Heat stress can have severe deleterious effects on embryo development and survival. The present study evaluated whether CSF2 can protect the developmental competence of the bovine embryo following exposure to a heat shock of 41°C at the zygote and morula stages. In the first experiment, putative zygotes and 2-cell embryos were assigned to receive either 10 ng/ml CSF2 or vehicle, and then cultured for 15 h at either 38.5°C or 41°C and then at 38.5°C until day 7.5. Heat shock reduced blastocyst development for embryos treated with vehicle but not for embryos cultured with CSF2. In the second experiment, day 5 embryos (morula) were treated with CSF2 or vehicle and then cultured for 15 h at either 38.5°C or 41°C and then at 38.5°C until day 7.5. Temperature treatment did not affect development to the blastocyst stage and there was no effect of CSF2 treatment or the interaction. Results indicate that CSF2 can reduce the deleterious effects of heat shock at the zygote or two-cell stage when the embryo is transcriptionally inactive.

Cistus species have seeds with hard coats which impose physical seed dormancy that can be released after seed scarification. In fire-prone habitats, the break of physical seed dormancy is usually related to the heat produced during fires. It is commonly accepted that most hard-seeded species, including those of the genus Cistus, are able to germinate under a wide range of temperatures in light as well as in darkness, once the seed becomes permeable. However, although many studies have focused on the release of physical dormancy only, a few have done so on the effect of environmental factors once dormancy is released. In this research, through a factorial experiment, we analysed the effects of light (light and darkness) and a range of temperatures (10, 15, 20, 25 and 30°C) on the seed germination of eight Cistus species after a heat shock. On average, almost 60% of the seeds did not germinate despite being viable, and this lack of germination increased with higher temperatures during the treatment. Although an idiosyncratic germination response emerged, temperature had a significant effect in all the species, reaching the highest levels of germination between 10 and 20°C. Light interacted with temperature in four cases by increasing the germination, especially under the least favourable temperatures. Environmental factors, such as temperature and light, appear to modulate the germination of the studied Cistus species after the release of physical seed dormancy.

Cadellia pentastylis (Surianaceae) is an Australian endemic threatened rainforest tree. Irregular flowering and fruiting events coupled with high rates of infertility and insect predation has meant that seed testing has not been possible for this species. Seeds were opportunistically collected from a wild population in early 2021, which allowed for the first germination tests to be conducted. In this study, the presence of physical dormancy was examined by performing an imbibition test using scarified and non-scarified seeds. We also investigated whether a 5-min heat shock treatment at temperatures ranging from 60 to 120°C improved germination success. The presence of physiological dormancy was also examined by recording germination success following a gibberellic acid or smoke-water pre-treatment. Both scarified and non-scarified seeds readily imbibed water over a 72-h period, and several seeds had germinated in both treatments after 48 h. Final germination proportion and t50 following a heat shock, gibberellic acid or smoke-water pre-treatment did not significantly differ from the controls. We conclude that C. pentastylis seeds are non-dormant. Although a palisade cell layer has been reported in the endocarp, our results suggest that this layer may not be sufficiently formed to restrict germination. We recommend that seeds are collected from populations following dispersal and propagated shortly after or stored as conservation collections in ex situ Seedbanks.

Triploidization plays an important role in aquaculture and surrogate technologies. In this study, we induced triploidy in the matrinxã fish (Brycon amazonicus) using a heat-shock technique. Embryos at 2 min post fertilization (mpf) were heat shocked at 38°C, 40°C, or 42°C for 2 min. Untreated, intact embryos were used as a control. Survival rates during early development were monitored and ploidy status was confirmed using flow cytometry and nuclear diameter analysis of erythrocytes. The hatching rate reduced with heat-shock treatment, and heat-shock treatments at 42°C resulted in no hatching events. Optimal results were obtained at 40°C with 95% of larvae exhibiting triploidy. Therefore, we report that heat-shock treatments of embryos (2 mpf) at 40°C for 2 min is an effective way to induce triploid individuals in B. amazonicus.

