duroquinone in the injured grains was low, indicating that lesions at the substrate side of ubiquinone determine the rate of 02 consumption. Leakage of NAD', and also of adenylate phosphates and Krebs cycle substrates out ofthe injured gains, was considerable. Increasing the initial moisture content of the grains strongly enhanced their resistance to cold hydration. Below 17% moisture content (fresh weight basis), the decrease in vigor closely matched the loss of NAD' and adenosine phosphates. Vitality was irreversibly lost by cold hydration below 10 to 12% initial moisture content. Injury to dry pollen was prevented by imbibition at 27°C. Decrease of vigor and increased leakage, however, started below 20°C, and complete loss of vitality occurred below 10°C. These results are interpreted as evidence that loss of membrane integrity is the primary cause of imbibitional chilling injury. Imbibition of various seed species at unfavorably low temperatures leads to loss of vitality or reduced vigor during further germination at favorable temperatures (6). Seeds of low initial moisture content are particularly sensitive to cold hydration. Cold imbibition periods as short as 5 min reduce germination and subsequent elongation of embryonic axes of soybean seeds (2). Rapid imbibition by dry seed tissues, even at nonchilling temperatures, can cause cellular damage ( 19). Protection against the damaging effect of rapid uptake of cold liquid water may be obtained by first allowing the initial moisture content to increase by exposure to water vapor (3, 17, 18), or by slowing the water uptake by an osmoticum (19, 29). Similarly, imbibitional lysis of low moisture yeast cells has been prevented by equilibration in water vapor prior to cold hydration (27). Permanent address: Department of Plant Physiology of the Agricultural University, Arboretumlaan 4, 6703 BD Wageningen, The Netherlands. Reduction of the initial moisture content of fresh lily pollen by freeze-drying leads to reduced vitality and respiration upon reimbibition in germination medium (7). Germination percentages of many air-dry pollen species at room temperature are considerably increased by equilibration in moist air prior to incubation in a suitable in vitro medium (10, 14, 23). It is evident, then, that pollen, like seeds, can be damaged by rapid water entry. Besides a marked stimulation of the leakage of solutes, imbibitional chilling is reflected in a low rate of respiration in seeds during their poor germination at 25°C (13, 28). The low respiration is mainly due to reduction of the activity of the Cyt route; both the leakage and the low respiration are attributed to membrane lesions (13). In an attempt to determine whether the site of the injury was at the mitochondrial membrane, Cohn and Obendorf (6) measured radicle growth, 02 uptake, ATP contents, and some mitochondrial parameters of corn embryos, but they could not demonstrate an involvement of mitochondrial properties. In contrast, Duke et al. (8) found that imbibitional chilling caused changes in the RC ratio and in the activity of several dehydrogenases in soybean embryo mitochondria, isolated after 48 h of germination. These discrepancies are most likely due to the complexity of the seed, consisting of different multicellular tissues which take up water at different rates. Pollen offers substantial advantages for chilling experiments. Because of its small size, imbibition occurs very rapidly. When mitochondria are isolated, a homogeneous population is obtained, practically completely originating from the vegetative cell. Further, techniques of estimating mitochondrial capacities in situ have been recently developed, allowing for the detection of mitochondrial failure without isolation (1 1). In the present study, we have analyzed the extent of the injury to the respiratory system resulting from imbibition of dry pollen in cold germination media. Respiration in isolated mitochondria as well as mitochondrial functioning in situ were determined in relation to the leakage patterns of some important respiratory components. The measurements indicate that the low respiratory activity in injured pollen is a consequence of the loss of integrity of mitochondrial membranes and plasmalemma. MATERIAILS AND METHODS Plant Material and Treatments. Pollen was sieved from the male flowers of Typha latifolia L., collected from field populations in the neighborhood of Wijchen, the Netherlands, in JuneJuly. Pollen was air-dried to water contents of about 7% (on a fresh weight basis) and then stored at -25°C. If not otherwise stated, the pollen was rehydrated to about 0.5 g water/g dry weight by exposure to a humid atmosphere for 16 h at 4°C, prior to germination in vitro. The pollen was generally germinated at a density of 10 mgml-' in germination medium with vigorous shaking at 23°C. The germination medium of Hoekstra and van Roekel (1 1) was used. Tube emergence and growth were examined by light microscopy on 100 grains/sample at intervals during germination. If necessary, samples were temporarily stored in fixative. 