Expression of the Arabidopsis redox-related LEA protein, SAG21 is regulated by ERF, NAC and WRKY transcription factors.

SAG21/LEA5 is an unusual late embryogenesis abundant protein in Arabidopsis thaliana, that is primarily mitochondrially located and may be important in regulating translation in both chloroplasts and mitochondria. SAG21 expression is regulated by a plethora of abiotic and biotic stresses and plant growth regulators indicating a complex regulatory network. To identify key transcription factors regulating SAG21 expression, yeast-1-hybrid screens were used to identify transcription factors that bind the 1685 bp upstream of the SAG21 translational start site. Thirty-three transcription factors from nine different families bound to the SAG21 promoter, including members of the ERF, WRKY and NAC families. Key binding sites for both NAC and WRKY transcription factors were tested through site directed mutagenesis indicating the presence of cryptic binding sites for both these transcription factor families. Co-expression in protoplasts confirmed the activation of SAG21 by WRKY63/ABO3, and SAG21 upregulation elicited by oligogalacturonide elicitors was partially dependent on WRKY63, indicating its role in SAG21 pathogen responses. SAG21 upregulation by ethylene was abolished in the erf1 mutant, while wound-induced SAG21 expression was abolished in anac71 mutants, indicating SAG21 expression can be regulated by several distinct transcription factors depending on the stress condition.

Roadmap for the next decade of plant programmed cell death research.

Programmed cell death (PCD) is fundamentally important for plant development, abiotic stress responses and immunity, but our understanding of its regulation remains fragmented. Building a stronger research community is required to accelerate progress in this area through knowledge exchange and constructive debate. In this Viewpoint, we aim to initiate a collective effort to integrate data across a diverse set of experimental models to facilitate characterisation of the fundamental mechanisms underlying plant PCD and ultimately aid the development of a new plant cell death classification system in the future. We also put forward our vision for the next decade of plant PCD research stemming from discussions held during the 31st New Phytologist workshop, 'The Life and Death Decisions of Plant Cells' that took place at University College Dublin in Ireland (14-15 June 2023). We convey the key areas of significant progress and possible future research directions identified, including resolving the spatiotemporal control of cell death, isolation of its molecular and genetic regulators, and harnessing technical advances for studying PCD events in plants. Further, we review the breadth of potential impacts of plant PCD research and highlight the promising new applications of findings from this dynamically evolving field.

Biochemical and molecular changes in peach fruit exposed to cold stress conditions.

Storage or transportation temperature is very important for preserving the quality of fruit. However, low temperature in sensitive fruit such as peach can induce loss of quality. Fruit exposed to a specific range of temperatures and for a longer period can show chilling injury (CI) symptoms. The susceptibility to CI at low temperature varies among cultivars and genetic backgrounds. Along with agronomic management, appropriate postharvest management can limit quality losses. The importance of correct temperature management during postharvest handling has been widely demonstrated. Nowadays, due to long-distance markets and complex logistics that require multiple actors, the management of storage/transportation conditions is crucial for the quality of products reaching the consumer.Peach fruit exposed to low temperatures activate a suite of physiological, metabolomic, and molecular changes that attempt to counteract the negative effects of chilling stress. In this review an overview of the factors involved, and plant responses is presented and critically discussed. Physiological disorders associated with CI generally only appear after the storage/transportation, hence early detection methods are needed to monitor quality and detect internal changes which will lead to CI development. CI detection tools are assessed: they need to be easy to use, and preferably non-destructive to avoid loss of products.

How far can omics go in unveiling the mechanisms of floral senescence?

Floral senescence is of fundamental interest in understanding plant developmental regulation, it is of ecological and agricultural interest in relation to seed production, and is of key importance to the production of cut flowers. The biochemical changes occurring are well-studied and involve macromolecular breakdown and remobilisation of nutrients to developing seeds or other young organs in the plant. However, the initiation and regulation of the process and inter-organ communication remain to be fully elucidated. Although ethylene emission, which becomes autocatalytic, is a key regulator in some species, in other species it appears not to be as important. Other plant growth regulators such as cytokinins, however, seem to be important in floral senescence across both ethylene sensitive and insensitive species. Other plant growth regulators are also likely involved. Omics approaches have provided a wealth of data especially in ornamental species where genome data is lacking. Two families of transcription factors: NAC and WRKY emerge as major regulators, and omics information has been critical in understanding their functions. Future progress would greatly benefit from a single model species for understanding floral senescence; however, this is challenging due to the diversity of regulatory mechanisms. Combining omics data sets can be powerful in understanding different layers of regulation, but in vitro biochemical and or genetic analysis through transgenics or mutants is still needed to fully verify mechanisms and interactions between regulators.

