ABA Levels Research Articles

Integrated Methylome and Transcriptome Analysis Provides Insights into the DNA Methylation Underlying the Mechanism of Cytoplasmic Male Sterility in Kenaf (Hibiscus cannabinus L.).

Cytoplasmic male sterility (CMS) is widely exploited in hybrid seed production. Kenaf is an important fiber crop with high heterosis. The molecular mechanism of kenaf CMS remains unclear, particularly in terms of DNA methylation. Here, using the anthers of a kenaf CMS line (P3A) and its maintainer line (P3B), comparative physiological, DNA methylation, and transcriptome analyses were performed. The results showed that P3A had considerably lower levels of IAA, ABA, photosynthetic products and ATP contents than P3B. DNA methylome analysis revealed 650 differentially methylated genes (DMGs) with 313 up- and 337 down methylated, and transcriptome analysis revealed 1788 differentially expressed genes (DEGs) with 558 up- and 1230 downregulated genes in P3A compared with P3B. Moreover, 45 genes were characterized as both DEGs and DMGs, including AUX, CYP, BGL3B, SUS6, AGL30 and MYB21. Many DEGs may be regulated by related DMGs based on methylome and transcriptome studies. These DEGs were involved in carbon metabolism, plant hormone signal transduction, the TCA cycle and the MAPK signaling pathway and were shown to be important for CMS in kenaf. These results provide new insights into the epigenetic mechanism of CMS in kenaf and other crops.

Treatment of Winter Wheat (Triticum aestivum L.) Seeds with Electromagnetic Field Influences Germination and Phytohormone Balance Depending on Seed Size

Electromagnetic field (EMF) and its effect on crop plant growth and their quality parameters is increasingly gaining the interest of researchers in agronomic science. However, the exact mechanism of EMF action in plant cells is still unclear. Among the completely unexplored parameters is the relationship between the EMF effects and the seed size. Thus, the EMF effect was analyzed in winter wheat seeds categorized into two size groups, small and big. The study focused on the germination kinetics, early growth parameters, and phytohormone concentrations (indole-3-acetic acid, IAA and abscisic acid, ABA) in seeds, roots, and coleoptiles after exposure to EMFs (50 Hz, 7 mT) and their controls. EMF exposure resulted in faster germination and the more rapid early growth of organs, especially in big seeds in dark conditions. The faster germination and seedling growth of small seeds in control conditions, and of big seeds after EMF exposure, corresponds largely to the decline in IAA and ABA levels. This study confirms that presowing treatment with an EMF is a promising tool for sustainable seed crop improvement, but detailed studies on the EMF mechanism of action, including phytohormones, are necessary to better control future crop yield, especially considering the factor of seed size.

How do plants remember drought?

Plants develop both short-term and transgenerational memory of drought stress through epigenetic regulation of transcription for a better response to subsequent exposure. Recurrent spells of droughts are more common than a single drought, with intermittent moist recovery intervals. While the detrimental effects of the first drought on plant structure and physiology are unavoidable, if survived, plants can memorize the first drought to present a more robust response to the following droughts. This includes a partial stomatal opening in the watered recovery interval, higher levels of osmoprotectants and ABA, and attenuation of photosynthesis in the subsequent exposure. Short-term drought memory is regulated by ABA and other phytohormone signaling with transcriptional memory behavior in various genes. High levels of methylated histones are deposited at the drought-tolerance genes. During the recovery interval, the RNA polymerase is stalled to be activated by a pause-breaking factor in the subsequent drought. Drought leads to DNA demethylation near drought-response genes, with genetic control of the process. Progenies of the drought-exposed plants can better adapt to drought owing to the inheritance of particular methylation patterns. However, a prolonged watered recovery interval leads to loss of drought memory, mediated by certain demethylases and chromatin accessibility factors. Small RNAs act as critical regulators of drought memory by altering transcript levels of drought-responsive target genes. Further studies in the future will throw more light on the genetic control of drought memory and the interplay of genetic and epigenetic factors in its inheritance. Plants from extreme environments can give queues to understanding robust memory responses at the ecosystem level.

Dynamics of endogenous levels and subcellular localization of ABA and cytokinins during pollen germination in spruce and tobacco.

