Long-term tropospheric ozone pollution disrupts plant-microbe-soil interactions in the agroecosystem.

Tropospheric ozone (O3 ) threatens agroecosystems, yet its long-term effects on intricate plant-microbe-soil interactions remain overlooked. This study employed two soybean genotypes of contrasting O3 -sensitivity grown in field plots exposed elevated O3 (eO3 ) and evaluated cause-effect relationships with their associated soil microbiomes and soil quality. Results revealed long-term eO3 effects on belowground soil microbiomes and soil health surpass damage visible on plants. Elevated O3 significantly disrupted belowground bacteria-fungi interactions, reduced fungal diversity, and altered fungal community assembly by impacting soybean physiological properties. Particularly, eO3 impacts on plant performance were significantly associated with arbuscular mycorrhizal fungi, undermining their contribution to plants, whereas eO3 increased fungal saprotroph proliferation, accelerating soil organic matter decomposition and soil carbon pool depletion. Free-living diazotrophs exhibited remarkable acclimation under eO3 , improving plant performance by enhancing nitrogen fixation. However, overarching detrimental consequences of eO3 negated this benefit. Overall, this study demonstrated long-term eO3 profoundly governed negative impacts on plant-soil-microbiota interactions, pointing to a potential crisis for agroecosystems. These findings highlight urgent needs to develop adaptive strategies to navigate future eO3 scenarios.

Shifts in the Composition and Activities of Denitrifiers Dominate CO2 Stimulation of N2O Emissions.

Elevated atmospheric CO2 (eCO2) often increases soil N2O emissions, but the underlying mechanisms remain largely unknown. One hypothesis suggests that high N2O emissions may stem from increased denitrification induced by CO2 enhancement of plant carbon (C) allocation belowground. However, direct evidence illustrating linkages among N2O emissions, plant C allocation, and denitrifying microbes under eCO2 is still lacking. We examined the impact of eCO2 on plant C allocation to roots and their associated arbuscular mycorrhizal fungi and its subsequent effects on N2O emissions and denitrifying microbes in the presence of two distinct N sources, ammonium nitrogen (NH4+-N) and nitrate nitrogen (NO3--N). Our results showed that the form of the N inputs dominated the effects of eCO2 on N2O emissions: eCO2 significantly increased N2O emissions with NO3--N inputs but had no effect with NH4+-N inputs. eCO2 increased plant biomass N more with NH4+-N than with NO3--N inputs, likely reducing microbial access to available N under NH4+-N inputs and/or contributing to higher N2O emissions under NO3--N inputs. eCO2 enhanced root and mycorrhizal N uptake and also increased N2O emissions under NO3--N inputs. Further, eCO2 enhancement of N2O emissions under NO3--N inputs concurred with a shift in the soil denitrifier community composition in favor of N2O-producing (nirK- and nirS-type) over N2O-consuming (nosZ-type) denitrifiers. Together, these results indicate that eCO2 stimulated N2O emissions mainly through altering plant N preference in favor of NH4+ over NO3- and thus stimulating soil denitrifiers and their activities. These findings suggest that effective management of N sources may mitigate N2O emissions by negating the eCO2 stimulation of soil denitrifying microbes and their activities.

Ethylenediurea (EDU) pretreatment alleviated the adverse effects of elevated O3 on Populus alba "Berolinensis" in an urban area.

Ethylenediurea (EDU) has been used as a chemical protectant against ozone (O3). However, its protective effect and physiological mechanisms are still uncertain. The present study aimed to investigate the changes of foliar visible injury, physiological characteristics and emission rates of volatile organic compounds (VOCs) in one-year-old Populus alba "Berolinensis" saplings pretreated with EDU and exposed to elevated O3 (EO, 120 µg/m3). The results showed that foliar visible injury symptoms under EO were significantly alleviated in plants with EDU application (p < 0.05). Under EO, net photosynthetic rate, the maximum photochemical efficiency of PSII and the photochemical efficiency of PSII of plants pretreated with 300 and 600 mg/L EDU were similar to unexposed controls and significantly higher compared to EO-stressed plants without EDU pretreatment, respectively. Malondialdehyde content was highest in EO without EDU and decreased significantly by 14.9% and 21.3% with 300 and 600 mg/L EDU pretreatment, respectively. EDU pretreatment alone increased superoxide dismutase activity by 10-fold in unexposed plants with further increases of 88.4% and 37.5% in EO plants pretreated with 300 and 600 mg/L EDU pretreatment, respectively (p < 0.05). Abscisic acid content declined under EO relative to unexposed controls with the effect partially reversed by EDU pretreatments. Similarly, VOCs emission rate declined under EO relative to unexposed plants with a recovery of emission rate observed with 300 and 600 mg/L EDU pretreatment. These findings provided significant evidence that EDU exerted a beneficial effect and protection on the tested plants against O3 stress.