Storage of halved strawberry fruits affects aroma, phytochemical content and gene expression, and is affected by pre-harvest factors.

Introduction: Strawberry fruit are highly valued for their aroma which develops during ripening. However, they have a short shelf-life. Low temperature storage is routinely used to extend shelf-life for transport and storage in the supply chain, however cold storage can also affect fruit aroma. Some fruit continue to ripen during chilled storage; however, strawberries are a non-climacteric fruit and hence ripening postharvest is limited. Although most strawberry fruit is sold whole, halved fruit is also used in ready to eat fresh fruit salads which are of increasing consumer demand and pose additional challenges to fresh fruit storage. Methods: To better understand the effects of cold storage, volatilomic and transcriptomic analyses were applied to halved Fragaria x ananassa cv. Elsanta fruit stored at 4 or 8°C for up to 12 days over two growing seasons. Results and discussion: The volatile organic compound (VOC) profile differed between 4 or 8°C on most days of storage. Major differences were detected between the two different years of harvest indicating that aroma change at harvest and during storage is highly dependent on environmental factors during growth. The major component of the aroma profile in both years was esters. Over 3000 genes changed in expression over 5 days of storage at 8°C in transcriptome analysis. Overall, phenylpropanoid metabolism, which may also affect VOCs, and starch metabolism were the most significantly affected pathways. Genes involved in autophagy were also differentially expressed. Expression of genes from 43 different transcription factor (TF) families changed in expression: mostly they were down-regulated but NAC and WRKY family genes were mainly up-regulated. Given the high ester representation amongst VOCs, the down-regulation of an alcohol acyl transferase (AAT) during storage is significant. A total of 113 differentially expressed genes were co-regulated with the AAT gene, including seven TFs. These may be potential AAT regulators.

Comparative transcriptomic profiling of peach and nectarine cultivars reveals cultivar-specific responses to chilled postharvest storage.

Introduction: Peach (Prunus persica (L.) Batsch,) and nectarine fruits (Prunus persica (L.) Batsch, var nectarine), are characterized by a rapid deterioration at room temperature. Therefore, cold storage is widely used to delay fruit post-harvest ripening and extend fruit commercial life. Physiological disorders, collectively known as chilling injury, can develop typically after 3 weeks of low-temperature storage and affect fruit quality. Methods: A comparative transcriptomic analysis was performed to identify regulatory pathways that develop before chilling injury symptoms are detectable using next generation sequencing on the fruits of two contrasting cultivars, one peach (Sagittaria) and one nectarine, (Big Top), over 14 days of postharvest cold storage. Results: There was a progressive increase in the number of differentially expressed genes between time points (DEGs) in both cultivars. More (1264) time point DEGs were identified in 'Big Top' compared to 'Sagittaria' (746 DEGs). Both cultivars showed a downregulation of pathways related to photosynthesis, and an upregulation of pathways related to amino sugars, nucleotide sugar metabolism and plant hormone signal transduction with ethylene pathways being most affected. Expression patterns of ethylene related genes (including biosynthesis, signaling and ERF transcription factors) correlated with genes involved in cell wall modification, membrane composition, pathogen and stress response, which are all involved later during storage in development of chilling injury. Discussion: Overall, the results show that common pathways are activated in the fruit of 'Big Top' nectarine and 'Sagittaria' peach in response to cold storage but include also differences that are cultivar-specific responses.

From laboratory to industrial storage - Translating volatile organic compounds into markers for assessing garlic storage quality.