We used the enzyme-linked immunosorbent assay (ELISA) to assess the level of endogenous hormones in spruce pollen, and immunolocalization and confocal microscopy to study hormone localization in spruce and tobacco pollen. During pollen activation, the levels of ABA, zeatin, and its riboside significantly decreased. After the initiation of polar growth, the levels of all cytokinins increased sharply; ABA level also increased. In dormant spruce pollen grains, zeatin and ABA were localized uniformly throughout the cytoplasm. Zeatin was not detected in the nuclei, and the antheridial cell showed higher levels than the vegetative cell; ABA signal was detected in the cytoplasm and the nuclei. In germinating pollen, both hormones were detected mainly in plastids. The similar pattern was found in growing pollen tubes; signal from ABA also had a noticeable level in the cytosol of the tube cell, and was weaker in the antheridial cell. Zeatin fluorescence, on the other hand, was more pronounced in the antheridial cell. In non-germinated grains of tobacco, zeatin was localized mainly in organelles. ABA in dormant pollen grains demonstrated uniform localization, including the nuclei and cytoplasm of both cells. After germination, zeatin was accumulated in the plasmalemma or cell wall. ABA signal in the cytoplasm decreased; in the nuclei, it remained high. In growing tubes, the strongest zeatin and ABA signals were observed at the plasma membrane. The differences in ABA and cytokinin localization between species and dynamic changes in their level in spruce pollen highlight the key spatial and temporal parameters of hormonal regulation of gymnosperm pollen germination.

Transcription factor ANAC004 enhances Cd tolerance in Arabidopsis thaliana by regulating cell wall fixation, translocation and vacuolar detoxification of Cd, ABA accumulation and antioxidant capacity

Cadmium (Cd) is toxic to plants, which have evolved multiple strategies to cope with Cd stress. In this study, we identified a nucleus-localized NAC-type transcription factor, ANAC004, which is induced by Cd and involved in regulating Cd resistance in Arabidopsis. First, anac004 mutants exhibited Cd sensitive phenotype and accumulated more Cd (12–23% higher than wild type in roots and shoots); plants overexpressing ANAC004 showed the opposite phenotype and with lower Cd accumulation. Second, ANAC004 enhanced Cd fixation in cell wall hemicellulose, thus reducing Cd2+ influx into root cells. Third, ANAC004 was involved in the process of vacuolar Cd compartmentalization by regulating the genes associated with Cd detoxification (PCS1/2, NAS4, ABCC1/2/3, MTP1/3, IREG2 and NRAMP3/4). Fourth, ANAC004 reduced root-to-shoot Cd translocation through down-regulated Cd translocation-related genes (HMA2 and HMA4). Finally, the expression of genes related to ABA synthesis (AAO3, MCSU, and NCED3) and the activities of antioxidant enzymes (SOD, POD and CAT) were all reduced in anac004 mutants, leading to reduced levels of endogenous ABA and increased accumulation of reactive oxygen species (O2.− and H2O2) and MDA, which ultimately weakened resistance to Cd. Our results suggest that ANAC004 decreases Cd accumulation in Arabidopsis through enhancing cell wall Cd immobilization, increasing vacuolar Cd detoxification, and inhibiting Cd translocation, thus improving Cd resistance, processes that might be mediated by ABA signaling and antioxidant defense systems.

Induction of polyploid Malus prunifolia and analysis of its salt tolerance.

The apple rootstock Malus prunifolia (Willd.) Borkh. is widely used for apple production. Because polyploid plants are often more tolerant to abiotic stress than diploids, we wondered whether polyploidy induction in M. prunifolia might improve its stress tolerance, particularly to high salinity. We used a combination of colchicine and dimethyl sulfoxide (DMSO) to induce chromosome doubling in M. prunifolia and identified the resulting polyploids by stomatal observations and flow cytometry. We found the best way to induce polyploidy in M. prunifolia was to use 2% DMSO and 0.05% colchicine for 2days for leaves or 0.02% colchicine for stem segments. The results of hydroponic salt treatment showed that polyploid plants were more salt tolerant and had greater photosynthetic efficiency, thicker leaf epidermis and palisade tissues, and shorter but denser root systems than diploids. During salt stress, the polyploid leaves and roots accumulated less Na+, showed upregulated expression of three salt overly sensitive (SOS) pathway genes, and produced fewer reactive oxygen species. The polyploid plants also had considerably higher ABA and jasmonic acid levels than diploid plants under salt stress. Under normal growth conditions, gibberellins (GAs) levels were much lower in polyploid leaves than in diploid leaves; however, after salt treatment, polyploid leaves showed upregulation of essential GAs synthesis genes. In summary, we developed a system for the induction of polyploidy in M. prunifolia and response to salt stress of the resulting polyploids, as reflected in leaf and root morphology, changes in Na+ accumulation, antioxidant capacity and plant hormone levels.

