Most plants are adapted to their environments through generations of exposure to all elements. The adaptation process involves the best possible response to fluctuations in the environment based on the genetic and epigenetic make-up of the organism. Many plant species have the capacity to acclimate or adapt to certain stresses, allowing them to respond more efficiently, with fewer resources diverted from growth and development. However, plants can also acquire protection against stress across generations. Such a response is known as an intergenerational response to stress; typically, plants lose most of the tolerance in the subsequent generation when propagated without stress. Occasionally, the protection lasts for more than one generation after stress exposure and such a response is called transgenerational. In this review, we will summarize what is known about inter- and transgenerational responses to stress, focus on phenotypic and epigenetic events, their mechanisms and ecological and evolutionary meaning.

Probiotics are bacteria among the intestinal flora that are beneficial for human health. Bifidobacterium longum (BL) is a prototypical probiotic that is widely used in yogurt making, supplements and others. Although various physiological effects of BL have been reported, those associated with longevity and anti-ageing still remain elusive. Here we aimed to elucidate the physiological effects of killed BL (BR-108) on stress tolerance and longevity of Caenorhabditis elegans and their mechanisms. Worms fed killed BL in addition to Escherichia coli (OP50) displayed reduced body length in a BL dose-dependent manner. When compared with those fed E. coli alone, these worms had a higher survival rate following heat stress at 35°C and hydrogen peroxide-induced oxidative stress. A general decrease in motility was observed over time in all worms; however, killed BL-fed ageing worms displayed increased movement and longer life span than those fed E. coli alone. However, the longevity effect was suppressed in sir-2.1, daf-16 and skn-1-deficient worms. Killed BL induced DAF-16 nuclear localisation and increased the expression of the DAF-16 target gene hsp-12.6. These results revealed that the physiological effects of killed BL in C. elegans were mediated by DAF-16 activation. These findings contradict previous observations with different Bifidobacterium and Lactobacillus strains, which showed the role for SKN-1 independently of DAF-16.

The main purpose of the study was to compare the capacity of the major sheep breeds in Morocco to cope with climate changes through the ranges of several climate parameters in which they can be found. We first delimitated the climatic ‘domains’ of each breed by constructing a database including altitude and climatic parameters (minima mean of the coldest month, maxima mean of the hottest month, annual rainfall, pluviothermic coefficient of Emberger Q2, annual minima mean and annual maxima mean) on a 30-year period using the representative stations of each breed distribution. The overlap between each breed combination was quantified through a canonical analysis that extracted the most discriminant parameters. The variance analysis of each climatic parameter evidenced two breeds remarkable by their tolerance. The first one is the Timahdite, mainly settled in areas over 1100 m, which can tolerate the greatest variations in annual rainfall and pluviothermic coefficient. In spite of this feature, this breed is endangered owing to the decreasing quality of pastures. The second one is the D’man which apparently can support high variations in extreme temperatures. In fact, this breed is not well adapted to pastures and requires a special microclimate offered by oases. The information reported in this study will be the basis for the establishment of characterization and selection strategies for Moroccan sheep.

The water uptake by the seed in arid and semi-arid ecosystems may not be continuous, but may occur in hydration and dehydration cycles (HD). In order to investigate the benefits of discontinuous hydration on seed germination of Pilosocereus catingicola subsp. salvadorensis subjected to environmental stresses, an imbibition curve was determined and times X, Y and Z selected, corresponding to 25, 50 and 75% of the first phase of imbibition, respectively. Seeds of two populations (Caatinga and Restinga) were subjected to 0, 1, 2 and 3 HD cycles, with 48 h of dehydration. Water and saline stress responses were tested at five osmotic potentials (0.0, –0.1, –0.3, –0.6 and –0.9 MPa). Discontinuous hydration provided greater tolerance to water and saline stress in the seeds from the Caatinga region. When the seeds were subjected to water stress, the reduction of the mean germination time (MGT) provided by HD cycles decreased with increasing concentration of the solution. In saline stress, the reduction of MGT was greater in the solutions with the highest concentrations. We conclude that the seeds of the same species collected from populations located in different ecosystems have different germination responses after passage through discontinuous hydration and HD cycles. This provides greater tolerance to environmental stresses but with different responses among populations.

Plants respond to sub-optimal conditions by the synthesis of specific ‘stress’ proteins, and these are thought to play a role in stress tolerance. Some of these proteins accumulate during late seed development, arguably to protect against damage during post-maturation drying and subsequent imbibition, prior to germination. Seed vigour is also determined during this late stage of seed development. High vigour seeds are those that can withstand the desiccation required for storage and successfully germinate under sub-optimal conditions to establish healthy seedlings. If stress proteins are involved in tolerating stress conditions, then they are likely to be important determinants of seed vigour. In this work the relationship between seed vigour (measured by seed germination performance following rapid aging, or under water stress) in Brassica oleracea var. capitata and the content of two classes of stress protein (dehydrins and a low molecular weight heat shock protein HSP17.6) at maturity was examined. Dehydrins did not show a positive relationship with seed performance. However, the protein HSP17.6 showed a positive correlation with seed performance, and a treatment that reduced the amount of this protein in the seed also caused a reduction in subsequent seed performance.