Garlic (Allium sativum L.) has long been grown for its culinary and health-promoting qualities. The seasonal nature of garlic cropping requires that bulbs be stored for many months after harvest to ensure a year-round supply. During this time, quality is known to deteriorate, and efforts have been made to improve the longevity of stored bulbs. Cold temperatures within the stores prolong shelf life, but fine temperature control is needed to avoid freezing damage or cold induced stress. Here, quality traits (alliinase activity, firmness, and water content) are measured in response to a 96 h - 5 °C cold stress, to simulate the effect of non-isothermic temperature control in a - 1.5 °C warehouse. Volatile organic compounds (VOCs) are measured by thermal desorption gas chromatography time of flight mass spectrometry to identify markers of non-isothermic storage in garlic. 129 compounds were putatively identified and four (L-lactic acid, 2,6-dimethylhetpadecane, 4-methyldodecane, and methylcyclopentane) showed high predictive accuracy for cold stress. VOCs were also sampled directly from a cold storage facility and the whole profile discriminated between sampling time points. Five VOCS were highly predictive for storage time in the warehouse but were different to VOCs previously shown to discriminate between storage times in a laboratory setting. This indicates the need for realistic warehouse experiments to test quality markers.

Fruitomics: The Importance of Combining Sensory and Chemical Analyses in Assessing Cold Storage Responses of Six Peach (Prunus persica L. Batsch) Cultivars.

Cold storage is used to extend peach commercial life, but can affect quality. Quality changes are assessed through the content of nutritionally relevant compounds, aroma, physical characters and/or sensorially. Here, six peach and nectarine cultivars were sampled at commercial harvest and after 7 days of 1 °C storage. A trained panel was used to evaluate sensorial characters, while carotenoids, phenolics, vitamin C, total sugars, and qualitative traits including firmness, titrable acidity and soluble solid content were integrated with volatile organic compound (VOC) analysis previously reported. The different analyses reveal interesting patterns of correlation, and the six cultivars responded differently to cold storage. Sensory parameters were correlated with 64 VOCs and seven intrinsic characters. Acidity, firmness, and 10 VOCs were strongly negatively correlated with harmony and sweetness, but positively correlated with bitterness, astringency, and crunchiness. In contrast, Brix, b-carotene, and six VOCs were positively correlated with harmony and sweetness.

Cross-Cultural Differences between Italian and UK Consumer Preferences for 'Big Top' Nectarines in Relation to Cold Storage.

Nectarines are perishable fruits grown in Southern Europe, valued for their sensorial properties. Chilling is used in the supply chain for Northern European consumers, while Southern European consumers can access fresh, locally grown fruit or cold-stored supermarket fruit. Cold storage and fruit ripening affect texture and flavour. Here a consumer survey and hedonic testing compared the appreciation of nectarines (cv. Big Top) in Italy and at two UK sites (n = 359). Fruit was at the commercial harvest stage, or stored at 1 °C or 5 °C for seven days, then sampled after two days' (Italy and one UK site) or four days' (second UK site) ambient recovery. In the consumer survey, the most important factors involved in purchase decision were ripeness, texture, colour, taste and price. Named varieties were more important to Italian than UK respondents, whilst ripeness, price, taste, blemishes, aroma, and 'best before date' were more important in the UK. In sensory analyses, fruits at the commercial harvest stage were preferred to those stored at 1 °C. Preference for the 5 °C stored peaches depended on recovery time. Distinct clusters of peach sensorial attributes were positively or negatively linked to hedonic rating. Factors important in purchase decisions did not affect hedonic rating in the tasting.

Forage grass growth under future climate change scenarios affects fermentation and ruminant efficiency.

With an increasing human population access to ruminant products is an important factor in global food supply. While ruminants contribute to climate change, climate change could also affect ruminant production. Here we investigated how the plant response to climate change affects forage quality and subsequent rumen fermentation. Models of near future climate change (2050) predict increases in temperature, CO2, precipitation and altered weather systems which will produce stress responses in field crops. We hypothesised that pre-exposure to altered climate conditions causes compositional changes and also primes plant cells such that their post-ingestion metabolic response to the rumen is altered. This "stress memory" effect was investigated by screening ten forage grass varieties in five differing climate scenarios, including current climate (2020), future climate (2050), or future climate plus flooding, drought or heat shock. While varietal differences in fermentation were detected in terms of gas production, there was little effect of elevated temperature or CO2 compared with controls (2020). All varieties consistently showed decreased digestibility linked to decreased methane production as a result of drought or an acute flood treatment. These results indicate that efforts to breed future forage varieties should target tolerance of acute stress rather than long term climate.