Fire is a frequent disturbance in the Cerrado and is one of the major factors affecting vegetation structure and diversity. Fire events open gaps within the herbaceous layer and increase temperature fluctuation in the soil surface. In addition to being an important environmental filter for germination, fire is a germination cue for species with physiological dormancy. This study aimed to evaluate the germination of native grasses, using daily temperature fluctuations and heat shock to overcome physiological dormancy in native grasses. We also evaluated seed longevity after dispersal for some species. We conducted the daily temperature fluctuation experiments on seeds of ten native grass species, which were collected and then placed in germination chambers simulating thermal fluctuation throughout the day (19–55°C). We also subjected seeds to different heat-shock treatments: 100°C for 1 min, 100°C for 3 min and 200°C for 1 min. To determine seed longevity, we stored seeds for 6 and 12 months after collection and then set them to germinate (27°C, 12/12 h light). Non-germinated seeds from all experiments were tested for viability. Most species had low longevity and germination percentages. Those that had physiological dormancy were stimulated to germinate when exposed to temperature fluctuations. One species resisted temperatures up to 200°C. For all other species, neither treatment affected germination percentages. Our results indicate the importance of these environmental filters for seedling recruitment of these species, considering the low longevity and the presence of physiological dormancy.

Heat shock may disrupt oocyte function by increasing the generation of reactive oxygen species (ROS). We evaluated the capacity of the antioxidant melatonin to protect oocytes using two models of oxidative stress – heat shock and the pro-oxidant menadione. Bovine cumulus–oocyte complexes (COC) were exposed in the presence or absence of 1 µM melatonin to the following treatments during maturation: 38.5°C, 41°C and 38.5°C+5 µM menadione. In the first experiment, COC were matured for 3 h with 5 µM CellROX® and analyzed by epifluorescence microscopy to quantify production of ROS. The intensity of ROS was greater for oocytes exposed to heat shock and menadione than for control oocytes. Melatonin reduced ROS intensity for heat-shocked oocytes and oocytes exposed to menadione, but not for control oocytes. In the second experiment, COC were matured for 22 h. After maturation, oocytes were fertilized and the embryos cultured for 7.5 days. The proportion of oocytes that cleaved after fertilization was lower for oocytes exposed to heat shock and menadione than for control oocytes. Melatonin increased cleavage for heat-shocked oocytes and oocytes exposed to menadione, but not for control oocytes. Melatonin tended to increase the developmental competence of embryos from heat-shocked oocytes but not for embryos from oocytes exposed to menadione or from control oocytes. In conclusion, melatonin reduced production of ROS of maturing oocytes and protected oocytes from deleterious effects of both stresses on competence of the oocyte to cleave after coincubation with sperm. These results suggest that excessive production of ROS compromises oocyte function.

This study investigated the breeding parameters and embryogenic development of diploid and heat shock-induced triploid eggs of Anabas testudineus (Bloch, 1792). To this effect, broodstocks of A. testudineus were induced to spawn using the Ovaprim® hormone. After fertilization, the eggs were divided into two groups and one portion heat shocked at 41°C (for 3 min), at approximately 4 min after fertilization. Results of fertilization, hatchability, as well as the sequence and timing of embryogenic development were collated from three breeding trials. Fertilization percentages were similar in both treatments (≈90%) while hatchability was higher in the diploid eggs (79.56%) than the triploid induced eggs (50.04%). Both treatments had the same sequence of embryogenetic stages; however, the timing of development was significantly delayed in the triploids (i.e. beyond the 2-cell stages) as compared with the observations in the control group (diploid eggs). Consequently, hatching time was 5 h faster in the diploid eggs [i.e. 18 hours post fertilization (hpf)] compared with the triploid induced eggs (23 hpf). The most critical stage of embryonic development in which mass mortality occurred in the different treatments was the somite stage. The status of triploid hatchlings was affirmed using erythrocyte morphology in 2-month-old fingerlings.

