Strigolactones positively regulate Verticillium wilt resistance in cotton via crosstalk with other hormones.

Verticillium wilt caused by Verticillium dahliae is a serious vascular disease in cotton (Gossypium spp.). V. dahliae induces the expression of the CAROTENOID CLEAVAGE DIOXYGENASE 7 (GauCCD7) gene involved in strigolactone (SL) biosynthesis in Gossypium australe, suggesting a role for SLs in Verticillium wilt resistance. We found that the SL analog rac-GR24 enhanced while the SL biosynthesis inhibitor TIS108 decreased cotton resistance to Verticillium wilt. Knock-down of GbCCD7 and GbCCD8b genes in island cotton (Gossypium barbadense) decreased resistance, whereas overexpression of GbCCD8b in upland cotton (Gossypium hirsutum) increased resistance to Verticillium wilt. Additionally, Arabidopsis (Arabidopsis thaliana) SL mutants defective in CCD7 and CCD8 putative orthologs were susceptible, whereas both Arabidopsis GbCCD7- and GbCCD8b-overexpressing plants were more resistant to Verticillium wilt than wild-type (WT) plants. Transcriptome analyses showed that several genes related to the jasmonic acid (JA)- and abscisic acid (ABA)-signaling pathways, such as MYELOCYTOMATOSIS 2 (GbMYC2) and ABA-INSENSITIVE 5, respectively, were upregulated in the roots of WT cotton plants in responses to rac-GR24 and V. dahliae infection but downregulated in the roots of both GbCCD7- and GbCCD8b-silenced cotton plants. Furthermore, GbMYC2 suppressed the expression of GbCCD7 and GbCCD8b by binding to their promoters, which might regulate the homeostasis of SLs in cotton through a negative feedback loop. We also found that GbCCD7- and GbCCD8b-silenced cotton plants were impaired in V. dahliae-induced reactive oxygen species (ROS) accumulation. Taken together, our results suggest that SLs positively regulate cotton resistance to Verticillium wilt through crosstalk with the JA- and ABA-signaling pathways and by inducing ROS accumulation.

Differential metabolic rearrangements in the roots and leaves of Cicer arietinum caused by single or double nitrate and/or phosphate deficiencies.

Nitrate (NO3 - ) and phosphate (Pi) deficiencies are the major constraints for chickpea productivity, significantly impacting global food security. However, excessive fertilization is expensive and can also lead to environmental pollution. Therefore, there is an urgent need to develop chickpea cultivars that are able to grow on soils deficient in both NO3 - and Pi. This study focused on the identification of key NO3 - and/or Pi starvation-responsive metabolic pathways in the leaves and roots of chickpea grown under single and double nutrient deficiencies of NO3 - and Pi, in comparison with nutrient-sufficient conditions. A global metabolite analysis revealed organ-specific differences in the metabolic adaptation to nutrient deficiencies. Moreover, we found stronger adaptive responses in the roots and leaves to any single than combined nutrient-deficient stresses. For example, chickpea enhanced the allocation of carbon among nitrogen-rich amino acids (AAs) and increased the production of organic acids in roots under NO3 - deficiency, whereas this adaptive response was not found under double nutrient deficiency. Nitrogen remobilization through the transport of AAs from leaves to roots was greater under NO3 - deficiency than double nutrient deficiency conditions. Glucose-6-phosphate and fructose-6-phosphate accumulated in the roots under single nutrient deficiencies, but not under double nutrient deficiency, and higher glycolytic pathway activities were observed in both roots and leaves under single nutrient deficiency than double nutrient deficiency. Hence, the simultaneous deficiency generated a unique profile of metabolic changes that could not be simply described as the result of the combined deficiencies of the two nutrients.

The histidine phosphotransfer AHP4 plays a negative role in Arabidopsis plant response to drought.