Closing the global ozone yield gap: Quantification and cobenefits for multistress tolerance.

Increasing both crop productivity and the tolerance of crops to abiotic and biotic stresses is a major challenge for global food security in our rapidly changing climate. For the first time, we show how the spatial variation and severity of tropospheric ozone effects on yield compare with effects of other stresses on a global scale, and discuss mitigating actions against the negative effects of ozone. We show that the sensitivity to ozone declines in the order soybean > wheat > maize > rice, with genotypic variation in response being most pronounced for soybean and rice. Based on stomatal uptake, we estimate that ozone (mean of 2010-2012) reduces global yield annually by 12.4%, 7.1%, 4.4% and 6.1% for soybean, wheat, rice and maize, respectively (the "ozone yield gaps"), adding up to 227 Tg of lost yield. Our modelling shows that the highest ozone-induced production losses for soybean are in North and South America whilst for wheat they are in India and China, for rice in parts of India, Bangladesh, China and Indonesia, and for maize in China and the United States. Crucially, we also show that the same areas are often also at risk of high losses from pests and diseases, heat stress and to a lesser extent aridity and nutrient stress. In a solution-focussed analysis of these results, we provide a crop ideotype with tolerance of multiple stresses (including ozone) and describe how ozone effects could be included in crop breeding programmes. We also discuss altered crop management approaches that could be applied to reduce ozone impacts in the shorter term. Given the severity of ozone effects on staple food crops in areas of the world that are also challenged by other stresses, we recommend increased attention to the benefits that could be gained from addressing the ozone yield gap.

Contrasting Warming and Ozone Effects on Denitrifiers Dominate Soil N2O Emissions.

Nitrous oxide (N2O) in the atmosphere is a major greenhouse gas and reacts with volatile organic compounds to create ozone (an air pollutant) in the troposphere. Climate change factors such as warming and elevated ozone (eO3) affect N2O fluxes, but the direction and magnitude of these effects are uncertain and the underlying mechanisms remain unclear. We examined the impact of simulated warming (control + 3.6 °C) and eO3 (control + 45 ppb) on soil N2O fluxes in a soybean agroecosystem. Results obtained showed that warming significantly increased soil labile C, microbial biomass, and soil N mineralization, but eO3 reduced these parameters. Warming enhanced N2O-producing denitrifers ( nirS- and nirK-type), corresponding to increases in both the rate and sum of N2O emissions. In contrast, eO3 significantly reduced both N2O-producing and N2O-consuming ( nosZ-type) denitrifiers but had no impact on N2O emissions. Further, eO3 offsets the effects of warming on soil labile C, microbial biomass, and the population size of denitrifiers but still increased N2O emissions, indicating a direct effect of temperature on N2O emissions. Together, these findings suggest that warming may promote N2O production through increasing both the abundance and activities of N2O-producing microbes, positively feeding back to the ongoing climate change.

Atmospheric CO2 Enrichment and Reactive Nitrogen Inputs Interactively Stimulate Soil Cation Losses and Acidification.

