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.

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.

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.

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.

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.

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.

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.

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.

A new method to localise and quantify oxidative stress in live juvenile mussels.

Stress and survival of the juvenile New Zealand green-lipped mussel, Perna canaliculus, is a poorly understood bottleneck in the ecological and economic performance of a significant aquaculture crop. This species was therefore selected as a model organism for the development of a new method to quantify oxidative stress in whole individuals. An in vivo ROS-activated stain (CellROX™) was administered to anaesthetised, translucent juveniles that were subsequently formaldehyde fixed and then visualised using confocal microscopy. Subsequent application of image analysis to quantifying ROS-positive tissue areas was successfully used to detect stress differences in juvenile mussels exposed to varying levels of emersion. This integrated method can be used to localise and quantify ROS production in individual translucent bivalve life stages (larval and juvenile), while relative stability following fixation greatly expands potential practical field applications. This article has an associated First Person interview with the first and third authors of the paper.

Kinetics of Colour Development during Frying of Potato Pre-Treated with Pulsed Electric Fields and Blanching: Effect of Cultivar.

The current research aimed to investigate the effect of pulsed electric fields (1 kV/cm; 50 and 150 kJ/kg) followed by blanching (3 min., 100 °C) on the colour development of potato slices during frying on a kinetic basis. Four potato cultivars 'Crop77', 'Moonlight', 'Nadine', and 'Russet Burbank' with different content of glucose and amino acids were used. Lightness (L* values from colorimeter measurement) was used as a parameter to assess the colour development during frying. The implementation of PEF and blanching as sequential pre-treatment prior to frying for all potato cultivars was found effective in improving their lightness in the fried products. PEF pre-treatment did not change the kinetics of L* reduction during frying (between 150 and 190 °C) which followed first-order reaction kinetics. The estimated reaction rate constant (k) and activation energy (Ea based on Arrhenius equation) for non-PEF and PEF-treated samples were cultivar dependent. The estimated Ea values during the frying of PEF-treated 'Russet Burbank' and 'Crop77' were significantly (p < 0.05) lower (up to 30%) than their non-PEF counterparts, indicating that the change in k value of L* became less temperature dependence during frying. This kinetic study is valuable to aid the optimisation of frying condition in deep-fried potato industries when PEF technology is implemented.

Emersion and Relative Humidity Modulate Stress Response and Recovery Dynamics in Juvenile Mussels (Perna canaliculus).

The early stages of intertidal mussels, including the green-lipped mussel, Perna canaliculus, face both direct and indirect environmental threats. Stressors may influence physiological status and, ultimately, survival. An understanding of the nature of stress experienced is critical to inform conservation and aquaculture efforts. Here, we investigated oxidative stress dynamics in juvenile P. canaliculus in relation to emersion duration (1-20 h) and relative humidity (RH, 29-98%) by quantifying oxidative damage (protein carbonyls, lipid hydroperoxides, 8-hydroxydeoxyguanosine) and enzymatic antioxidants (superoxide dismutase, catalase, glutathione peroxidase and reductase). Mussels held in low RH during emersion experienced severe water loss (>70%), high mortality (>80%) and increased oxidative damage (35-45% increase compared to control conditions), while mussels held at high RH were not impacted, even after 20 h of air exposure. Following re-immersion, reoxygenation stress resulted in further increases in damage markers in mussels that had experienced dryer emersion conditions; protective action of antioxidants increased steadily during the 10 h re-immersion period, apparently supporting a reduction in damage markers after 1-5 h of immersion. Clearly, conditions during emersion, as well as duration, substantially influence physiological performance and recovery of juvenile mussels. Successful recruitment to intertidal beds or survival in commercial aquaculture operations may be mediated by the nature of emersion stress experienced by these vulnerable juveniles.

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.

Microplastic ingestion induces asymmetry and oxidative stress in larvae of the sea urchin Pseudechinus huttoni.

Determining the effects of microplastic (MP) ingestion by marine organisms, especially during the sensitive larval stages, is an important step in understanding wider ecosystem responses. We investigated the ingestion, retention (1-5 µm), and short-term exposure effects (1-4 µm) of spherical MPs by larvae of the sea urchin Pseudechinus huttoni. Larvae ingested MPs in a dose-dependent manner and successfully egested particles after a short retention period. Survival was not significantly affected by exposure to MPs over the 10-day experimental period, however, a teratogenic response in terms of delayed development resulted in an increase of larval arm asymmetry. Additionally, MP exposure resulted in oxidative damage to lipids and proteins in larval body tissue despite a significant upregulation of antioxidant defences. The findings indicate MP exposure may impair cellular function, leading to negative consequences for an organism's fitness and survival.