Little information is published related to seed germination and seedling emergence of Japanese foxtail, a troublesome annual grass weed widely distributed in winter wheat fields in China. Three Japanese foxtail populations were studied under laboratory and greenhouse conditions, to determine the effects of different environmental factors on seed germination or seedling emergence. Chemical control is absolutely necessary in integrated management, and efficacy of POST herbicides against different growth stages of Japanese foxtail was evaluated. Germination rate was 90% or more when temperature ranged from 5 to 25 C, with germination onset shortened as temperature increased. Light was not required for germination to occur. For pH values ranging from 5 to10 there was no effect on seed germination. Japanese foxtail seed germination was sensitive to osmotic stress and completely inhibited at an osmotic potential of -1.1 MPa. The ‘1513’ population of Japanese foxtail demonstrated tolerance to soil salinity, with 98% germination at 80 mM NaCl compared with 25 and 40% germination for populations ‘1532’ and ‘1544’, respectively. High amounts of crop residue (10 t ha−1) suppressed Japanese foxtail emergence 38 to 55%. Germination of seeds placed at 160 C for 5 min was completely inhibited for dry seeds, with a similar effect at 130 C for pre-soaked seeds. Seed burial in the soil from 0 to 4 cm had no effect on seedling emergence, but burial at 7 cm completely inhibited seedling emergence. POST herbicides mesosulfuron-methyl (13.5 g ai ha−1), clodinafop-propargyl (67.5 g ai ha−1), pyroxsulam (13.5 g ai ha−1), pinoxaden (67.5 g ai ha−1) and isoproturon (1125 g ai ha−1) reduced plant dry weight 80% or more when applied at three- to seven-leaf stage, but control declined with application at later growth stages. The information from this study helps to develop an integrated approach to Japanese foxtail management.

Many researchers are investigating the extreme resilience of bacterial endospores against chemical and physical inactivating agents. The presence of vegetative cells in spore suspensions can result in overly optimistic assessment of inactivating agents; therefore, various spore purification methods have been applied to separate spores from vegetative cells prior to testing. The present study was undertaken to evaluate the effect of two widely used spore purification methodologies on spore integrity and susceptibility to ultraviolet-A (UVA) radiation and free radicals generated from photocatalysts. Bacillus subtilis and Bacillus cereus spores were purified by procedures that involved heat shock alone or chemical washes, lysozyme treatment and heat shock (CLH). The purified spores were exposed to UVA radiation or free radicals generated by photocatalyst and susceptibility were evaluated in terms of survival ratio. The effect of purification procedure on the spore morphology was investigated with electron microscopy. The CLH purification process significantly damages spore coats and increases the susceptibility of Bacillus spores to UVA radiation and photocatalytic inactivation. It is therefore likely that the survival of CLH treated spores in extra-terrestrial environments would be less than that of the same spores purified by a less aggressive procedure.

The use of winter cover crops is beneficial to agriculture. We have tried to design a cover crop that self-destructs in response to an environmental cue, thereby eliminating the use of herbicides and tillage to remove the cover crop in late spring. Here, this novel concept is tested in a model system. The onset of summer brings with it elevated temperatures. Using this as the environmental cue, a self-destruction cassette was designed and tested in tobacco. A heat-shock–responsive promoter was used to direct expression of the ribonuclease Barnase. Because Barnase is extremely toxic to cells, it was necessary to coexpress its inhibitor, Barstar, whose expression was under the control of the CaMV 35S promoter. The wild-type and two mutated Barnase genes, one missense and one translation attenuated, were tested. Our results indicated that the translation-attenuated version of the Barnase gene was most effective in causing heat-shock–regulated plant death. Analysis of the T2 progeny of a transgenic plant carrying this Barnase mutant showed that the Barnase gene expression was sixfold higher in heat-shock–treated plants compared with untreated plants. This level of Barnase gene expression was sufficient to kill transgenic plants.

Although several invasive species have induced changes to the fire regime of invaded communities, potential intraspecific shifts in fire-related traits that might enhance the invasion success of these species have never been addressed. We assumed that traits conferring persistence and competitiveness in postfire conditions to downy brome, a quintessential invasive species of the Great Basin (North America), might be under selection in areas with recurrent fires. Therefore, we hypothesized that populations from frequently burned regions of the Great Basin would have (1) greater tolerance to fire at seed level, (2) higher relative seedling performance in postfire environments, and (3) greater flammability than unburned Central European populations that evolved without fire. Seeds were collected from three introduced populations from frequently burned regions in North America and three introduced populations of rarely or never burned sites from Central Europe. We performed (1) germination experiments with seeds subjected to the effect of different fire components (heat shocks, smoke, flame, ash), (2) pot experiments analyzing the effect of postfire conditions on the early growth of the seedlings, and (3) a series of flammability tests on dry biomass of plants reared in a common garden. All seeds tolerated the low-temperature treatments (40 to 100 C), but were destroyed at high heat shocks (140 and 160 C). Only the 100 C heat treatment caused a difference in reaction of seeds from different continents, as the European seeds were less tolerant to this heat shock. We found significantly increased seedling height and biomass after 4 wk of growth under postfire conditions in American populations, but not in European ones. American populations had enhanced flammability in three out of five measured parameters compared to European populations. In summary, these intraspecific differences in fire-related traits might contribute to the persistence and perhaps invasiveness of the frequently burned North American downy brome populations.