Reactive N inputs (Nr) may alleviate N-limitation of plant growth and are assumed to help sustain plant responses to the rising atmospheric CO2 (eCO2). However, Nr and eCO2 may elicit a cascade reaction that alters soil chemistry and nutrient availability, shifting the limiting factors of plant growth, particularly in acidic tropical and subtropical croplands with low organic matter and low nutrient cations. Yet, few have so far examined the interactive effects of Nr and eCO2 on the dynamics of soil cation nutrients and soil acidity. We investigated the cation dynamics in the plant-soil system with exposure to eCO2 and different N sources in a subtropical, acidic agricultural soil. eCO2 and Nr, alone and interactively, increased Ca2+ and Mg2+ in soil solutions or leachates in aerobic agroecosystems. eCO2 significantly reduced soil pH, and NH4+-N inputs amplified this effect, suggesting that eCO2-induced plant preference of NH4+-N and plant growth may facilitate soil acidification. This is, to our knowledge, the first direct demonstration of eCO2 enhancement of soil acidity, although other studies have previously shown that eCO2 can increase cation release into soil solutions. Together, these findings provide new insights into the dynamics of cation nutrients and soil acidity under future climatic scenarios, highlighting the urgency for more studies on plant-soil responses to climate change in acidic tropical and subtropical ecosystems.

Quantitative trait loci associated with soybean seed weight and composition under different phosphorus levels.

Seed size and composition are important traits in food crops and can be affected by nutrient availability in the soil. Phosphorus (P) is a non-renewable, essential macronutrient, and P deficiency limits soybean (Glycine max) yield and quality. To investigate the associations of seed traits in low- and high-P environments, soybean recombinant inbred lines (RILs) from a cross of cultivars Fiskeby III and Mandarin (Ottawa) were grown under contrasting P availability environments. Traits including individual seed weight, seed number, and intact mature pod weight were significantly affected by soil P levels and showed transgressive segregation among the RILs. Surprisingly, P treatments did not affect seed composition or weight, suggesting that soybean maintains sufficient P in seeds even in low-P soil. Quantitative trait loci (QTLs) were detected for seed weight, intact pods, seed volume, and seed protein, with five significant QTLs identified in low-P environments and one significant QTL found in the optimal-P environment. Broad-sense heritability estimates were 0.78 (individual seed weight), 0.90 (seed protein), 0.34 (seed oil), and 0.98 (seed number). The QTLs identified under low P point to genetic regions that may be useful to improve soybean performance under limiting P conditions.

Protecting the photosynthetic performance of snap bean under free air ozone exposure.

Tropospheric ozone (O3) is a major air pollutant and causes serious injury to vegetation. To protect sensitive plants from O3 damage, several agrochemicals have been assessed, including cytokinin (e.g., kinetin, KIN) and ethylenediurea (EDU) with cytokinin-like activity. In higher plant, leaves are primarily injured by O3 and protective agrochemicals are often applied by leaf spraying. To our knowledge, the mitigating abilities of EDU and KIN have not been compared directly in a realistic setup. In the present research, impacts of elevated O3 (2× ambient O3, 24hr per day, for 8days) on an O3 sensitive line (S156) of snap bean (Phaseolus vulgaris), which is often used for biomonitoring O3 pollution, were studied in a free air controlled exposure system. The day before starting the O3 exposure, plants were sprayed with a solution of EDU (300ppm), KIN (1mmol/L) or distilled water, to compare their protective abilities. The results demonstrated that 2× ambient O3 inhibited net photosynthetic rate and stomatal conductance, increased the minimal fluorescence yield of the dark-adapted state, decreased the maximal quantum yield of PSII photochemistry, and led to visible injury. KIN and EDU alleviated the reduction of the photosynthetic performance, and visible injury under O3 fumigation. The plants sprayed with EDU showed greater ability to mitigate the O3 damage than those sprayed with KIN. Chlorophyll fluorescence imaging may have detected more precisely the differences in O3 response across the leaf than the conventional fluorometer.

RNA-Seq study reveals genetic responses of diverse wild soybean accessions to increased ozone levels.