Cytokinin plays an important role in plant stress responses via a multistep signaling pathway, involving the histidine phosphotransfer proteins (HPs). In Arabidopsis thaliana, the AHP2, AHP3 and AHP5 proteins are known to affect drought responses; however, the role of AHP4 in drought adaptation remains undetermined. In the present study, using a loss-of-function approach we showed that AHP4 possesses an important role in the response of Arabidopsis to drought. This is evidenced by the higher survival rates of ahp4 than wild-type (WT) plants under drought conditions, which is accompanied by the downregulated AHP4 expression in WT during periods of dehydration. Comparative transcriptome analysis of ahp4 and WT plants revealed AHP4-mediated expression of several dehydration- and/or abscisic acid-responsive genes involved in modulation of various physiological and biochemical processes important for plant drought acclimation. In comparison with WT, ahp4 plants showed increased wax crystal accumulation in stems, thicker cuticles in leaves, greater sensitivity to exogenous abscisic acid at germination, narrow stomatal apertures, heightened leaf temperatures during dehydration, and longer root length under osmotic stress. In addition, ahp4 plants showed greater photosynthetic efficiency, lower levels of reactive oxygen species, reduced electrolyte leakage and lipid peroxidation, and increased anthocyanin contents under drought, when compared with WT. These differences displayed in ahp4 plants are likely due to upregulation of genes that encode enzymes involved in reactive oxygen species scavenging and non-enzymatic antioxidant metabolism. Overall, our findings suggest that AHP4 plays a crucial role in plant drought adaptation.

Phosphate or nitrate imbalance induces stronger molecular responses than combined nutrient deprivation in roots and leaves of chickpea plants.

The negative effects of phosphate (Pi) and/or nitrate (NO3- ) fertilizers on the environment have raised an urgent need to develop crop varieties with higher Pi and/or nitrogen use efficiencies for cultivation in low-fertility soils. Achieving this goal depends upon research that focuses on the identification of genes involved in plant responses to Pi and/or NO3- starvation. Although plant responses to individual deficiency in either Pi (-Pi/+NO3- ) or NO3- (+Pi/-NO3- ) have been separately studied, our understanding of plant responses to combined Pi and NO3- deficiency (-Pi/-NO3- ) is still very limited. Using RNA-sequencing approach, transcriptome changes in the roots and leaves of chickpea cultivated under -Pi/+NO3- , +Pi/-NO3- or -Pi/-NO3- conditions were investigated in a comparative manner. -Pi/-NO3- treatment displayed lesser effect on expression changes of genes related to Pi or NO3- transport, signalling networks, lipid remodelling, nitrogen and Pi scavenging/remobilization/recycling, carbon metabolism and hormone metabolism than -Pi/+NO3- or +Pi/-NO3- treatments. Therefore, the plant response to -Pi/-NO3- is not simply an additive result of plant responses to -Pi/+NO3- and +Pi/-NO3- treatments. Our results indicate that nutrient imbalance is a stronger stimulus for molecular reprogramming than an overall deficiency.

Genotype- and tissue-specific physiological and biochemical changes of two chickpea (Cicer arietinum) varieties following a rapid dehydration.

In nature, plants may suffer rapid dehydration (RD), which causes significant loss of the annual global chickpea production. Thus, ascertaining more knowledge concerning the RD-tolerance mechanisms in chickpea is crucial for developing high drought-tolerant varieties to assure sustainable chickpea production under sudden water deficit. Here, we focused on genotype-driven variation in leaf relative water content (RWC) and associated differences in RD-responsive physiological and biochemical attributes in roots and leaves of two chickpea varieties, FLIP00-21C and FLIP02-89C, subjected to well-watered and RD conditions. FLIP00-21C showed higher RD-tolerance than FLIP02-89C, evident by its higher leaf RWC during RD. Consistently, FLIP00-21C exhibited lower membrane injury due to lower hydrogen peroxide (H2 O2 ) accumulation than FLIP02-89C during RD, indicating reduced RD-induced oxidative damage. Under RD conditions, total phenolics in roots and flavonoids in roots and leaves increased more in FLIP02-89C compared to FLIP00-21C; however, the increased activities of superoxide dismutase and H2 O2 -scavenging enzymes were more properly coordinated in FLIP00-21C than in FLIP02-89C, which might contribute to more efficient antioxidant defense in FLIP00-21C than in FLIP02-89C. The higher leaf RWC of FLIP00-21C versus FLIP02-89C under RD might be associated with greater increases in the levels of the osmo-regulators proline and total free amino acids (TFAAs) in FLIP00-21C than in FLIP02-89C. Collectively, the higher RD-tolerance of FLIP00-21C is mainly associated with the maintenance of higher RWC, stronger antioxidant defense, and greater accumulation of proline and TFAAs. These traits could be useful for evaluating the drought-tolerance of chickpea varieties and further marker-assisted breeding approaches for improvement of chickpea productivity.

