Changing desiccation tolerance of pea embryo protoplasts during germination

Protoplasts were isolated from pea (Pisum sativum L. cv. Alaska) embryonic axes during and after germination to determine whether the loss of desiccation tolerance in the embryos also occurs in the protoplasts. At all times studied, protoplast survival decreased as water content decreased; however, the sensitivity to dehydration was less when the protoplasts were isolated from embryos that were still desiccation-tolerant (12 h and 18 h of imbibition) than when protoplasts were derived from axes that were sensitive (24 h and 36 h of imbibition). The water content at which 50% of the population was killed (WC50) increased throughout germination and early seedling growth for both the intact tissue and the protoplasts derived from them. Prior to radicle emergence, protoplasts were less desiccation-tolerant than the intact axes; however, protoplasts isolated from radicles shortly after emergence had lower WC50s than the intact radicles. A comparison of protoplast survival after isolation and dehydration in either 500 mM sucrose/raffinose or 700 mM sucrose revealed no difference in tolerance except at 24 h of imbibition, when protoplasts treated in the more concentrated solution had improved tolerance of dehydration. Although intact epicotyls are generally more desiccation-tolerant than radicles, protoplasts isolated separately from epicotyls and radicles did not differ in tolerance. Collectively, these data suggest that protoplasts gradually lose desiccation tolerance during germination, as do the orthodox embryos from which they were derived. However, even prior to radicle emergence, protoplasts display a sensitivity to progressive dehydration that is similar to that shown by recalcitrant and ageing embryos.     

Spatio-temporal changes in germination and radical elongation of barley seeds tracked by proteome analysis of dissected embryo, aleurone layer, and endosperm tissues

Germination of barley is accompanied by changes in water-soluble seed proteins. 2-DE was used to describe spatio-temporal proteome differences in dissected seed tissues associated with germination and the subsequent radicle elongation. Protein identification by MS enabled assignment of proteins and functions to the seed embryo, aleurone, and endosperm. Abundance in 2-DE patterns was monitored for 48 different proteins appearing in 79 gel spots at 8 time-points up to 72 h post imbibition (PI). In embryo, a beta-type proteasome subunit and a heat shock protein 70 fragment were among the earliest proteins to appear (at 4 h PI). Other early changes were observed that affected spots containing desiccation stress-associated late embryogenesis abundant and abscisic acid (ABA)-induced proteins. From 12 h PI proteins characteristic for desiccation stress disappeared rapidly, as did a putative embryonic protein and an ABA-induced protein, suggesting that these proteins are also involved in desiccation stress. Several redox-related proteins differed in spatio-temporal patterns at the end of germination and onset of radicle elongation. Notably, ascorbate peroxidase that was observed only in the embryo, increased in abundance at 36 h PI. The surprisingly early changes seen in the protein profiles already 4 h after imbibition indicate that germination is programmed during seed maturation.     

Changes in chromatin structure associated with germination of maize and their relation with desiccation tolerance

Morphological and physicochemical measurements of chromatin condensation were made on germinating maize (Zea mays L.) radicles to determine whether the loss of genetic activities that occurs during the loss of desiccation tolerance is linked to irreversible changes in chromatin condensation. Chromatin samples were compared at different stages of germination (0, 24 and 72 h after imbibition), before (control) and after 24 h of desiccation. Morphological changes in chromatin structure and condensation were characterized by a qualitative and quantitative electron microscope study of chromatin which was allowed to spread in 0.2 mol m^?3 EDTA and then laid on coated microscope grids. The experiments showed similar levels of chromatin condensation in quiescent embryos and 24-h-old radicles (desiccation-tolerant material). After 72 h of imbibition, when radicle emergence and desiccation intolerance had ceased, the chromatin underwent a major decondensation towards various lower order folded structures. Regardless of the desiccation tolerance stage, an in vivo drying treatment of 24- and 72-h-old radicles before chromatin extraction did not induce significant changes in the extent of condensation compared to their respective controls. Similar conclusions were drawn from measurements of several spectroscopy properties (absorbance ratios: A₂₆₀/A₂₄₀, A₂₆₀/A4₀₀; thermal denaturation, and linear electric dichroism) of chromatin fragments that were obtained after nuclease digestion and then dissolved in 0-2 mol m^?3 EDTA. In quiescent and 24-h-old material, chromatin fragments were poorly soluble but highly stable during thermal denaturation. Chromatin fragments were 3-5-fold more soluble and less thermally stable in 72-h-old material than in 24-h-old material. In vivo desiccation had no significant effects on these properties compared to the respective controls. Collectively these data suggest that desiccation did not induce irreversible changes in the condensation properties of chromatin. The likelihood that the decondensation process occurring during germination is linked to the loss of desiccation tolerance is discussed.     