BACKGROUND: Ozone is an air pollutant widely known to cause a decrease in productivity in many plant species, including soybean (Glycine max (L.) Merr). While the response of cultivated soybean to ozone has been studied, very little information is available regarding the ozone response of its wild relatives. RESULTS: Ozone-resistant wild soybean accessions were identified by measuring the response of a genetically diverse group of 66 wild soybean (Glycine soja Zucc. and Sieb.) accessions to elevated ozone levels. RNA-Seq analyses were performed on leaves of different ages from selected ozone-sensitive and ozone-resistant accessions that were subjected to treatment with an environmentally relevant level of ozone. Many more genes responded to elevated ozone in the two ozone-sensitive accessions than in the ozone-resistant accessions. Analyses of the ozone response genes indicated that leaves of different ages responded differently to ozone. Older leaves displayed a consistent reduction in expression of genes involved in photosynthesis in response to ozone, while changes in expression of defense genes dominated younger leaf tissue in response to ozone. As expected, there is a substantial difference between the response of ozone-sensitive and ozone-resistant accessions. Genes associated with photosystem 2 were substantially reduced in expression in response to ozone in the ozone-resistant accessions. A decrease in peptidase inhibitors was one of several responses specific to one of the ozone resistant accessions. CONCLUSION: The decrease in expression in genes associated with photosynthesis confirms that the photosynthetic apparatus may be an early casualty in response to moderate levels of ozone. A compromise of photosynthesis would substantially impact plant growth and seed production. However, the resistant accessions may preserve their photosynthetic apparatus in response to the ozone levels used in this study. Older leaf tissue of the ozone-resistant accessions showed a unique down-regulation of genes associated with endopeptidase inhibitor activity. This study demonstrates the existence of significant diversity in wild soybean for ozone response. Wild soybean accessions characterized in this study can be used by soybean breeders to enhance ozone tolerance of this important food crop.

Phenotypic variation and identification of quantitative trait loci for ozone tolerance in a Fiskeby III × Mandarin (Ottawa) soybean population.

KEY MESSAGE: Soybean quantitative trait loci for ozone response. Ground-level ozone reduces yield in crops such as soybean (Glycine max (L.) Merr.). Phenotypic variation has been observed for this trait in multiple species; however, breeding for ozone tolerance has been limited. A recombinant inbred population was developed from soybean genotypes differing in tolerance to ozone: tolerant Fiskeby III and sensitive Mandarin (Ottawa). Plants were exposed to ozone treatment for 5 days in greenhouse chambers followed by visual scoring for foliar injury. Mean injury score in the mid-canopy was 16 % for Fiskeby III, and 81 % for Mandarin (Ottawa). Injury scores were lower in younger leaves for both parents and progeny, compared to scores in the older leaves. Segregation was consistent with multigenic inheritance. Correlation coefficients for injury between leaf positions ranged from 0.34 to 0.81, with the closer leaf positions showing the greater correlation. Narrow sense heritability within an ozone treatment chamber was 0.59, 0.40, 0.29, 0.30, 0.19, and 0.35 for the 2nd, 3rd, 4th, 5th, 6th, and combined 3rd-5th main stem leaf positions (numbered acropetally), respectively, based on genotypic means over three independent replications. Quantitative trait loci (QTL) analysis showed that loci were associated with distinct leaf developmental stages. QTL were identified on Chromosome 17 for the 2nd and 3rd leaf positions, and on Chromosome 4 for the 5th and 6th leaf positions. Additional loci were identified on Chromosomes 6, 18, 19, and 20. Interacting loci were identified on Chromosomes 5 and 15 for injury on trifoliate 4. The ozone sensitive parent contributed one favorable allele for ozone response.

RNA-seq analysis reveals genetic response and tolerance mechanisms to ozone exposure in soybean.