The use of metabolomic quantitative trait locus mapping and osmotic adjustment traits for the improvement of crop yields under environmental stresses.

The sustainable production of food to feed an increasing world population is a major challenge for plant scientists, especially due to the unpredictable and dynamic nature of global climatic conditions. Heat waves, drought, increased soil salinity, unseasonal cold and flooding are all becoming more common climate-related causes of stress for crop plants, and are already affecting yields and the geographical distributions of optimal growing regions for many crops. Therefore, the development and application of multi-faceted strategies, including sustainable agricultural practices and the development and cultivation of new varieties containing genetic traits associated with abiotic stress tolerance, will either alone or together be essential to sustainably grow high-yielding crops under increasingly stressful environmental conditions. The development of abiotic stress-resilient crops requires an in-depth knowledge of plant development and of the biological processes that enable plants to survive in stressful environments, and this knowledge can be obtained from "omic" studies, such as bioinformatics, genomics, transcriptomics, proteomics and metabolomics. The plant metabolome can provide a snapshot of the physiological and biochemical status of a plant cell under normal or stressful conditions, and thus it is closely related to the plant phenotypes. Analysis of the metabolomes of plants grown under stressful conditions can be used to identify stress resistance-associated metabolites or biomarkers, which can then be used by plant breeders as selective markers to help identify the phenotypes, resulted from the complex interactions between genotype and environment. Osmotic adjustment is an important metabolic adaptation mechanism which helps plants survive abiotic stress and can support higher crop yield under stressful environmental conditions. This review highlights the recent advances in our understanding of the functions of abiotic stress-responsive metabolites, with an emphasis on the use of metabolomic quantitative trait locus mapping and osmotic adjustment agronomic traits, for the improvement of crop yields under environmental stresses.

Heat stress effects on source-sink relationships and metabolome dynamics in wheat.

Crops such as wheat (Triticum spp.) are predicted to face more frequent exposures to heat stress as a result of climate change. Increasing the yield and sustainability of yield under such stressful conditions has long been a major target of wheat breeding, and this goal is becoming increasingly urgent as the global population increases. Exposure of wheat plants in their reproductive or grain-filling stage to high temperature affects the duration and rate of grain filling, and hence has a negative impact on wheat productivity. Therefore, understanding the plasticity of the response to heat stress that exists between wheat genotypes, especially in source-sink relationships at the reproductive and grain-filling stages, is critical for the selection of germplasm that can maintain high yields under heat stress. A broad understanding of metabolic dynamics and the relationships between metabolism and heat tolerance is required in order to achieve this goal. Here, we review the current literature concerning the effects of heat stress on sink-source relationships in a wide range of wheat genotypes, and highlight the current metabolomic approaches that are used to investigate high temperature responses in wheat.

Phytohormones regulate convergent and divergent responses between individual and combined drought and pathogen infection.

Plants exposed to the combination of drought and pathogen infections are in a unique state, different from that of plants exposed to each stress alone. Plants undergo major hormonal changes during drought and/or pathogen infection, highlighting the importance of hormones as crucial mediators of plant stress responses. Evidence from individual stress studies has shown that drought and pathogen infection have both different and overlapping impacts on hormone metabolism and hormone-associated signal transduction pathways. Thus, under the combination of drought and pathogen infection, a reprograming of hormone levels and related signaling networks is inevitable. This process delivers data from plants exposed to individual stressors inadequate for predicting how hormone levels and related signaling networks will change in plants exposed to a combination of stressors. Furthermore, the yield of crop plants, determined by their capacity for stress acclimatization and resistance to pathogen infection, will be underpinned by interactions among the hormone pathways. Although many studies have been conducted to understand the molecular mechanisms associated with plant responses to combinations of stressors, the interactions that occur among hormones are far from being well-understood. We provide here an overview and evaluation of various reports on crosstalk or overlapping hormonal responses from individual stress studies and how the combination of drought and pathogen infection modulates hormone levels and their associated signaling pathways in plant responses to these combined stresses. We also give a brief overview of the importance of overlapping plant responses for the production of crop plants resistant to individual and combined stressors under natural environmental conditions.