Respiratory pathways in aged soybean seeds

Respiratory activities in soybean seeds (Glvcine max (L.) Merr. cv. Chippewa 64) have been examined in the first minutes after water imbibition and after three hours of imbibition, using either particles or intact axes. Cyanide and azide were utilized as inhibitors of the cytochrome oxidase pathway of respiration, and SHAM inhibitions were interpreted as effects on the alternative pathway, since in unaged axes inhibitions by SHAM were obtained only when respiratory activity had either been restricted with inhibitors of the cytochrome oxidase pathway or expanded by an uncoupler (CCCP). From the experiments with these inhibitors, it is suggested that unaged seeds utilize both respiration pathways in the cotyledon but only the cytochrome pathway in the axis. Accelerated aging causes a marked deterioration of respiration, especially that through the cytochrome pathway, and there is an associated engagement of the alternative pathway in the seed axis. It is suggested that the lowering of respiratory activity and the shift in respiratory pathways may play a major role in the decline of germinability and vigor.     

Respiratory changes with chilling injury of soybeans

The leakage of solutes from cotyledons of soybeans (cv. Chippewa 64) was markedly stimulated by a chilling treatment (1 to 4 C) during the 1st minute of imbibition, but chilling after even 1 minute of water uptake resulted in little or no leakage increase. The respiratory rate of soybean particles was reduced more than 60% if a chilling treatment (15 minutes at 1 to 4 C) was given during the first minutes of imbibition, and little or no reduction was obtained if the chilling treatment was begun at 5 to 15 minutes after the start of imbibition. Using KCN as an inhibitor of cytochrome oxidase pathway of respiration and salicylhydroxamic acid as an inhibitor of the alternative pathway, it was found that the chilling injury involved a major reduction in the cytochrome pathway in whole axes and cotyledons and an engagement of the alternative pathway of respiration in cotyledon tissue. The suggestion is made that the chilling injury involves lesions resulting from temperature stress during the reorganization of membranes with water entry, and that both the leakage and the respiratory effects are consequences of these membrane lesions.     

Cyanide-sensitive and Cyanide-resistant Respiration in the Germination of Cocklebur Seeds

Interrelation between the CN-sensitive cytochrome path and the CN-resistant, benzohydroxamic acid (BHM)-sensitive, or n-propylgallate (nPG)-sensitive alternative path in seed respiration during germination was examined using the nondormant upper and lower seeds of Xanthium pensylvanicum Wallr. The operation of both paths was required not only for normal germination of the lower seed but also for KCN- or NaN₃-induced germination of both. From the sensitivity to BHM of the germination response, it became obvious that the alternative path exerts its physiological activity as soon as it develops during the early period of water imbibition. Pretreatments with KCN and NaN₃ for promoting germination, strikingly decreased only the engagement of the cytochrome path in the subsequent respiration without affecting that of the alternative path. Nevertheless, no germination occurred without the operation of the cytochrome path. This suggested that excess operation of the cytochrome path is detrimental to germination, being maximal following the BHM-sensitive phase.     

Respiration during seed maturation

The contribution of cytochrome and alternative pathways to respiration during soybean [ Glycine max (L.) Merr. cv. Chippewa 64] seed formation and maturation was measured by oxygen consumption of embryonic axis and cotyledon tissues and of mitochondria isolated from immature seeds. Respiration rates (dry weight basis) declined duing the last month of seed maturation. Cyanide sensitive respiration (cytochrome pathway) accounted for 80 to 90% of total oxygen consumption throughout seed formation and maturation. The proportion of the total respiration resistant to 1 m M cyanide and inhibited by 1 m M salicylhydroxamic acid (alternative pathway) did not exceed 6%. A residual oxygen consumption, accounting for 5 to 15% of total respiration, persisted in the presence of both inhibitors. Likewise, virtually no alternative respiration was found in mitochondria prepared from immature seeds. The alternative respiratory pathway seems absent during soybean seed formation and maturation regardless of whether grown in the greenhouse or field.     

The effect of cold treatment during seed imbibition on lettuce radicle length and the uniformity of seedling emergence

Two experiments were conducted to determine how a period of exposure to 0 ^oC, between two periods of imbibition at 20 ^oC, affected radicle development and the uniformity of seedling emergence. In both experiments cold treatments, given 12 h after seed imbibition began, produced shorter radicles than those given at 0, 4, 8, 16 and 20 h. Increasing the duration of exposure to 0 ^oC gave longer radicles and reduced the spread of seedling emergence. It is concluded that lettuce seeds are particularly sensitive to a temperature change about 12 h after seed imbibition starts.     