Water availability regulates timing of germination, especially in those environments where it is a limiting factor. However, the water requirements for germination of most wild Mediterranean species are unknown. In this work we analysed the germination response to water stress of eight Cistaceae species with hard-coated seeds, which are typical for the Mediterranean shrublands. Seeds were exposed to a heat shock in order to overcome physical dormancy and then incubated under five water potentials (0, −0.2, −0.4, −0.6 and −0.8 MPa). Ungerminated seeds following these treatments were transferred to water in order to evaluate the recovery of germination. Additionally, at the end of the experiment, viability of still ungerminated seeds was examined. We analysed final germination percentage, time to reach 50% of the final germination (t 50), recovery of germination and seed viability in relation to water stress treatments. Furthermore, hydrotime analysis was performed; sensitivity to water stress, as characterized by Ψb(50), was checked in relation to seed size of species. Overall, water stress delayed and decreased germination but species showed different germination sensitivities, which were not related to seed size. Recovery of germination was high after disappearance of water stress but seed viability decreased in some species. Consequently, post-fire germination of Cistaceae must be strongly regulated by water availability and their seeds can recover germination capacity when sufficient water is available after long periods of incomplete hydration. However, some seeds die during the wait.

Heat shock may affect different aspects of oocyte maturation and its subsequent development to the blastocyst stage. A series of in vitro experiments was performed to determine whether physiologically heat shock (41°C) disrupts the progression of the ovine oocytes through meiosis, activation and blastocyst formation. Cumulus–oocyte complexes (COCs) were aspirated from 2–6-mm follicles and cultured at 38.5°C (control) or 41°C (heat shock) for the first 12 h of maturation. The oocytes were incubated at 38.5°C during the last 10 h of maturation and 8 days after activation. Results showed that most of the oocytes matured under heat-shock conditions remained at the germinal vesicle breakdown (GVBD) stage and they showed an aberrant chromatin configuration. After heat shock, oocyte diameter and time spent for zona pellucida dissolution increased (P < 0.05). The heat-shocked group had a higher percentage of oocytes with incomplete migration of cortical granules (P < 0.05). The heat-shock condition decreased (P < 0.05) cleavage rates (56.19 versus 89.28%) and morula formation (26.85 versus 37.81%). However, there was no significant difference in blastocyst formation and percentage of hatched blastocysts. At 12 h, heat shock had an adverse effect on embryo quality and reduced inner cell mass number (P < 0.05). Quantitative gene expression analysis showed greater transcripts (P < 0.05) for Na/K-ATPase mRNA in heat-shocked oocytes. To sum up, heat shock has disruptive effects on ovine oocyte maturation and can impair cellular and molecular factors that are important for embryo development.

An in vitro model of embryo production was used to examine the effects of insulin-like growth factor (IGF)-I on maturation and developmental competence of oocytes exposed to heat shock. Cumulus–oocyte complexes were matured at 38.5°C or exposed to acute heat shock (HS; 41.5°C), with or without 100 ng/ml IGF-I, for 22 h through in vitro maturation. The experimental groups were control (C), C + IGF-I, HS, and HS + IGF-I. Oocytes were fertilized at the end of maturation, and the proportion of cleaved embryos was recorded 44 h later. HS during maturation increased the proportion of TUNEL-positive oocytes (P < 0.05). HS did not have any effect on cortical granule translocation but impaired resumption of meiosis, expressed as a decreased proportion of oocytes with nuclei in metaphase I (P < 0.05) and metaphase II (MII; P < 0.05). HS decreased the proportion of oocytes that cleaved (P < 0.05), in particular those oocytes that further developed to 4-cell-stage embryos (P < 0.05). IGF-I alleviated, to some extent, the deleterious effects of HS on the oocytes as reflected by a reduced proportion of TUNEL-positive oocytes (P < 0.03). While not significant, IGF-I tended to increase the proportion of MII-stage oocytes (P < 0.08) and 4-cell-stage cleaved embryos (P < 0.06). Further examination is required to explore whether IGF-I also affects the developmental competence of oocytes exposed to HS.