The CRISPR/Cas9 system and its applications in crop genome editing.

The CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR associated protein9) system is an RNA-guided genome editing tool that consists of a Cas9 nuclease and a single-guide RNA (sgRNA). By base-pairing with a DNA target sequence, the sgRNA enables Cas9 to recognize and cut a specific target DNA sequence, generating double strand breaks (DSBs) that trigger cell repair mechanisms and mutations at or near the DSBs sites. Since its discovery, the CRISPR/Cas9 system has revolutionized genome editing and is now becoming widely utilized to edit the genomes of a diverse range of crop plants. In this review, we present an overview of the CRISPR/Cas9 system itself, including its mechanism of action, system construction strategies, and the screening methods used to identify mutants containing edited genes. We evaluate recent examples of the use of CRISPR/Cas9 for crop plant improvement, and research into the function(s) of genes involved in determining crop yields, quality, environmental stress tolerance/resistance, regulation of gene transcription and translation, and the construction of mutant libraries and production of transgene-free genome-edited crops. In addition, challenges and future opportunities for the use of the CRISPR/Cas9 system in crop breeding are discussed.

Comparative transcriptome analysis of nodules of two Mesorhizobium-chickpea associations with differential symbiotic efficiency under phosphate deficiency.

Phosphate (Pi) deficiency is known to be a major limitation for symbiotic nitrogen fixation (SNF), and hence legume crop productivity globally. However, very little information is available on the adaptive mechanisms, particularly in the important legume crop chickpea (Cicer arietinum L.), which enable nodules to respond to low-Pi availability. Thus, to elucidate these mechanisms in chickpea nodules at molecular level, we used an RNA sequencing approach to investigate transcriptomes of the nodules in Mesorhizobium mediterraneum SWRI9-(MmSWRI9)-chickpea and M. ciceri CP-31-(McCP-31)-chickpea associations under Pi-sufficient and Pi-deficient conditions, of which the McCP-31-chickpea association has a better SNF capacity than the MmSWRI9-chickpea association during Pi starvation. Our investigation revealed that more genes showed altered expression patterns in MmSWRI9-induced nodules than in McCP-31-induced nodules (540 vs. 225) under Pi deficiency, suggesting that the Pi-starvation-more-sensitive MmSWRI9-induced nodules required expression change in a larger number of genes to cope with low-Pi stress than the Pi-starvation-less-sensitive McCP-31-induced nodules. The functional classification of differentially expressed genes (DEGs) was examined to gain an understanding of how chickpea nodules respond to Pi starvation, caused by soil Pi deficiency. As a result, more DEGs involved in nodulation, detoxification, nutrient/ion transport, transcriptional factors, key metabolic pathways, Pi remobilization and signalling were found in Pi-starved MmSWRI9-induced nodules than in Pi-starved McCP-31-induced nodules. Our findings have enabled the identification of molecular processes that play important roles in the acclimation of nodules to Pi deficiency, ultimately leading to the development of Pi-efficient chickpea symbiotic associations suitable for Pi-deficient soils.

Legume genetic resources and transcriptome dynamics under abiotic stress conditions.

Grain legumes are an important source of nutrition and income for billions of consumers and farmers around the world. However, the low productivity of new legume varieties, due to the limited genetic diversity available for legume breeding programmes and poor policymaker support, combined with an increasingly unpredictable global climate is resulting in a large gap between current yields and the increasing demand for legumes as food. Hence, there is a need for novel approaches to develop new high-yielding legume cultivars that are able to cope with a range of environmental stressors. Next-generation technologies are providing the tools that could enable the more rapid and cost-effective genomic and transcriptomic studies for most major crops, allowing the identification of key functional and regulatory genes involved in abiotic stress resistance. In this review, we provide an overview of the recent achievements regarding abiotic stress resistance in a wide range of legume crops and highlight the transcriptomic and miRNA approaches that have been used. In addition, we critically evaluate the availability and importance of legume genetic resources with desirable abiotic stress resistance traits.