Changes of Fruit Abscission and Carbohydrates, Hormones, Related Gene Expression in the Fruit and Pedicel of Macadamia under Starvation Stress

In order toexplore the regulation mechanism of macadamia fruitlet abscission induced by ‘starvation stress’, a treatment of girdling and defoliation was applied to the bearing shoots of macadamia cultivar ‘H2’ at the early stage of fruit development, simulating the starvation stress induced by interrupting carbon supply to fruit. The levels of carbohydrates, hormones, and related gene expression in the different tissues (husk, seed, and pedicel) were investigated after treatment. The results showed that a severe fruit drop occurred 3~5 d after starvation stress treatment. The contents of glucose, fructose, and sucrose in both the husk and the seed were significantly decreased, as well as the fructose and sucrose in the pedicel; this large reduction occurred prior to the massive fruit shedding. Starvation stress significantly reduced the GA3 and ZR contents and enhanced the ABA level in the pedicel and the seed, whereas it did not obviously change these hormones in the husk. After treatment, IAA content decreased considerably in both the husk and seed but increased remarkably in the pedicel. In the husk, the expression of genes related to sugar metabolism and signaling (NI, HXK2, TPS, and TPP), as well as the biosynthesis of ethylene (ACO2 and ACS) and ABA (NCED1.1 and AAO3), was significantly upregulated by starvation stress, as well as the stress-responsive transcription factors (AP2/ERF, HD-ZIP12, bZIP124, and ABI5), whereas the BG gene associated with ABA accumulation and the early auxin-responsive genes (Aux/IAA22 and GH3.9) were considerably suppressed during the period of massive fruit abscission. Similar changes in the expression of all genes occurred in the pedicel, except for NI and AP2/ERF, the expression of which was significantly upregulated during the early stage of fruit shedding and downregulated during the period of severe fruit drop. These results suggest that complicated crosstalk among the sugar, IAA, and ABA signaling may be related to macadamia fruitlet abscission induced by carbohydrate starvation.

Intervention of Fluency and Anxiety in Mindfulness Training of Shooting.

Introduction Mindfulness cognitive therapy is based on mindfulness decompression, integrating the elements of cognitive behavioral therapy and related psychological education components, a set of mindfulness group courses designed. Objective In order to explore the influence of mindfulness training on fluency and anxiety in shooting sports training. Methods There are 22 athletes in a provincial shooting team, 12 in the experimental group and 10 people in the control group, grouped according to the random principle. A single-participant experiment design with multiple baseline levels of ABA was adopted. Results The fluency state of the athletes has increased from 28.75 to 30.63; the average value before the intervention increased by 6.5%, PEM = 88%, explaining that the previous intervention has a moderate-intensity effect. The average value of athletes' sports competition anxiety state 205 before intervention was reduced to 171.25, reduced by 16.5%, PEM = 100%, showing that the intervention effect is very effective. Conclusions After the shooting athletes received the intervention of the mindfulness cognitive intervention method MBCT, the state of fluency is improved, the level of competition anxiety is reduced, and the experimental intervention basically confirmed the research hypothesis. This study confirms the moderating role of emotion regulation self-efficacy between mindfulness and the fluency of shooters and provides further impetus for the refinement and development of a push-up spiral model that explains mindfulness mechanisms.

Long-Term High-Temperature Stress Impacts on Embryo and Seed Development in Brassica napus.