Senescence in dahlia flowers is regulated by a complex interplay between flower age and floret position.

Mechanisms regulating flower senescence are not fully understood in any species and are particularly complex in composite flowers. Dahlia (Dahlia pinnata Cav.) florets develop sequentially, hence each composite flower head includes florets of different developmental stages as the whole flower head ages. Moreover, the wide range of available cultivars enables assessment of intraspecific variation. Transcriptomes were compared amongst inner (younger) and outer (older) florets of two flower head ages to assess the effect of floret vs. flower head ageing. More gene expression, including ethylene and cytokinin pathway expression changed between inner and outer florets of older flower heads than between inner florets of younger and older flower heads. Additionally, based on Arabidopsis network analysis, different patterns of co-expressed ethylene response genes were elicited. This suggests that changes occur in young inner florets as the whole flower head ages that are different to ageing florets within a flower head. In some species floral senescence is orchestrated by the plant growth regulator ethylene. However, there is both inter and intra-species variation in its importance. There is a lack of conclusive data regarding ethylene sensitivity in dahlia. Speed of senescence progression, effects of ethylene signalling perturbation, and patterns of ethylene biosynthesis gene expression differed across three dahlia cultivars ('Sylvia', 'Karma Prospero' and 'Onesta') suggesting differences in the role of ethylene in their floral senescence, while effects of exogenous cytokinin were less cultivar-specific.

Molecular Analysis of the E2F/DP Gene Family of Daucus carota and Involvement of the DcE2F1 Factor in Cell Proliferation.

E2F transcription factors are key components of the RB/E2F pathway that, through the action of cyclin-dependent kinases, regulates cell cycle progression in both plants and animals. Moreover, plant and animal E2Fs have also been shown to regulate other cellular functions in addition to cell proliferation. Based on structural and functional features, they can be divided into different classes that have been shown to act as activators or repressors of E2F-dependent genes. Among the first plant E2F factors to be reported, we previously described DcE2F1, an activating E2F which is expressed in cycling carrot (Daucus carota) cells. In this study, we describe the identification of the additional members of the E2F/DP family of D. carota, which includes four typical E2Fs, three atypical E2F/DEL genes, and three related DP genes. Expression analyses of the carrot E2F and DP genes reveal distinctive patterns and suggest that the functions of some of them are not necessarily linked to cell proliferation. DcE2F1 was previously shown to transactivate an E2F-responsive promoter in transient assays but the functional role of this protein in planta was not defined. Sequence comparisons indicate that DcE2F1 could be an ortholog of the AtE2FA factor of Arabidopsis thaliana. Moreover, ectopic expression of the DcE2F1 cDNA in transgenic Arabidopsis plants is able to upregulate AtE2FB and promotes cell proliferation, giving rise to polycotyly with low frequency, effects that are highly similar to those observed when over-expressing AtE2FA. These results indicate that DcE2F1 is involved in the control of cell proliferation and plays important roles in the regulation of embryo and plant development.

Fruit volatilome profiling through GC × GC-ToF-MS and gene expression analyses reveal differences amongst peach cultivars in their response to cold storage.

Peaches have a short shelf life and require chilling during storage and transport. Peach aroma is important for consumer preference and determined by underlying metabolic pathways and gene expression. Differences in aroma (profiles of volatile organic compounds, VOCs) have been widely reported across cultivars and in response to cold storage. However, few studies used intact peaches, or used equilibrium sampling methods subject to saturation. We analysed VOC profiles using TD-GC × GC-ToF-MS and expression of 12 key VOC pathway genes of intact fruit from six cultivars (three peaches, three nectarines) before and after storage at 1 °C for 7 days including 36 h shelf life storage at 20 °C. Two dimensional GC (GC × GC) significantly enhances discrimination of thermal desorption gas chromatography time-of-flight mass spectrometry (TD-GC-ToF-MS) and detected a total of 115 VOCs. A subset of 15 VOCs from analysis with Random Forest discriminated between cultivars. Another 16 VOCs correlated strongly with expression profiles of eleven key genes in the lipoxygenase pathway, and both expression profiles and VOCs discriminated amongst cultivars, peach versus nectarines and between treatments. The cultivar-specific response to cold storage underlines the need to understand more fully the genetic basis for VOC changes across cultivars.