The "STAY-GREEN" trait and phytohormone signaling networks in plants under heat stress.

The increasing demand for food and the heavy yield losses in primary crops due to global warming mean that there is an urgent need to improve food security. Therefore, understanding how plants respond to heat stress and its consequences, such as drought and increased soil salinity, has received much attention in plant science community. Plants exhibit stress tolerance, escape or avoidance via adaptation and acclimatization mechanisms. These mechanisms rely on a high degree of plasticity in their cellular metabolism, in which phytohormones play an important role. "STAY-GREEN" is a crucial trait for genetic improvement of several crops, which allows plants to keep their leaves on the active photosynthetic level under stress conditions. Understanding the physiological and molecular mechanisms concomitant with "STAY-GREEN" trait or delayed leaf senescence, as well as those regulating photosynthetic capability of plants under heat stress, with a certain focus on the hormonal pathways, may be a key to break the plateau of productivity associated with adaptation to high temperature. This review will discuss the recent findings that advance our understanding of the mechanisms controlling leaf senescence and hormone signaling cascades under heat stress.

Polyamines in macroalgae: advances and future perspectives.

Polyamines (PA) are ubiquitous, small, aliphatic cations found in all living cells. In recent years the importance of these molecules for macroalgae has become evident and a substantial body of knowledge has been accumulated over the last three decades. This review summarizes research on the PAs found in macroalgae, their transport and metabolism, and their biological significance in processes such as cell division, chloroplast development, and reproduction. The involvement of PAs in environmental stress responses in macroalgae is also addressed. The discussion of PAs in this review not only demonstrates that PAs play an important role in physiological processes in macroalgae, but also clearly demonstrates the similarities and differences between PA metabolism in macroalgae and higher plants. Key areas for future research are also discussed.

Opportunities and Implications of Pasture-Based Lamb Fattening to Enhance the Long-Chain Fatty Acid Composition in Meat.

Health-conscious consumers following dietary fat recommendations require meat that is low in saturated fat with preferably high levels of long-chain omega-3 fatty acids. This review summarizes the influence of dietary lipids from red meat on human health and examines the potential to enhance lipid composition through pasture-feeding. The role of fatty acids in plant and ruminant metabolism is discussed to highlight the complexity of ruminal digestion when trying to enhance fatty acids in meat. Generally, ruminants that consume pasture diets have been shown to produce a more desirable fatty acid composition than those fed grain and offer potential to be further enhanced by using specific plant species. Elevated polyunsaturated fat content in meat, however, tends to increase susceptibility to oxidation, which influences other meat quality characteristics including shelf-life and color. The use of specific plant species may mitigate these negative effects due to vitamin E or other antioxidants in these plants, which protect polyunsaturated fats from oxidation. When assessing the potential of plants as a natural dietary fat source, consideration must be given to environmental influences on plant fatty acid composition to ensure consistent production of meat products with high nutritive value under a range of management practices. This review also explores the potential impact of climate change on plant fatty acid composition, and the potential implications of this for meat quality.

Regulation of polyamine metabolism in Pyropia cinnamomea (W.A. Nelson), an important mechanism for reducing UV-B-induced oxidative damage.