Brassica napus (rapeseed) is the second most important oilseed crop worldwide. Global rise in average ambient temperature and extreme weather severely impact rapeseed seed yield. However, fewer research explained the phenotype changes caused by moderate-to-high temperatures in rapeseed. To investigate these events, we determined the long-term response of three spring cultivars to different temperature regimes (21/18°C, 28/18°C, and 34/18°C) mimicking natural temperature variations. The analysis focused on the plant appearance, seed yield, quality and viability, and embryo development. Our microscopic observations suggest that embryonic development is accelerated and defective in high temperatures. Reduced viable seed yield at warm ambient temperature is due to a reduced fertilization rate, increased abortion rate, defective embryonic development, and pre-harvest sprouting. Reduced auxin levels in young seeds and low ABA and auxin levels in mature seeds may cause embryo pattern defects and reduced seed dormancy, respectively. Glucosinolates and oil composition measurements suggest reduced seed quality. These identified cues help understand seed thermomorphogenesis and pave the way to developing thermoresilient rapeseed.

OsHIPL1, a hedgehog-interacting protein-like 1 protein, increases seed vigour in rice.

SummaryThe cultivation of rice varieties with high seed vigour is vital for the direct seeding of rice, and the molecular basis of regulation of seed vigour remains elusive. Here, we cloned a new gene OsHIPL1, which encodes hedgehog‐interacting protein‐like 1 protein as a causal gene of the major QTL qSV3 for rice seed vigour. OsHIPL1 was mainly localized in the plasma membrane and nucleus. RNA sequencing (RNA‐seq) revealed that the ABA‐related genes were involved in the OsHIPL1 regulation of seed vigour in rice. The higher levels of endogenous ABA were measured in germinating seeds of OsHIPL1 mutants and NIL‐qsv3 line compared to IR26 plants, with two up‐regulated ABA biosynthesis genes (OsZEP and OsNCED4) and one down‐regulated ABA catabolism gene OsABA8ox3. The expression of abscisic acid‐insensitive 3 (OsABI3), OsABI4 and OsABI5 was significantly up‐regulated in germinating seeds of OsHIPL1 mutants and NIL‐qsv3 line compared to IR26 plants. These results indicate that the regulation of seed vigour of OsHIPL1 may be through modulating endogenous ABA levels and altering OsABIs expression during seed germination in rice. Meanwhile, we found that OsHIPL1 interacted with the aquaporin OsPIP1;1, then affected water uptake to promote rice seed germination. Based on analysis of single‐nucleotide polymorphism data of rice accessions, we identified a Hap1 haplotype of OsHIPL1 that was positively correlated with seed germination. Our findings showed novel insights into the molecular mechanism of OsHIPL1 on seed vigour.

The Effective Role of Nano-Silicon Application in Improving the Productivity and Quality of Grafted Tomato Grown under Salinity Stress

This study aims to determine the influence of grafting and nano-silicon fertilizer on the growth and production of tomatoes (Solanumlycopersicum L.) under salinity conditions. A commercial tomato hybrid (cv. Strain B) was used as a scion and two tomato phenotypes were used as rootstocks: S. pimpinellifolium and Edkawy. The rootstock effect was evaluated by growing plants at two NaCl concentrations plus the control (0, 4000, and 8000 ppm NaCl). Nano-silicon foliar application (0.5 ppm) after 20, 28, and 36 days from transplanting was also used to mitigate salinity stress. Antioxidants, hormones, and proline were evaluated for a better understanding of the physiological changes induced by salinity and grafting. The results showed that grafting either on S. pimpinellifolium or Edkawy combined with nano-silicon application enhanced shoot and root growth, fruit yield, and fruit quality. The Edkawy rootstock was more effective than the S. pimpinellifolium rootstock in terms of counteracting the negative effect of salinity. Higher levels of mineral contents, GA3, ABA, and proline were detected in shoots that were subjected to grafting and nano-silicon application compared to the control treatment. This study indicates that grafting and nano-silicon application hold potential as alternative techniques to mitigate salt stress in commercial tomato cultivars.