Mutation of Arabidopsis Copper-Containing Amine Oxidase Gene AtCuAOδ Alters Polyamines, Reduces Gibberellin Content and Affects Development.

Polyamines (PAs) are essential metabolites in plants performing multiple functions during growth and development. Copper-containing amine oxidases (CuAOs) catalyse the catabolism of PAs and in Arabidopsis thaliana are encoded by a gene family. Two mutants of one gene family member, AtCuAOδ, showed delayed seed germination, leaf emergence, and flowering time. The height of the primary inflorescence shoot was reduced, and developmental leaf senescence was delayed. Siliques were significantly longer in mutant lines and contained more seeds. The phenotype of AtCuAOδ over-expressors was less affected. Before flowering, there was a significant increase in putrescine in AtCuAOδ mutant leaves compared to wild type (WT), while after flowering both spermidine and spermine concentrations were significantly higher than in WT leaves. The expression of GA (gibberellic acid) biosynthetic genes was repressed and the content of GA1, GA7, GA8, GA9, and GA20 was reduced in the mutants. The inhibitor of copper-containing amine oxidases, aminoguanidine hydrochloride, mimicked the effect of AtCuAOδ mutation on WT seed germination. Delayed germination, reduced shoot height, and delayed flowering in the mutants were rescued by GA3 treatment. These data strongly suggest AtCuAOδ is an important gene regulating PA homeostasis, and that a perturbation of PAs affects plant development through a reduction in GA biosynthesis.

MSH2 and MSH6 in Mismatch Repair System Account for Soybean (Glycine max (L.) Merr.) Tolerance to Cadmium Toxicity by Determining DNA Damage Response.

Our aim was to investigate DNA mismatch repair (MMR) genes regulating cadmium tolerance in two soybean cultivars. Cultivars Liaodou 10 (LD10, Cd-sensitive) and Shennong 20 (SN20, Cd-tolerant) seedlings were grown hydroponically on Murashige and Skoog (MS) media containing 0-2.5 mg·L-1 Cd for 4 days. Cd stress induced less random amplified polymorphism DNA (RAPD) polymorphism in LD10 than in SN20 roots, causing G1/S arrest in LD10 and G2/M arrest in SN20 roots. Virus-induced gene silencing (VIGS) of MLH1 in LD10-TRV-MLH1 plantlets showed markedly diminished G1/S arrest but enhanced root length/area under Cd stress. However, an increase in G1/S arrest and reduction of G2/M arrest occurred in SN20-TRV-MSH2 and SN20-TRV-MSH6 plantlets with decreased root length/area under Cd stress. Taken together, we conclude that the low expression of MSH2 and MSH6, involved in the G2/M arrest, results in Cd-induced DNA damage recognition bypassing the MMR system to activate G1/S arrest with the assistance of MLH1. This then leads to repressed root growth in LD10, explaining the intervarietal difference in Cd tolerance in soybean.

Short-Term Post-Harvest Stress that Affects Profiles of Volatile Organic Compounds and Gene Expression in Rocket Salad During Early Post-Harvest Senescence.

Once harvested, leaves undergo a process of senescence which shares some features with developmental senescence. These include changes in gene expression, metabolites, and loss of photosynthetic capacity. Of particular interest in fresh produce are changes in nutrient content and the aroma, which is dependent on the profile of volatile organic compounds (VOCs). Leafy salads are subjected to multiple stresses during and shortly after harvest, including mechanical damage, storage or transport under different temperature regimes, and low light. These are thought to impact on later shelf life performance by altering the progress of post-harvest senescence. Short term stresses in the first 24 h after harvest were simulated in wild rocket (Diplotaxis tenuifolia). These included dark (ambient temperature), dark and wounding (ambient temperature), and storage at 4 °C in darkness. The effects of stresses were monitored immediately afterwards and after one week of storage at 10 °C. Expression changes in two NAC transcription factors (orthologues of ANAC059 and ANAC019), and a gene involved in isothiocyanate production (thiocyanate methyltransferase, TMT) were evident immediately after stress treatments with some expression changes persisting following storage. Vitamin C loss and microbial growth on leaves were also affected by stress treatments. VOC profiles were differentially affected by stress treatments and the storage period. Overall, short term post-harvest stresses affected multiple aspects of rocket leaf senescence during chilled storage even after a week. However, different stress combinations elicited different responses.