Hsp90 (a.k.a. Hsp83) plays a significant role in the life cycle control of the protozoan parasite Leishmania donovani. Rather than protecting Leishmania spp. against adverse and stressful environs, Hsp90 is required for the maintenance of the motile, highly proliferative insect stage, the promastigote. However, Hsp90 is also essential for survival and proliferation of the intracellular mammalian stage, the amastigote. Moreover, recent evidence shows Hsp90 and other components of large multi-chaperone complexes as substrates of stage-specific protein phosphorylation pathways, and thus as likely effectors of the signal transduction pathways in Leishmania spp. Future efforts should be directed towards the identification of the protein kinases and the critical phosphorylation sites as targets for novel therapeutic approaches.

Ageing of wheat seeds (Triticum aestivum cv. Galahad, 1986 harvest) during storage is accompanied by an increase in the proportion of abnormal seedlings produced when aged seed lots germinate. The response of aleurone layers from normally and abnormally germinated seeds to heat shock has been investigated. [14C]-amino acid incorporation into aleurone layers from 3-d normally and abnormally germinated seeds diminished after 4 h at 42°C but the aleurone layers were able to recover significantly during a 5-h period at 20°C following heat shock. [35S]-methionine pulse-labelling of aleurone proteins showed that α-amylase isoenzyme synthesis was abolished upon heat shock in aleurone layers for both normally and abnormally germinated seeds and that only aleurone layers from normally germinated seeds were capable of recommencing substantial α-amylase synthesis during the recovery period. Heat shock caused aleurone layers from abnormally germinated seeds to synthesize polypeptides not synthesized by aleurone layers from normally germinated seeds under the same conditions. The synthesis of certain polypeptides by aleurone layers from abnormally germinated seeds was enhanced at 20°C, 42°C and during recovery compared with normally germinated seeds. These results are discussed in terms of the control of gene expression during heat shock and recovery during seed ageing.

Aleurone layers of mature germinating barley (Hordeum vulgare, cv. Himalaya) grains respond to heat shock by synthesizing heat-shock proteins (HSPs) and by selectively suppressing the synthesis of proteins normally translated by endoplasmic reticulum (ER)-bound ribosomes. To determine if this also was the case during seed development, we investigated the synthesis of proteins translated by ER-bound ribosomes in heat-shocked aleurone layers isolated from developing barley grains. The optimal induction temperature for the heat shock response in developing aleurone layers was 37.5°C, and temperatures above 42°C inhibited translation. HSPs with apparent molecular masses of 71.1, 66.2, 57.8, 19.1 and 18.8 kDa were induced. Other studies have shown that, in gibberellic acid (GA)-induced aleurone layers from mature barley grains, these temperatures were 40°C and 45°C, respectively. Furthermore, in developing aleurone layers, mRNAs encoding proteins translated by ER-bound ribosomes (mRNAs for a lipid transfer protein and a putative amylase/protease inhibitor) remained stable during heat shock. The ER membranes themselves remained in stacks, but the lumen became distended with electron-dense material. Heat shock prevented the movement of proteins from the ER into the rest of the endomembrane pathway. In contrast, other studies show that in mature, GA-induced aleurone layers, heat shock dissociates ER stacks and blocks translation, but the processing of secretory proteins in the endomembrane pathway is not inhibited. The observation that the same tissue at different developmental stages may respond differently to heat shock indicates that components of the heat-shock response are developmentally regulated. This system provides an opportunity to better understand the nuances of the heat-shock response, especially the post-transcriptional gene regulatory mechanisms that occur.