Enhancement in Seed Priming-Induced Starch Degradation of Rice Seed Under Chilling Stress via GA-Mediated \u03b1-Amylase Expression

Chilling stress is the major abiotic stress that severely limited the seedling establishment of direct-seeded rice in temperate and sub-tropical rice production regions. While seed priming is an efficient pre-sowing seed treatment in enhancing crop establishment under abiotic stress. Our previous research has identified two seed priming treatments, selenium priming (Se) and salicylic priming (SA) that effectively improved the seed germination and seedling growth of rice under chilling stress. To further explore how seed priming enhance the starch degradation of rice seeds under chilling stress, the present study evaluated the effects of Se and SA priming on germination and seedling growth, α-amylase activity, total soluble sugar content, hormone content and associated gene relative expression under chilling stress. The results showed that both Se and SA priming significantly increased the seed germination and seedling growth attributes, and enhanced the starch degradation ability by increasing α-amylase activity and total soluble sugar content under chilling stress. Meanwhile, seed priming increased the transcription level of OsRamy1A, OsRamy3B that regulated by GA, and increased the transcription level of OsRamy3E that regulated by sugar signals. Furthermore, seed priming significantly improved the GA3 contents in rice seeds by up-regulating the expression of OsGA3ox1 and OsGA20ox1, and decreased the ABA content and the expression of OsNCED1, indicating that the improved starch degradation ability in primed rice seeds under chilling stress might be attributed to the increased GA3 and decreased ABA levels in primed rice seeds, which induced the expression of GA-mediated α-amylase. However, studies to explore how seed priming mediate hormonal metabolism and the expression of OsRamy3E are desperately needed.

Beta-Amylase and Phosphatidic Acid Involved in Recalcitrant Seed Germination of Chinese Chestnut.

Chinese chestnut (Castanea mollissima), a species with recalcitrant seeds, is an important source of nuts and forest ecosystem services. The germination rate of recalcitrant seeds is low in natural habitats and decreases under conditions of desiccation and low temperature. The germination rate of cultivated Chinese chestnut seeds is significantly higher than that of wild seeds. To explore the reasons for the higher germination rate of cultivated seeds in Chinese chestnut, 113,524 structural variants (SVs) between the wild and cultivated Chinese chestnut genomes were detected through genome comparison. Genotyping these SVs in 60 Chinese chestnut accessions identified allele frequency changes during Chinese chestnut domestication, and some SVs are overlapping genes for controlling seed germination. Transcriptome analysis revealed downregulation of the abscisic acid synthesis genes and upregulation of the beta-amylase synthesis genes in strongly selected genes of cultivated seeds. On the other hand, hormone and enzyme activity assays indicated a decrease in endogenous ABA level and an increase in beta-amylase activity in cultivated seeds. These results shed light on the higher germination rate of cultivated seeds. Moreover, phosphatidic acid synthesis genes are highly expressed in seed germination stages of wild Chinese chestnut and may play a role in recalcitrant seed germination. These findings provide new insight into the regulation of wild seed germination and promote natural regeneration and succession in forest ecosystems.

Identification of Key Gene Networks and Deciphering Transcriptional Regulators Associated With Peanut Embryo Abortion Mediated by Calcium Deficiency.

Peanut embryo development is easily affected by a variety of nutrient elements in the soil, especially the calcium level. Peanut produces abortive embryos in calcium-deficient soil, but underlying mechanism remains unclear. Thus, identifying key transcriptional regulators and their associated regulatory networks promises to contribute to a better understanding of this process. In this study, cellular biology and gene expression analyses were performed to investigate peanut embryo development with the aim to discern the global architecture of gene regulatory networks underlying peanut embryo abortion under calcium deficiency conditions. The endomembrane systems tended to disintegrate, impairing cell growth and starch, protein and lipid body accumulation, resulting in aborted seeds. RNA-seq analysis showed that the gene expression profile in peanut embryos was significantly changed under calcium deficiency. Further analysis indicated that multiple signal pathways were involved in the peanut embryo abortion. Differential expressed genes (DEGs) related to cytoplasmic free Ca2+ were significantly altered. DEGs in plant hormone signaling pathways tended to be associated with increased IAA and ethylene but with decreased ABA, gibberellin, cytokinin, and brassinosteroid levels. Certain vital genes, including apoptosis-inducing factor, WRKYs and ethylene-responsive transcription factors, were up-regulated, while key regulators of embryo development, such as TCP4, WRI1, FUS3, ABI3, and GLK1 were down-regulated. Weighted gene co-expression network analysis (WGCNA) identified 16 significant modules associated with the plant hormone signaling, MAPK signaling, ubiquitin mediated proteolysis, reserve substance biosynthesis and metabolism pathways to decipher regulatory network. The most significant module was darkolivegreen2 and FUS3 (AH06G23930) had the highest connectivity among this module. Importantly, key transcription factors involved in embryogenesis or ovule development including TCP4, GLK1, ABI3, bHLH115, MYC2, etc., were also present in this module and down regulated under calcium deficiency. This study presents the first global view of the gene regulatory network involved in peanut embryo abortion under calcium deficiency conditions and lays foundation for improving peanut tolerances to calcium deficiency by a targeted manipulation of molecular breeding.