Expression of Arabidopsis WEE1 in tobacco induces unexpected morphological and developmental changes.

WEE1 regulates the cell cycle by inactivating cyclin dependent protein kinases (CDKs) via phosphorylation. In yeast and animal cells, CDC25 phosphatase dephosphorylates the CDK releasing cells into mitosis, but in plants, its role is less clear. Expression of fission yeast CDC25 (Spcdc25) in tobacco results in small cell size, premature flowering and increased shoot morphogenetic capacity in culture. When Arath;WEE1 is over-expressed in Arabidopsis, root apical meristem cell size increases, and morphogenetic capacity of cultured hypocotyls is reduced. However expression of Arath;WEE1 in tobacco plants resulted in precocious flowering and increased shoot morphogenesis of stem explants, and in BY2 cultures cell size was reduced. This phenotype is similar to expression of Spcdc25 and is consistent with a dominant negative effect on WEE1 action. Consistent with this putative mechanism, WEE1 protein levels fell and CDKB levels rose prematurely, coinciding with early mitosis. The phenotype is not due to sense-mediated silencing of WEE1, as overall levels of WEE1 transcript were not reduced in BY2 lines expressing Arath;WEE1. However the pattern of native WEE1 transcript accumulation through the cell cycle was altered by Arath;WEE1 expression, suggesting feedback inhibition of native WEE1 transcription.

Nuclear Migration: An Indicator of Plant Salinity Tolerance in vitro.

In order to understand the mechanisms underlying acquisition of tolerance to salinity, we recently produced callus tissues of tobacco and Medicago truncatula resistant to NaCl-induced salt stress following application of a step-up recurrent selection method. The effects of salinity on cell size are known, but those on cell morphometry including cell and nuclear surface area and position of nuclei within salt stress resistant cells were never studied before. This work fills that gap, using suspension cultured cells of M. truncatula A17 initiated from callus, and Nicotiana tabacum BY-2 cell line resistant to increasing NaCl concentrations up to 150 mM NaCl. The surface area of salinity resistant cells of M. truncatula A17 and N. tabacum BY2 and their nuclei, produced by step-up recurrent selection, were reduced, and cells elongated as NaCl increased, but these parameters proved to be unreliable in explaining cell survival and growth at high NaCl. Conversely, nuclei of resistant cells migrated from the center to the periphery of the cytoplasm close to the walls. Nuclear marginalization was for the first time observed as a result of salt stress in plant cells, and could be a novel helpful morphological marker of acquisition of salinity tolerance.

5-Azacytidine promotes shoot regeneration during Agrobacterium-mediated soybean transformation.

Agrobacterium-mediated soybean transformation has been greatly improved in recent years, however the transformation efficiency is still low and highly genotype-dependent when compared to other species. Here, we characterized seventeen soybean genotypes based on their genetic transformation efficiencies, i.e., high and low, during Agrobacterium-mediated transformation. To reveal the molecular basis of this transformation difference, we constructed a highly efficient transient transgene expression system using soybean cotyledon protoplasts and then assess the methylation levels of promoter and coding regions of an EYFP (enhanced yellow fluorescent protein) gene introduced into the protoplast cultures of various soybean genotypes using BSP (bisulfite sequencing PCR). Increased methylation was found to be associated with the considerably decreased transfection efficiency (as percentage of EYFP fluorescent protoplasts) in low-efficacy genotypes as compared with those in high-efficacy on three DAT (day after transfection). 5-Azacytidine (5-Azac), a demethylating reagent commonly applied in epigenetic researches, significantly improved the transient transfection efficiency and transgene expression level in low-efficiency genotypes. Furthermore, the shoot regeneration efficiency in low-efficiency genotypes was substantially increased by 5-Azac treatment in an Agrobacterium-mediated soybean transformation system. Taken together, we concluded that lower methylation level in transgene contributed to enhanced shoot regeneration in Agrobacterium-mediated soybean transformation.