Triadimefon reduces transpiration and increases yield in water stressed plants

The total free amino acid pools in radicles of watermelon seeds, investigated during imbibition of water at 25°C, were higher under the most (darkness) than under the least (continuous broad spectrum far-red light) favourable light regime for germination. When seeds were imbibed in an appropriate osmotic solution of PEG-6000 (fully suppressing germination), in darkness or under continuous red or far-red light, the biochemical analyses of the radicles after 1,2,3 and 4 days from the onset of imbibition show that while the total soluble sugar content remains rather constant in all treatments, significant changes are observed in the total free amino acid pools. After the first day, a considerable increase characterizes the "darkness" pool in contrast to a moderate one under red, while the "far-red" pool remains constant. Ultimately, at 4 days, the three pools are 190,142 and 123% of the 0 day radicle one. The qualitative free amino acid determination of the 4 day darkness and far-red pools shows a considerably increased percentage contribution of glutamic acid, arginine and citrulline in the "darkness" pool. The free amino acid increase in non-illuminated radicles may be correlated to germinability; moreover, it is evidently a phytochrome-mediated, pre-germinatory event, probably due to the hydrolysis of proteins (known to be rich in glutamic acid and arginine), stored in the radicle.     

The role of recovery of mitochondrial structure and function in desiccation tolerance of pea seeds

Mitochondrial repair is of fundamental importance for seed germination. When mature orthodox seeds are imbibed and germinated, they lose their desiccation tolerance in parallel. To gain a better understanding of this process, we studied the recovery of mitochondrial structure and function in pea (Pisum sativum cv. Jizhuang) seeds with different tolerance to desiccation. Mitochondria were isolated and purified from the embryo axes of control and imbibed-dehydrated pea seeds after (re-)imbibition for various times. Recovery of mitochondrial structure and function occurred both in control and imbibed-dehydrated seed embryo axes, but at different rates and to different maximum levels. The integrity of the outer mitochondrial membrane reached 96% in all treatments. However, only the seeds imbibed for 12 h and then dehydrated recovered the integrity of the inner mitochondrial membrane (IMM) and State 3 (respiratory state in which substrate and ADP are present) respiration (with NADH and succinate as substrate) to the control level after re-imbibition. With increasing imbibition time, the degree to which each parameter recovered decreased in parallel with the decrease in desiccation tolerance. The tolerance of imbibed seeds to desiccation increased and decreased when imbibed in CaCl(2) and methylviologen solution, respectively, and the recovery of the IMM integrity similarly improved and weakened in these two treatments, respectively. Survival of seeds after imbibition-dehydration linearly increased with the increase in ability to recover the integrity of IMM and State 3 respiration, which indicates that recovery of mitochondrial structure and function during germination has an important role in seed desiccation tolerance.     

Cyanide-Resistant Respiration in Relation to Fruit Respiratory Climacteric

Changes in respiratory rate and the effects of respiratory inhibitorson respiration were determined in apple (Malus sylvestris cv. Delicious) and red pepper (Capsicum fructescens) fruits dusting different stages of development and ripening.The results showed that there was an abrupt rise in respiration daring ripening inapple fruit, but the respiration of the red pepper declined continuously throughout theripening period. Thus the apple is climacteric and the red pepper is non-climacteric fruit. The respiration of apple fruit was sensitive to KCN (1 mM) during the period ofdevelopment but changed to CLAM-sensitive and CN-resistant during preclimactericand climacteric phases, indicating that a diversion of respiratory pathways from the cy-tochrome path to the alternative path has occurred. The respiration of the red pepperfruit was CN-sensitive thoughout the whole period of fruit ripening, suggesting thatthe operation of the CN-resistant path was insignificant. Slices from climacteric apple fruits developed induced .respiration after aging, bothKCN and CLAM (1 mM) inhibited the induced respiratic considerably. However, slices from red pepper fruits showed no evidence of induced respiration after aging. Slices from climacteric apple fruits infiltrated with 3 mM CLAM before aging, reducedthe peak of the induced respiration by about 30%, indicating that the development ofinduced respiration was suppressed by the presence of CLAM. The above results indicated that the: climacteric fruits were characterized by diversion of traffic from the cytochrome path to the alternative path during ripening andby the development of induced respiration after slicing and aging. While in nonclimacteric fruits no .diversion of electron transport path was observed during ripening andno induced respiration occurred after aging. Although both the eytochrome and alternative pathways were present in the tissue of red pepper fruits, the alternative pathwas not operating except when the cytochrome path was blocked or was saturated by electron flow.     