It is generally accepted that ultraviolet (UV) radiation can have adverse affects on phototrophic organisms, independent of ozone depletion. The red intertidal seaweed Pyropia cinnamomea W.A. Nelson (previously Porphyra cinnamomea Sutherland et al. 2011), similar to many other intertidal macrophytes, is exposed to high levels of UV radiation on a daily basis due to emersion in the upper littoral zone. It has been shown that seaweeds, like higher plants, respond to an increased activity of antioxidative enzymes when exposed to stress. However, earlier investigations have shown that P. cinnamomea also compensates for stress due to UV radiation by increasing polyamine (PA) levels, especially bound-soluble and bound-insoluble PAs. The PA precursor putrescine (PUT) can be synthesized via two enzymatic pathways: arginine decarboxylase (ADC) and ornithine decarboxylase (ODC). Both of these enzymes showed increased activity in P. cinnamomea under UV stress. In higher plants, ADC is the enzyme responsible for increased PA levels during stress exposure, while ODC is correlated with cell division and reproduction. However, there are contrary findings in the literature. Using two irreversible inhibitors, we identified the enzyme most likely responsible for increased PUT synthesis and therefore increased stress tolerance in P. cinnamomea. Our results show that changes in the PA synthesis pathway in P. cinnamomea under UV stress are based on an increased activity of ADC. When either inhibitor was added, lipid hydroperoxide levels increased even under photosynthetically active radiation, suggesting that PAs are involved in protection mechanisms under normal light conditions as well. We also show that under optimum or low-stress conditions, ODC activity is correlated with PUT synthesis.

Oxidative Damage and Antioxidants as Markers for the Selection of Emersion Hardening Treatments in GreenshellTM Mussel Juveniles (Perna canaliculus).

Transport out of the water is one of the most challenging events for juvenile Perna canaliculus and can be a highly inefficient process, with many juveniles subsequently being lost following extended periods of emersion. Hardening techniques offer a possible method for reducing transport-related stress. In this study, different hardening treatments (short, long and intermittent sub-lethal emersion) were used to prepare ~1.2 mm P.canaliculus for transport (20 h) and subsequent reoxygenation stress during re-immersion (i.e., recovery). The oxidative stress responses, resettlement behaviour, respiration rates and survival of the mussels after transport and during recovery were all assessed. Short emersion (1 h) as a hardening treatment prior to transport did not cause major stress to the mussels, which maintained respiration at control levels, showed significantly stimulated antioxidant defences during recovery, showed greater resettlement behaviour and remained viable after 24 h of recovery. In comparison, the long and intermittent emersion treatments negatively impacted oxidative stress responses and affected the viability of the mussels after 24 h of recovery. This study showed that exposing juvenile P.canaliculus to a mild stress prior to transport may stimulate protective mechanisms, therefore eliciting a hardening response, but care must be taken to avoid overstressing the mussels. Improving the management of stress during the transport of juvenile mussels may be key to minimising mussel losses and increasing harvest production, and biomarkers associated with oxidative stress/antioxidant metabolism could be valuable tools to ensure emersion hardening does not overstress the mussels and reduce survival.

Improvement of qualitative and quantitative traits in cotton under normal and stressed environments using genomics and biotechnological tools: A review.

Due to the increasing demand for high-quality and high fiber-yielding cotton (Gossypium spp.), research into the development of stress-resilient cotton cultivars has acquired greater significance. Various biotic and abiotic stressors greatly affect cotton production and productivity, posing challenges to the future of the textile industry. Moreover, the content and quality of cottonseed oil can also potentially be influenced by future environmental conditions. Apart from conventional methods, genetic engineering has emerged as a potential tool to improve cotton fiber quality and productivity. Identification and modification of genome sequences and the expression levels of yield-related genes using genetic engineering approaches have enabled to increase both the quality and yields of cotton fiber and cottonseed oil. Herein, we evaluate the significance and molecular mechanisms associated with the regulation of cotton agronomic traits under both normal and stressful environmental conditions. In addition, the importance of gossypol, a toxic phenolic compound in cottonseed that can limit consumption by animals and humans, is reviewed and discussed.

Unraveling the genetic enigma of rice submergence tolerance: Shedding light on the role of ethylene response factor-encoding gene SUB1A-1.