H2O2-Responsive Hormonal Status Involves Oxidative Burst Signaling and Proline Metabolism in Rapeseed Leaves.

Drought alters the level of endogenous reactive oxygen species (ROS) and hormonal status, which are both involved in the regulation of stress responses. To investigate the interplay between ROS and hormones in proline metabolism, rapeseed (Brassica napus L.) plants were exposed to drought or exogenous H2O2 (Exo-H2O2) treatment for 10 days. During the first 5 days, the enhanced H2O2 concentrations in drought treatment were associated with the activation of superoxide dismutase (SOD) and NADPH oxidase, with enhanced ABA and SA levels, while that in Exo-H2O2 treatment was mainly associated with SA-responsive POX. During the latter 5 days, ABA-dependent ROS accumulation was predominant with an upregulated oxidative signal-inducible gene (OXI1) and MAPK6, leading to the activation of ABA synthesis and the signaling genes (NCED3 and MYC2). During the first 5 days, the enhanced levels of P5C and proline were concomitant with SA-dependent NDR1-mediated signaling in both drought and Exo-H2O2 treatments. In the latter 5 days of drought treatment, a distinct enhancement in P5CR and ProDH expression led to higher proline accumulation compared to Exo-H2O2 treatment. These results indicate that SA-mediated P5C synthesis is highly activated under lower endogenous H2O2 levels, and ABA-mediated OXI1-dependent proline accumulation mainly occurs with an increasing ROS level, leading to ProDH activation as a hypersensitive response to ROS and proline overproduction under severe stress.

Synergistic Modulation of Seed Metabolites and Enzymatic Antioxidants Tweaks Moisture Stress Tolerance in Non-Cultivated Traditional Rice Genotypes during Germination.

Traditional rice landraces are treasures for novel genes to develop climate-resilient cultivars. Seed viability and germination determine rice productivity under moisture stress. The present study evaluated 100 rice genotypes, including 85 traditional landraces and 15 improved cultivars from various agro-ecological zones of Tamil Nadu, along with moisture-stress-susceptible (IR 64) and moisture-stress-tolerant (IR 64 Drt1) checks. The landraces were screened over a range of osmotic potentials, namely (−) 1.0 MPa, (−) 1.25 MPa and (−) 1.5 MPa, for a period of 5 days in PEG-induced moisture stress. Physio-morphological traits, such as rate of germination, root and shoot length, vigor index, R/S ratio and relative water content (RWC), were assessed during early moisture stress at the maximum OP of (−) 1.5 MPa. The seed macromolecules, phytohormones (giberellic acid, auxin (IAA), cytokinin and abscisic acid), osmolytes and enzymatic antioxidants (catalase and superoxide dismutase) varied significantly between moisture stress and control treatments. The genotype Kuliyadichan registered more IAA and giberellic acid (44% and 35%, respectively, over moisture-stress-tolerant check (IR 64 Drt1), whereas all the landraces showed an elevated catalase activity, thus indicating that the tolerant landraces effectively eliminate oxidative damages. High-performance liquid chromatography analysis showed a reduction in cytokinin and an increase in ABA level under induced moisture stress. Hence, the inherent moisture-stress tolerance of six traditional landraces, such as Kuliyadichan, Rajalakshmi, Sahbhagi Dhan, Nootripathu, Chandaikar and Mallikar, was associated with metabolic responses, such as activation of hydrolytic enzymes, hormonal crosstalk, ROS signaling and antioxidant enzymes (especially catalase), when compared to the susceptible check, IR 64. Hence, these traditional rice landraces can serve as potential donors for introgression or pyramiding moisture-stress-tolerance traits toward developing climate-resilient rice cultivars.