The effects of desiccation and temperature on the germination capability of Carica papaya L. were investigated for seeds extracted from three commercial fruit batches. More than 50% of freshly isolated, cleaned (sarcotesta removed) but undried seeds germinated at 26°C. However, desiccation to approx. 20%seed RH reduced germination at this temperature to < 10%. A substantial increase in germination at alternating temperatures (33/19°C) indicated that desiccation induced seed dormancy rather than viability loss. Dormancy could be removed in a large proportion of the population by the application of a single heat shock to rehydrated seeds for 4 h at 36°C, with subsequent return to 26°C for germination. Longer (days) and shorter (minutes) periods of heat shock were less effective for releasing dormancy. Heat shock was generally applied 5 or 14 d after rehydration had started, but the treatment was equally effective after imbibition for only 1 d. Light was always applied during both imbibition and the post-heat shock treatment, but was not essential during the actual heat shock treatment. Rehydration and post-heat shock temperature treatments in the range of 16°C to 36°C revealed the same optima of 26°C. Dormancy was re-imposed in heat-shocked seeds when they were subsequently dried to seed relative humidities of 25 to 75%(5 to 11% moisture content [fresh weight basis]), but this state could be removed by a further heat shock. The longer heat-shocked seeds were held on agar-water at 26°C prior to re-drying, the greater the level of desiccation intolerance.

Summary 37

I. INTRODUCTION 37

II. DIGESTION OF PLANTS IN THE RUMEN: OLD AND NEW CONCEPTS 39

III. RUMEN-INDUCED PLANT METABOLISM: CELL DEGRADATION AND DEATH 41

1. Summary of plant cell death processes 41

2. Anaerobic stress and flooding tolerance of plants 42

3. Plant cell responses to elevated temperatures 45

4. Wounding responses/pathogen attachment 45

5. Senescence in the rumen? 47

IV. FUTURE PROSPECTS 50

Acknowledgements 51

References 51

It is generally assumed that breakdown of plant material in the rumen is a process mediated by gut microorganisms. This view arose because of the identification of a pre-gastric fermentation in the rumen, brought about by a large and diverse microbial population. The extensive use of dried and ground feed particles in forage evaluation might have helped to promote this assumption. However, although the assumption might be correct in animals feeding on conserved forage (hay and silage) where the cells of ingested forage are dead, it is possible that with grazed (living) forage, the role played by plant enzymes in the rumen has been overlooked. In a grazing situation, plant cells that remain intact on entering the rumen are not inert, but will respond to the perceived stresses of the rumen environment for as long as they are metabolically viable. Metabolic adjustments could include anaerobic and heat-shock responses that could promote premature senescence, leading to remobilization of cell components, especially proteins. Moreover, contact of plant cells with colonizing microorganisms in the rumen might promote a type of hypersensitive response, in much the same way as it does outside the rumen. After fresh plant material enters the rumen and prior to extensive plant cell-wall degradation, there is often a phase of rapid proteolysis providing N in excess of that required to maintain the rumen microbial population. The inefficient use of this ingested N results in generation of ammonia and urea in exhaled breath and urine, which promotes welfare and environmental pollution concerns. Therefore an important research goal in livestock agriculture is to find ways of decreasing this initial rate of proteolysis in the rumen. This will benefit the farmer financially (through decreased use of feed supplements), but will also benefit the environment, as N pollution can adversely affect pasture diversity and ecology. This review considers the possible responses of plant metabolism to the rumen environment, and how such considerations could alter current thinking in ruminant agriculture.

We analyzed the roles of the three domains of a Drosophila hnRNP A1 homolog by expression of wild-type and mutant versions of HRB87F/hrp36 in Drosophila melanogaster. HRB87F/hrp36 is one of two Drosophila proteins that is most similar to mammalian A1 hnRNP, and like A1, consists of two copies of the RNA-binding domain (RBD) motif followed by a glycine-rich domain (GRD). The role of the domains in nuclear localization and RNA binding to polytene chromosomal sites was determined. RBD-1 and the GRD were largely responsible for both the cellular location of the protein and for the typical chromosomal distribution pattern of the protein at sites of Pol II transcription. RBD-1 also provided a role in the exon-skipping activity of the protein that was not provided by RBD-2. On the other hand, RBD-2 and the GRD were responsible for the very limited chromosomal distribution pattern seen upon heat shock, when HRB87F/hrp36 is sequestered at heat-shock puff 93D, which encodes a long nucleus-restricted RNA. Thus, these studies indicate that the two RBDs function independently of each other but in concert with the GRD. In addition, the self-association property of the GRD was strikingly evident in these overexpressed proteins.