The world's low-lying rice (Oryza sativa) cultivation areas are under threat of submergence or flash flooding due to global warming. Rice plants manifest a variety of physiological and morphological changes to cope with submergence and hypoxia, including lowering carbohydrate consumption, inhibiting shoot elongation, and forming a thicker leaf gas film during submergence. Functional studies have revealed that submergence tolerance in rice is mainly determined by an ethylene response factor (ERF) transcription factor-encoding gene, namely SUBMERGENCE 1A-1 (SUB1A-1) located in the SUB1 quantitative trait locus. The SUB1A-1-dependent submergence tolerance is manifested through hormonal signaling involving ethylene, gibberellic acid, brassinosteroid, auxin and jasmonic acid. Considerable progress has been made toward the introduction of SUB1A-1 into rice varieties through a conventional marker-assisted backcrossing approach. Here, we review the recent advances in the physiological, biochemical and molecular dynamics of rice submergence tolerance mediated by the 'quiescence strategy'. Thus, the present review aims to provide researchers with insights into the genetics of rice submergence tolerance and future perspectives for designing submergence-resilient plants for sustainable agriculture under the uncertainties of climate change.

Metabolic Regulation of Copper Toxicity during Marine Mussel Embryogenesis.

The development of new tools for assessing the health of cultured shellfish larvae is crucial for aquaculture industries to develop and refine hatchery methodologies. We established a large-volume ecotoxicology/health stressor trial, exposing mussel (Perna canaliculus) embryos to copper in the presence of ethylenediaminetetraacetic acid (EDTA). GC/MS-based metabolomics was applied to identify potential biomarkers for monitoring embryonic/larval health and to characterise mechanisms of metal toxicity. Cellular viability, developmental abnormalities, larval behaviour, mortality, and a targeted analysis of proteins involved in the regulation of reactive oxygen species were simultaneously evaluated to provide a complementary framework for interpretative purposes and authenticate the metabolomics data. Trace metal analysis and speciation modelling verified EDTA as an effective copper chelator. Toxicity thresholds for P. canaliculus were low, with 10% developmental abnormalities in D-stage larvae being recorded upon exposure to 1.10 µg·L-1 bioavailable copper for 66 h. Sublethal levels of bioavailable copper (0.04 and 1.10 µg·L-1) caused coordinated fluctuations in metabolite profiles, which were dependent on development stage, treatment level, and exposure duration. Larvae appeared to successfully employ various mechanisms involving the biosynthesis of antioxidants and a restructuring of energy-related metabolism to alleviate the toxic effects of copper on cells and developing tissues. These results suggest that regulation of trace metal-induced toxicity is tightly linked with metabolism during the early ontogenic development of marine mussels. Lethal-level bioavailable copper (50.3 µg·L-1) caused severe metabolic dysregulation after 3 h of exposure, which worsened with time, substantially delayed embryonic development, induced critical oxidative damage, initiated the apoptotic pathway, and resulted in cell/organism death shortly after 18 h of exposure. Metabolite profiling is a useful approach to (1) assess the health status of marine invertebrate embryos and larvae, (2) detect early warning biomarkers for trace metal contamination, and (3) identify novel regulatory mechanisms of copper-induced toxicity.

Impacts of Rapid Desiccation on Oxidative Status, Ultrastructure and Physiological Functions of Syzygium maire (Myrtaceae) Zygotic Embryos in Preparation for Cryopreservation.

Syzygium maire is a highly threatened Myrtaceae tree species endemic to New Zealand. Due to its recalcitrant seed storage behaviour, cryopreservation is the only viable long-term ex situ conservation option for this species. This study investigated viability, oxidative stress, thermal properties, and ultrastructure of zygotic embryo axes (EAs) desiccated to various moisture contents (MC). Fresh EAs had a MC of c. 1.9 g/g with 100% viability but rapid desiccation to MC < 0.3 g/g significantly reduced viability and decreased the activities of the enzymatic antioxidants superoxide dismutase, catalase and glutathione peroxidase, with a sevenfold increase in the production of protein carbonyls and lipid peroxides. Differential Scanning Calorimetry analysis showed no thermal events in EAs desiccated to a MC of <0.2 g/g, indicating that all freezable water had been removed, but this was lethal to both EAs and enzymatic antioxidants. The ultrastructure of desiccated EAs showed signs of plasmolysis, while fully hydrated EAs exposed to cryogenic temperature had ultrastructural disintegration and membrane damage. The decline in enzymatic antioxidant activities and the increase in lipid peroxidation suggest that S. maire EA viability loss is due to oxidative stress rather than structural impacts.