After seven generations of self-crossing assisted by β-glucuronidase (GUS) detection, 26 homozygous transgenic rice lines were obtained. Transgene effects on iron content, agronomic traits, rice quality and stress tolerance were studied. The results indicated that the average iron content in the milled grain of the transgenic (pea ferritin, Fer) rice (Oryza sativa) was 10.37 μg/g among all the homozygous lines, and only four lines (Fer34, Fer36, Fer39 and Fer65) had significantly higher iron contents than that of the control, Xiushui11 (6.46 μg/g). Significant differences in main agronomic traits, such as days from sowing to heading, plant height, main panicle length, total grains of main panicle, seed setting rate, 1000-grain weight, and yield per plant were observed among some homozygous lines, but similar flag leaf length and number of panicles per plant were found. This showed that insertion of the foreign gene (Fer) had a slight impact on the main agronomic traits, but that the transgene had no negative effects. The quality traits of homozygous rice lines, such as the percentage of brown rice, milled rice and full milled rice, grain length, grain width, ratio of grain length and width, and amylose content were all similar to those of the control. However, variations were noticed in chalkiness, translucency, alkali spreading value and gel consistency among partial homozygous lines. Responses of homozygous lines to low and high temperatures were not significantly correlated with the iron contents, and resistance to rice blast (Pyricularia oryzae), bacterial blight (Xanthomonas oryzae) and brown planthopper (Niaparvata lugens) were enhanced with increased iron contents.

Infective juveniles (IJs) of the entomopathogenic nematode (EPN) Steinernema feltiae IS-6 can survive exposure to 24% glycerol solution by entering an osmotically desiccated state. Exposure of osmotically desiccated nematodes to extreme temperature assays (40 °C for 10 h and −20 °C for 360 h) resulted in gradual reduction in survival, whereas non-desiccated IJs died within a short exposure to the assay conditions. Through SDS-PAGE, a stress-related protein UNC-87 was found in osmotically desiccated IJs exposed to 40 °C for 3, 6, and 8 h, whose survival rates were 98·9±1·43, 78·5±5·87 and 20·9±4·93%, respectively. The protein was not found in IJs following exposure of osmotically desiccated individuals to 40 °C for 10 h, in which none of the IJs survived. After exposure to −20 °C for 360 h, the survival of osmotically desiccated EPNs with a weak band of UNC-87 was 13·0±3·32%. To identify other responsive proteins that are required for osmotic stress, we used 2-dimensional electrophoresis to analyse the proteins in osmotically desiccated EPNs. The results revealed that 10 novel protein spots and 10 up-regulated protein spots in osmotically desiccated IJs were detected by digital image analysis. Mass spectrometry analysis of 7 significant spots indicated that osmotic stress in desiccated IJs was associated with the induction of actin, Proteasome regulatory particle (ATPase-like), GroEL chaperonin, GroES co-chaperonin and transposase family member. It seems to show actin, UNC-87 and Proteasome regulatory particle may play distinct roles in specific aspects of organization of macromolecular structures under desiccation stress. GroEL and GroES are members of the Hsp60 family of chaperons.

The impact of seed size on germination and seedling growth, as affected by water stress, was studied for five tree species from tropical dry forest of India, viz. Albizia procera, Acacia nilotica, Phyllanthus emblica, Terminalia arjuna and Terminalia chebula. Germination tests were conducted under five osmotic potential levels. Seedlings from large (LS) and small (SS) seeds were grown at four soil moisture levels. Observations were made on height, leaf area, biomass and other growth traits such as relative growth rate (RGR), net assimilation rate (NAR), specific leaf area (SLA), and root:shoot (R:S) ratio. Seeds of pioneer species and large seeds, within species, germinated earlier, and with increasing water stress, per cent germination and germination velocity declined. RGR was inversely related with drought tolerance. R:S ratio increased, RGR and SLA declined, but NAR increased with water stress. Notwithstanding successional status, the slow-growing species registered minimum reduction in biomass due to water stress. The response of LS and SS seedlings also differed for some of the growth variables. Increase in NAR could be a compensatory response to water stress, and the marked allocational plasticity could help maximize capture of the limited resource. Seedlings from smaller seeds, particularly of fast-growing species, would be able to cope with mild drought by morphogenetic and physiological plastic response in a better way than those from large seeds. However, seedlings from large seeds had greater survival than those from smaller seeds under intense water stress.