SlDREB3, a negative regulator of ABA responses, controls seed germination, fruit size and the onset of ripening in tomato

SlDREB3 was identified as a ripening up-regulated gene of the AP2/ERF-domain family of transcription factors. Its manipulation affects processes primarily governed by ABA. It negatively regulates ABA responses in tomato by altering ABA levels/signaling and is, in turn, negatively regulated by ABA. SlDREB3 over-expression lines show higher transcript levels of the ABA metabolism genes CYP707A3 and UGT75C1 and an 85% reduction in ABA levels leading to early seed germination. In contrast, suppression lines show decreased CYP707A3/UGT75C1 expression, 3-fold higher ABA levels and delayed germination. The expression of other ABA signaling and response genes is also affected. Suppression of SlDREB3 accelerates the onset of ripening by 4–5 days while its over-expression delays it and also reduces final fruit size. SlDREB3 manipulation effects large scale changes in the fruit transcriptome with suppression lines showing early increase in ABA levels and activation of most ripening pathway genes that govern ethylene, carotenoids and softening. Strikingly, key transcription factors like CNR, NOR, RIN, FUL1, governing ethylene-dependent and ethylene-independent aspects of ripening, are activated early upon SlDREB3 suppression suggesting their control by ABA. The studies identify SlDREB3 as a negative regulator of ABA responses across tissues and a key ripening regulator controlling ethylene-dependent and ethylene-independent aspects.

Effect of exogenous application of salt stress and glutamic acid on lettuce (Lactuca sativa L.)

• The combined effect of salinity and glutamic acid (GA) has been investigated in lettuce. • Yield, PSII, chlorophyll, proline and ABA levels were affected by salt stress. • ROS-scavenging genes were down-regulated under stress and not affected by GA. Salinity is a serious environmental issue which can negatively affect crop growth and productivity worldwide. Lettuce is generally considered as a salt-sensitive crop; however, different cultivars may have different adaptive mechanisms to this environmental stress. The application of biostimulants has proven to be a strategic strategy to improve plant responses to abiotic stresses and to foster resilience of crops during cultivation. This study intended to explore the physiological mechanisms underlying Romaine lettuce plant responses to salt stress, also in combination with the exogenous application of glutamic acid. The glutamic acid treatment was applied as foliar spray for the first time before salt exposure, followed by three applications during the stress. To understand the effect of salinity and glutamic acid treatment, different physiological and molecular analytical determinations were performed. High salinity induced a general stimulation of PSII and chlorophyll content. In particular, the performance index (+102%) and the number of reaction centres per cross section (+75,7%) increased, whereas the energy dissipation as heat per reaction centres (-32,1%) and the net rate of the centres’ closure (Mo) (-39.4%) decreased. Moreover, a reduction of yield (-26,5%) was observed in plants grown under high salinity. The concentration of proline was stimulated by salinity whereas ABA levels were reduced. The analyses of the genes encoding for ROS scavenging enzymes showed a general downregulation in response to salinity with the only exception of LsSOD . The application of the glutamic acid did not show a clear effect of the amino acid on lettuce plants, regardless the different growing conditions.

Infection by Moniliophthora perniciosa reprograms tomato Micro-Tom physiology, establishes a sink, and increases secondary cell wall synthesis.

Witches' broom disease of cacao is caused by the pathogenic fungus Moniliophthora perniciosa. By using tomato (Solanum lycopersicum) cultivar Micro-Tom (MT) as a model system, we investigated the physiological and metabolic consequences of M. perniciosa infection to determine whether symptoms result from sink establishment during infection. Infection of MT by M. perniciosa caused reductions in root biomass and fruit yield, a decrease in leaf gas exchange, and down-regulation of photosynthesis-related genes. The total leaf area and water potential decreased, while ABA levels, water conductance/conductivity, and ABA-related gene expression increased. Genes related to sugar metabolism and those involved in secondary cell wall deposition were up-regulated upon infection, and the concentrations of sugars, fumarate, and amino acids increased. 14C-glucose was mobilized towards infected MT stems, but not in inoculated stems of the MT line overexpressing CYTOKININ OXIDASE-2 (35S::AtCKX2), suggesting a role for cytokinin in establishing a sugar sink. The up-regulation of genes involved in cell wall deposition and phenylpropanoid metabolism in infected MT, but not in 35S::AtCKX2 plants, suggests establishment of a cytokinin-mediated sink that promotes tissue overgrowth with an increase in lignin. Possibly, M. perniciosa could benefit from the accumulation of secondary cell walls during its saprotrophic phase of infection.