Pepper seed germination assessed by combined X-radiography and computer-aided imaging analysis

A lot of pepper seeds having 87 % germination were subjected to X-ray inspection using a non lethal dose of radiation. Seeds with less than 2.7 % (on the basis of total seed area) of free space area, i.e. the spaces between embryo and endosperm, were classified as highly viable seeds (97–100 % germination) with the lowest level of abnormal seedlings. Seeds X-ray classified as good were subjected to a computerised image analysis to study seed imbibition and radicle elongation. The patterns of seed area increase, chosen as the most accurate indicator of seed swelling, resembled the triphasic curve of water uptake. The first phase was completed at 9 h followed by a second phase that varied widely in time until completion of germination between 52 and 96 h. The proportion of seeds with radicle protrusion between 52–56 h and 64–72 h assessed with the image analysis was significantly higher than that recorded using a conventional germination test. In addition, the rate of increase of seed area during the third phase of imbibition, mostly due to protrusion of the radicle tip and its growth, was highly correlated with the corresponding radicle elongation rate.     

能量对香石竹切花在瓶插期间呼吸电子途径的影响

采用外加0.1 mmol/L ATP或0.5 mmol/L二硝基苯酚(DNP)的方法,研究了香石竹(Dianthus caryophyllus L.)切花在25±1℃和80%～90%相对湿度下瓶插期间呼吸电子传递途径的变化情况.结果表明,香石竹切花在瓶插7 d时的呼吸速率达到高峰;ATP处理明显加快切花的呼吸速率,在瓶插7 d时呼吸速率高峰值较对照(未用ATP处理)升高1倍.细胞色素途径与总呼吸活性存在显著正相关.细胞色素途径占总呼吸的比重在切花瓶插4 d后上升,并且线粒体电子传递主要依靠细胞色素主路途径进行.经ATP处理后香石竹切花的交替呼吸途径的容量、实际运行活性和运行系数明显增加;交替呼吸途径占总呼吸活性比重在瓶插4 d后迅速上升,并且交替呼吸途径容量与总呼吸活性存在显著正相关.而DNP处理则降低交替呼吸途径容量.这说明外源ATP处理加强了香石竹切花在整个瓶插期间的呼吸作用,增加了呼吸速率的高峰值,提高了抗氰呼吸作用.     

The appearance and characterization of cyanide-resistant respiration in the fungus Candida albicans

The respiration of yeast-form cells of the dimorphic fungus Candida albicans became resistant to cyanide during aging treatment in the resting state. An alternative, cyanide-resistant respiratory pathway was found to develop fully in cells aged at a concentration of 0.75 X 10(9)/ml or more at 25 C, but did not appear at 5 C. Chloramphenicol did not prevent the appearance of the alternative respiratory pathway. The effects of inhibitors, salicylhydroxamic acid (SHAM) and disulfiram (tetraethylthiuram disulfide), on respiration of aged cells were examined, and results indicated that SHAM binds at a site on the alternative respiratory pathway whereas disulfiram binds at two sites, one on the conventional respiratory pathway and the other on the alternative pathway. Thus, SHAM is a more selective inhibitor of the alternative respiration of C. albicans cells. SHAM-titration of the alternative respiration revealed that less than 10% of the maximal activity of the alternative respiratory pathway was utilized under normal conditions, indicating that the alternative respiratory pathway makes a small contribution to the total respiration. It was therefore concluded that the alternative, cyanide-resistant respiratory pathway operates fully when the cyanide-sensitive, cytochrome pathway is blocked although aged cells possess both respiratory pathways.     

The Effect of Changing Temperatures During Imbibition on Ultrastructure in Germinating Pea Embryonic Radicles