Gene expression in Fucus vesiculosus L. from the Ria Formosa, Faro, Portugal was investigated by screening a cDNA library generated by suppression subtractive hybridization (SSH) for algae undergoing mild desiccation stress (60–70% tissue water content). The subtractive library was constructed from small amounts (1 μg) of total RNA using PCR-based techniques. Screening by reverse Northern analysis (cDNA Southerns) revealed that between 60% and 70% of clones randomly selected from the library were differentially regulated in desiccated and hydrated algae. Most genes could not be directly identified based on sequence homology with known gene sequences. However, several cDNAs for chloroplast-encoded transcripts were identified and shown to be up-regulated or differentially regulated in desiccated algae relative to hydrated controls. These included partial sequences for ribulose-l,5-bisphosphate carboxylase/oxygenase (Rubisco; rbcL/rbcS), chloroplast coupling factor ATPase (atpH/atpI) and a photosystem I P700 chlorophyll a binding protein (psaA). The usefulness of reverse Northern analysis was confirmed with conventional Northern analysis of transcript abundance for Rubisco, which varied rapidly in response to light and the hydration status of the algae. These results show that SSH is a useful technique for analysing the responses of gene expression to environmental stress, and as a starting point for the subsequent identification of stress-responsive genes in macroalgae.

Summary 185

I. INTRODUCTION 186

II. EFFECT OF ZINC ON PRODUCTION OF REACTIVE OXYGEN SPECIES 186

1. Superoxide-generating NADPH oxidase 186

2. Zinc deficiency potentiates iron-mediated free radical production 189

(a) Iron accumulation in zinc-deficient plants 189

(b) Iron-induced production of free radicals 189

3. Zinc deficiency-enhanced photooxidation 191

(a) Decrease in photosynthesis 191

(b) Light-induced leaf chlorosis 192

(c) Decrease in indole-3-acetic acid 192

III. MEMBRANE DAMAGE BY REACTIVE OXYGEN SPECIES 193

1. Impairments in membrane structure 193

2. Phospholipids and –SH groups 195

3. Alterations in ion absorption 195

(a) Membrane-bound ATPases 195

(b) Nutrient uptake 197

(c) Changes in activity of ion channels 197

IV. DETOXIFICATION OF REACTIVE OXYGEN SPECIES 198

1. Superoxide dismutases 198

2. H2O2-scavenging enzymes 198

V. INVOLVEMENT OF ZINC IN PLANT STRESS TOLERANCE 199

VI. CONCLUSIONS 199

Acknowledgements 200

References 200

Zinc deficiency is one of the most widespread micronutrient deficiencies in plants and causes severe reductions in crop production. There are a number of physiological impairments in Zn-deficient cells causing inhibition of the growth, differentiation and development of plants. Increasing evidence indicates that oxidative damage to critical cell compounds resulting from attack by reactive O2 species (ROS) is the basis of disturbances in plant growth caused by Zn deficiency. Zinc interferes with membrane-bound NADPH oxidase producing ROS. In Zn-deficient plants the iron concentration increases, which potentiates the production of free radicals. The Zn nutritional status of plants influences photooxidative damage to chloroplasts, catalysed by ROS. Zinc-deficient leaves are highly light-sensitive, rapidly becoming chlorotic and necrotic when exposed to high light intensity. Zinc plays critical roles in the defence system of cells against ROS, and thus represents an excellent protective agent against the oxidation of several vital cell components such as membrane lipids and proteins, chlorophyll, SH-containing enzymes and DNA. The cysteine, histidine and glutamate or aspartate residues represent the most critical Zn- binding sites in enzymes, DNA-binding proteins (Zn-finger proteins) and membrane proteins. In addition, animal studies have shown that Zn is involved in inhibition of apoptosis (programmed cell death) which is preceded by DNA and membrane damage through reactions with ROS.

The concept of the ‘nucleotype’ embraces aspects of the nuclear DNA that affect the phenotype other than through the coding sequences. In this context, we consider one particular situation, namely DNA variation due to the presence of supernumerary B chromosomes (Bs); these Bs, which are additional to the basic complement of A chromosomes, generate a spectrum of DNA amounts, and thus nucleotypes, within many species. We consider the physiological consequences of carrying extra chromosomes that appear by and large to lack coding sequences, and focus on two species with contrasting case histories: rye (Secale cereale L.) and chives (Allium schoenoprasum L.), which illustrate ‘selfish’ and ‘adaptive’ explanations of the persistence of B chromosomes.

Dehydrin proteins (late embryogenesis abundant (LEA) D11 family) are produced in a wide variety of plant species in response to environmental stimuli with a dehydrative component, including drought, low temperature, salinity, and developmental stages such as seed and pollen maturation. Despite their widespread occurrence and abundance in cells under dehydrative conditions, the biochemical role of dehydrins remains elusive. The subcellular location of dehydrins is consistent with a biochemical role as an intracellular stabilizer, possibly with surfactant characteristics, acting upon targets in both the nucleus and cytoplasm. In some species, dehydrin loci are located within quantitative trait loci (QTL) intervals for important phenotypic traits including winter hardiness in barley (Hordeum vulgare L.) and anthesis-silking interval in maize (Zea mays L.). Dehydrin loci tend to be multigenic and occur in clusters on more than one chromosome. Investigations are currently under way in our laboratory and others' to move beyond protein accumulation studies and correlations with QTL to uncover direct cause-and-effect relationships between dehydrin (dhn) genes and phenotypes associated with physiological responses to stress.