Abstract OT1-18-05: A multicenter, open-label, phase 2 study of Imprime PGG and pembrolizumab in patients with metastatic breast cancer who have progressed through prior hormonal therapy

Abstract Immune checkpoint inhibitors (ICI) have shown limited response in breast cancers, especially the hormone receptor (HR) positive/human epidermal growth factor receptor 2-negative (Her2-) subtype that is considered to be ‘immunologically cold’ in comparison to the TNBC and Her2+ subtypes. Results from Keynote-028 study showed a ORR of 12% to anti-PD1 antibody, pembrolizumab (KEYTRUDA®; PEM) in previously treated HR+/HER2-/programmed death ligand 1-positive (PD-L1-positive) advanced breast cancer patients. With limited options available for these patients, there is a significant unmet need to expand the clinical benefit from ICI to increased numbers of patients. Imprime PGG (Imprime) is a novel beta glucan acting as a pathogen-associated molecular pattern (PAMP) that drives a cascade of immune activating events. Imprime treatment repolarizes the immunosuppressive microenvironment, activates the maturation of antigen presenting cells, and has significantly enhanced the efficacy of ICI therapy in several preclinical tumor models. Imprime has been studied in combination with PEM, in metastatic TNBC and advanced melanoma in a recently completed Ph2 trial (IMPRIME 1) where in TNBC pts the objective response rate (ORR) = 15.9%, disease control rate (DCR) = 54.5%, median duration of response (mDoR)=12.7mo, median progression free survival (mPFS) =2.86 mo, 12-month overall survival (OS) rate = 57.6%, and median OS= 16.4 months. Clinical benefit in IMPRIME 1 was particularly evident in a subset of pts, mTNBC “converters” (12/44 total TNBC pts) who had originally been diagnosed with ER/PR+ disease and progressed through endocrine therapies +/- CDK4/6 inhibitors. In these 12 TNBC converters, the ORR = 50%, DCR = 83%, mDoR=11.2, mPFS=5.6 mo, 12-month OS rate=64.8%, and mOS= 17.4 months. Imprime is now being explored in pts with hormone-resistant metastatic breast cancer (MBC) in combination with PEM. This is a phase 2, Simon’s 2-Stage study of pts with MBC who have progressed through prior hormonal therapy with at least one CDK4/6 inhibitor. All pts will receive Imprime 4 mg/kg/wk + PEM 200 mg Q3wk. A total of 23 pts will be enrolled into Stage 1. If ≥4 pts in Stage 1 have an objective response after 12 wks of treatment, the study will proceed into Stage 2. Twenty-four (24) pts will be enrolled in Stage 2 for a total combined population of 47 pts. Rejection of the null hypothesis will require documenting at least 10 objective responses in the total population. Main eligibility criteria include a diagnosis of MBC having failed prior hormonal therapy with at least one CDK4/6 inhibitor, ≤1 prior line of chemotherapy, serum ABA level of ≥20 µg/mL, and no prior ICI exposure. The primary endpoint is ORR (per RECIST v1.1); secondary endpoints are mPFS, PFS, mOS, OS, DCR, DoR, and safety. Exploratory objectives will assess the impact of the treatment combination on immune activating events in peripheral blood and at the tumor level, and correlate changes in PD-L1 status, the tumor microenvironment, and serum ABA with response. Point estimates with the exact 95% confidence intervals of ORR and DCR will be computed. For mPFS, PFS, mOS, OS, and DoR, medians, first, and third quartiles with 95% confidence intervals (CIs) will be estimated using Kaplan-Meier method. Safety parameters will be summarized. The trial is sponsored by HiberCell, Inc. in collaboration with Merck Sharp & Dohme Corp., a subsidiary of Merck & Co., Inc., Kenilworth, NJ, USA. Approximately 25 US sites will participate in the study. For information, contact Nick Niles nniles@hibercell.com. 612.230.5846. Citation Format: Alison Stopeck, Michele Gargano, Nandita Bose, Nick Niles, Michael Chisamore, Jose Iglesias. A multicenter, open-label, phase 2 study of Imprime PGG and pembrolizumab in patients with metastatic breast cancer who have progressed through prior hormonal therapy [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr OT1-18-05.