815 www.plantphysiol.org on December 30, 2017 Published by Downloaded from Copyright © 1984 American Society of Plant Biologists. All rights reserved. Plant Physiol. Vol. 74, 1984 Isolation of Mitochondria. Isolation medium, disruption method, and filtration of the crude suspension were performed as described earlier (1 1). Mitochondria were obtained by differential centrifugation following procedures previously outlined in (1 1). Damaged mitochondria, however, required the higher centrifugal force of 30,000g for 10 min for pelleting. Testing of Segments of the ETC.' The standard assay medium for mitochondrial activity was as reported earlier (1 1). TMPDmediated ascorbate oxidation, DHQ oxidation, NADH oxidation, succinate oxidation, and malate oxidation were measured polarographically, using a Clark-type O2 electrode in a 2-ml water-jacketed cell at 24°C. 02 uptake by intact grains was determined similarly, generally at a pollen density of 2 mg-ml-'. Methods of application of the various substrates and correction for nonspecific 02 uptake were similar as outlined previously (11). When the TMPD + ascorbate couple was employed in intact pollen, an extra 1 mm EGTA was added to the germination medium. Succinic dehydrogenase activity was measured spectrophotometrically as the decrease in A at 600 nm due to the reduction of DCIP (0.06 mM). Potassium succinate was the substrate (10 mM) and phenazine methosulfate (I mM) was used as the mediator. Azide and AA were used to block electron transport at the second and third site of phosphorylation. The thenoyltrifluoroacetone-sensitive activity of succinate-DQ oxidoreductase was also measured spectrophotometrically at 600 nm, using DCIP as the final electron acceptor. The concentration of DQ, which mediated the electron transfer to DCIP, was 0.5 mm, injected as an acetone solution (final concentration of the acetone, 0.5%, v/v). Azide and AA were also present in the reaction medium. Enzyme activities were calculated using an extinction coefficient of Erc'd,, = 21 mM-' *cm' for DCIP at 600 nm. Activities of NADH-DQ oxidoreductase, malate-DQ oxidoreductase, and Cyt oxidase were assayed as described earlier (1 1). An in situ estimate of the electron flow through ubiquinone was obtained by following the reduction of DCIP at 600 nm in the presence ofAA and azide with DQ as the electron mediator. The easy penetration ofDQ into the inner mitochondrial membrane, and of DHQ through exine, intine, plasmalemma, and outer mitochondrial membrane was demonstrated earlier (1 1). Omission of DQ in the assay resulted in no further reduction of DCIP after a short initial decrease of absorbance due to reducing compounds leaking from the pollen. During the assay the pollen in the germination medium was stirred continuously. Determination of Nicotinamide Adenine Dinucleotides. Extraction and determination ofNAD+ and NADH were accomplished as previously described (1 1), using 8 mg of pollen or medium equivalents for each extraction. Determination of Adenosine Phosphates. Extraction of adenosine phosphates and enzymic conversion of ADP and AMP into ATP were essentially as outlined by Hoekstra (9), except for the use of Tricine buffer instead of Hepes. ATP was determined using the luciferin-luciferase method, by measuring bioluminescence in a LKB 1250 luminometer over 10 s. In each extract ATP (=XI), and the converted mixtures of [ATP + ADP] (=X2), and [ATP + ADP + AMP] (=X3) were assayed three times. Samples with and without internal standard were counted alternately. Care was taken so that the ATP concentration of the internal standard exceeded that of the extract in order to avoid 2 Abbreviations: ETC, electron transport chain; AA, antimycin A; BSTFA, bis(trimethylsilyl)trifluoroacetamide; CCCP, carbonyl cyanide in-chlorophenylhydrazone; DCIP, 2,6-dichlorophenolindophenol; DQ, duroquinone-2,3,5,6-tetramethyl-1,4-benzoquinone; DHQ, durohydroquinone, reduced form of DQ; EC, energ charge; TMPD, N,N,N',N'tetramethyl p-phenylenediamine dihydrochloride. inaccuracy due to statistical reasons. The EC is defined as (ATP + '/2ADP)/(ATP + ADP + AMP), and was calculated as (X, + X2)/2X3). Determination of Succinate, Fumarate, Malate, and Citrate. Samples containing 300 mg of pollen, or equivalent amounts of freeze-dried germination medium (containing phosphate buffer instead of citrate buffer) were boiled in 80% ethanol for 20 min, and then cooled on ice. After addition of glutaric acid as the internal standard, pollen and debris were removed by centrifugation at 1000g, and the ethanol was evaporated in vacuo. The watery fraction was passed through a 5-ml QAE-Sephadex column (plastic hypodermic), previously equilibrated according to Redgwell (21), and eluted with 30 ml H2O. The eluate contained the sugars. Organic acids were collected by eluting with 30 ml 4% HCOOH. After freeze-drying, the desiccated organic acids were dissolved in 1 ml dry acetone and transferred to a small glass vial. Following evaporation of the acetone, a further 30 ,l dry acetone and 10 ,l BSTFA were added for silylation at room temperature (minimum time, 10 min). A I-Ml sample was injected into a Hewlett-Packard 5730 A gas chromatograph equipped with a flame ionization detector and coupled to an electronic integrator. The glass column used (4 mm in diameter x 180 cm) was packed with 3% Dexsil 300 on 80/100 mesh CWAW-DMCS (12). The temperature program was 100 to 1600C at 2°C min-', total run time of 45 min. The carrier gas was N2 at 20 mlmin-'. The detector temperature was 300°C and the injector temperature was 1 50°C. Quantitation was achieved by a standard mixture of succinic acid, fumaric acid, glutaric acid, malic acid, and citric acid. Adjustment of Initial Moisture Content. Elevated moisture contents in pollen were established by equilibration for 17 h in a humid atmosphere at 20°C, followed by slow drying to the desired level. This procedure was preferred over moisture adjustment using glycerol-water mixtures, as glycerol vapor was suspected to interfere with the samples. Moisture contents were determined by weighing 250-mg samples before and after drying at 1 10°C for 4 h. All moisture contents are expressed on a fresh weight basis. Protein Determination. Protein was determined according to Bradford (1), using Coomassie Brilliant Blue G-250. Dilutions were made in phosphate-buffered saline containing 0.1% Triton X-100. Chemicals. Dexsil 300 on 80/100 mesh CW-AW-DMCS was obtained from Science Laboratories Inc., State College, PA, and BSTFA was from Aldrich. Durohydroquinone was from Pfaltz & Bauer. Other chemicals were obtained from Sigma.