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.

Enhancing Salt Tolerance of Plants: From Metabolic Reprogramming to Exogenous Chemical Treatments and Molecular Approaches.

Plants grow on soils that not only provide support for root anchorage but also act as a reservoir of water and nutrients important for plant growth and development. However, environmental factors, such as high salinity, hinder the uptake of nutrients and water from the soil and reduce the quality and productivity of plants. Under high salinity, plants attempt to maintain cellular homeostasis through the production of numerous stress-associated endogenous metabolites that can help mitigate the stress. Both primary and secondary metabolites can significantly contribute to survival and the maintenance of growth and development of plants on saline soils. Existing studies have suggested that seed/plant-priming with exogenous metabolites is a promising approach to increase crop tolerance to salt stress without manipulation of the genome. Recent advancements have also been made in genetic engineering of various metabolic genes involved in regulation of plant responses and protection of the cells during salinity, which have therefore resulted in many more basic and applied studies in both model and crop plants. In this review, we discuss the recent findings of metabolic reprogramming, exogenous treatments with metabolites and genetic engineering of metabolic genes for the improvement of plant salt tolerance.

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.

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.

Structural Changes Induced by Pulsed Electric Fields Increase the Concentration of Volatiles Released in Red Onion (Allium cepa L. var. Red Pearl) Bulbs.

This study investigated whether pulsed electric field (PEF) treatment can induce structural changes of whole, intact red onion bulb (Allium cepa L. var. Red Pearl). Onion bulbs were treated at electric field strengths of 0.6 and 1.2 kV/cm combined with energy inputs of 6 and 60 kJ/kg at different onion orientations with respect to the high voltage electrode. Results showed that onion cells across all fleshy scales experienced uniform cell damage with a higher proportion (>80%) of non-metabolically viable cells after PEF treatment at 1.2 kV/cm when the root end was positioned facing toward the PEF electrode. The findings were supported by cryogenic-scanning electron micrographs (cryo-SEM), where the underlying storage circular cells were completely damaged owing to the PEF treatment. In this study, it was found that the treatment intensity of PEF to induce structural damage across all the scale layers of an onion bulb coincided with an increase in dipropyl disulfide (DPDS) released from the onion bulbs. Therefore, DPDS was used as a volatile marker indicating cellular disruption within whole, intact onion bulbs. A considerable increase of DPDS, up to 52-fold, was detected from PEF-treated onion bulbs compared to untreated bulbs.

Genome-wide identification and characterization of the metal tolerance protein (MTP) family in grape (Vitis vinifera L.).

Metal tolerance proteins (MTPs) play an important role in the transport of metals at the cellular, tissue and whole plant levels. In the present study, 11 MTP genes were identified and these clustered in three major sub-families Fe/Zn-MTP, Zn-MTP, and Mn-MTP, and seven groups, which are similar to the grouping of MTP genes in both Arabidopsis and rice. Vitis vinifera metal tolerance proteins (VvMTP) ranged from 366 to 1092 amino acids, were predicted to be located in the cell vacuole, and had four to six putative TMDs, except for VvtMTP12 and VvMTP1. The VvMTPs had putative cation diffusion facilitator (CDF) domains and the putative Mn-MTPs also had zinc transporter dimerization domains (ZD-domains). V. vinifera Mn-MTPs had gene structures and motif distributions similar to those of the Fe/Zn-MTP and Zn-MTP sub-families. The upstream regions of VvMTP genes had variable frequencies of cis-regulatory elements that could indicate regulation at different developmental stages and/or differential regulation in response to stress. Comparison of the VvMTP coding sequences with known miRNAs found in various plant species indicated the presence of 13 putative miRNAs, with 7 of these associated with VvMTPs. Temporal and spatial expression profiling indicates a potential role for VvMTP genes during growth and development in grape plants, as well as the involvement of these genes in plant responses to environmental stress, especially osmotic stress. The data generated from this study provides a basis for further investigation of the roles of MTP genes in grapes.

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.