Hodson, M. J., Di Nola, L. and Mayer, A. M. 1987. The effectof changing temperatures during imbibition on ultrastructurein germinating pea embryonic radicles.—J. exp. Bot. 38:525–534. Pea seeds were imbibed at 5 °C or at 25 °C for 5–6h and then germinated at the same temperature or transferredto the other temperature for 15 h. After imbibition or germinationthe embryonic axis was removed, fixed and the ultrastructureof the radicle tip examined. Exposure of the seeds to 5 °Cduring imbibition resulted in the cessation of almost all ultrastructuralchanges during subsequent germination at 5 °C. When theseeds were transferred to 25 °C development of ultrastructurewas far slower than expected. In particular, mitochondrial structurefailed to develop, there were few Golgi and little ER, and lipidbodies located near the plasma membrane disappeared or fusedmuch more slowly with the membrane. An attempt is made to relatethe observations on ultrastructure with data previously reportedon the metabolism of phospholipids in the plasma membrane andER of the embryonic axis. A tentative hypothesis to accountfor the effect of temperature of imbibition on subsequent seedgermination is proposed. Key words: Pisum sativum L. cv. Alaska, germination, imbibition, temperature, ultrastructure, lipid bodies, protein bodies, vacuolation, mitochondria     

小麦幼苗叶片抗氰呼吸对轻度水分胁迫的响应

小麦幼苗经－０．５ＭＰa聚乙二醇（ＰＥＧ－６０００）溶液暗中渗透迫０、６、１２、１８、２４、３０、３６、４２、４８h,叶片的总呼吸速率（Ｖt)呈现先上升后降低的趋势,交替途径呼吸也表现出相同的变化模式。水分胁迫初期（０－１２h),交替途径容量（Ｖalt)、实际运行活性（ρValt)及运行系数（ρ值）均上升,此后（１８－４８h）逐渐下降,水分胁迫也影响了呼吸电子流在２条呼吸途径中的分配比例,胁迫初期的０－１２h内,流经交替途径的电子流增多,而流向细胞色素主路的电子流减少,但随着胁迫时间的延长,交替途径的贡献降低,而细胞色素主路的贡献增加,说明小麦叶片的抗氰呼吸在水分胁迫初期被诱导增加,而随着胁迫进行的延长又表现为下降。     

The regulation of respiration and growth of potato tuber callus by endogenous ethylene. Suppression of ethylene action by 2,5-norbornadiene

The growth (fresh and dry weight increase) of potato tuber ( Solanum tuberosum L. cv. Bintje) callus discs was stimulated by incubation in air with 500 ppm 2,5-norbornadiene (NBD, a competitive inhibitor of ethylene action) and inhibited by incubation in air with 4 000 ppm NBD. Ethylene formation by the callus was stimulated by NBD. The development of the alternative pathway, measured in isolated mitochondria was inhibited by NBD in a concentration-dependent way. The alternative pathway capacity, measured in vivo, was inhibited by 4 000 ppm NBD, but not by 500 ppm. Uninhibited in vivo respiration, which consists of cytochrome path activity and alternative path activity, was stimulated by the treatment with 500 ppm NBD. The main contribution to this stimulation was made by the cytochrome pathway. In 4 000 ppm NBD-treated callus, uninhibited respiration seemed to be unaffected as a consequence of an inhibited cytochrome path activity, which was compensated by a stimulated alternative path activity. Both in 500 and 4 OIK) ppm NBD-treated callus the alternative path activity in vivo was stimulated.
The regulatory role for endogenous ethylene in potato tuber callus is discussed in relation to: 1) The induction of respiratory pathways, 2) the supply of reduction equivalents in vivo and 3) growth.     

Induction of phenylalanine ammonia-lyase (PAL) in germinating lettuce seeds (Lactuca sativa)

Dry lettuce seeds (achenes of Lactuca sativa L. cv. Grand Rapids) contain no detectable phenylalanine ammonia-lyase (PAL, EC 4.3.1.5) activity. Enzyme activity could be detected in these seeds within 4 h of imbibition under white light. The specific activity of PAL increased rapidly during the next 12–16 h of imbibition. Far-red light completely suppressed germination as well as the development of PAL. Gibberellic acid (GA₃, 0.1 m M ), although effective in causing almost 100% germination in dark, did not induce proportionate increases in PAL. Seed germination as well as PAL activity were substantially inhibited by cis -4-cyclohexene-l, 2-dicarboximide (CHDC, 1.0 m M ) both in light and dark. Both GA₃ and benzyladenine (BA, 0.1 m M ) retarded radicle elongation in light. Concomitantly, a decrease in PAL activity was observed. Benzyladenine was able to reverse the effects of CHDC on germination but PAL activity was still highly reduced, probably due to the inhibitory effects of BA on elongation of the radicle. More than 95% of the extractable PAL was found to be present in the radicle. When seeds incubated in white light for 10 h were transferred to FR, further increases in PAL activity as well as the growth of the radicle were severely inhibited. It is suggested that the induction of PAL in light-sensitive lettuce seeds is coincidental with the germination of seeds, and the amount of PAL per germinated seed is related to the extent of elongation of the embryonic axes.