BACKGROUND: Oxidative stress caused by ground level ozone is a contributor to yield loss in a number of important crop plants. Soybean (Glycine max) is considered to be ozone sensitive, and current research into its response to oxidative stress is limited. To better understand the genetic response in soybean to oxidative stress, an RNA-seq analysis of two soybean cultivars was performed comparing an ozone intolerant cultivar (Mandarin-Ottawa) and an ozone resistant cultivar (Fiskeby III) following exposure to ozone. RESULTS: Analysis of the transcriptome data revealed cultivar-specific expression level differences of genes previously implicated in oxidative stress responses, indicating unique cultivar-specific responses. Both Fiskeby III and Mandarin (Ottawa) exhibit an increased expression of oxidative response genes as well as glutathiones, phenylpropanoids, and phenylalanine ammonia-lyases. Mandarin (Ottawa) exhibited more general stress response genes whereas Fiskeby III had heightened expression of metabolic process genes. An examination of the timing of gene responses over the course of ozone exposure identified significantly more differentially expressed genes across all time points in Mandarin (Ottawa) than in Fiskeby III. The timing of expression was also considered to identify genes that may be indicative of a delayed response to ozone stress in Fiskeby III, We found that Mandarin (Ottawa) exhibits an higher level of expression in early time points for oxidative and general stress response genes while Fiskeby III seems to maintain expression of defense and stress response genes. Of particular interest was the expression of wax and cutin biosynthetic genes that we found to be expressed in Mandarin (Ottawa) in all sampled time points, whereas the expression of this pathway is only in the first time point for Fiskeby III. CONCLUSIONS: We were able to identify differentially expressed genes that correspond to each of the known or expected categories of genes previously implicated in other species for ozone stress. Our study shows evidence that at least part of the observed ozone tolerance of Fiskeby III may be due to its thicker, denser leaves providing passive resistance thereby limiting the degree of ozone exposure. The observed diminished genetic response is then likely a consequence of this reduced exposure.

Alteration of the alkaloid profile in genetically modified tobacco reveals a role of methylenetetrahydrofolate reductase in nicotine N-demethylation.

Methylenetetrahydrofolate reductase (MTHFR) is a key enzyme of the tetrahydrofolate (THF)-mediated one-carbon (C1) metabolic network. This enzyme catalyzes the reduction of 5,10-methylene-THF to 5-methyl-THF. The latter donates its methyl group to homocysteine, forming methionine, which is then used for the synthesis of S-adenosyl-methionine, a universal methyl donor for numerous methylation reactions, to produce primary and secondary metabolites. Here, we demonstrate that manipulating tobacco (Nicotiana tabacum) MTHFR gene (NtMTHFR1) expression dramatically alters the alkaloid profile in transgenic tobacco plants by negatively regulating the expression of a secondary metabolic pathway nicotine N-demethylase gene, CYP82E4. Quantitative real-time polymerase chain reaction and alkaloid analyses revealed that reducing NtMTHFR expression by RNA interference dramatically induced CYP82E4 expression, resulting in higher nicotine-to-nornicotine conversion rates. Conversely, overexpressing NtMTHFR1 suppressed CYP82E4 expression, leading to lower nicotine-to-nornicotine conversion rates. However, the reduced expression of NtMTHFR did not affect the methionine and S-adenosyl-methionine levels in the knockdown lines. Our finding reveals a new regulatory role of NtMTHFR1 in nicotine N-demethylation and suggests that the negative regulation of CYP82E4 expression may serve to recruit methyl groups from nicotine into the C1 pool under C1-deficient conditions.

Differential expression of a novel gene EaF82a in green and yellow sectors of variegated Epipremnum aureum leaves is related to uneven distribution of auxin.

EaF82, a gene identified in previous studies of the variegated plant Epipremnum aureum, exhibited a unique expression pattern with greater transcript abundance in yellow sectors than green sectors of variegated leaves, but lower abundance in regenerated pale yellow plants than in green plants derived from leaf tissue culture. Studies of its full-length cDNA and promoter region revealed two members with only the EaF82a expressed. Immunoblotting confirmed that EaF82a encodes a 12 kDa protein and its accumulation consistent with its gene expression patterns in different color tissues. Transient expression of EaF82a-sGFP fusion proteins in protoplasts showed that EaF82a seems to be present in the cytosol as unidentified spots. Sequence motif search reveals a potential auxin responsive element in promoter region. Using transgenic Arabidopsis seedlings carrying EaF82a promoter driving the bacterial uidA (GUS) gene, an increased GUS activity was observed when IAA (indole-3-acetic acid) concentration was elevated. In E. aureum, EaF82a is more abundant at the site where axillary buds emerge and at the lower side of bending nodes where more IAA accumulates relative to the upper side. The measurement of endogenous IAA levels in different color tissues revealed the same pattern of IAA distribution as that of EaF82a expression, further supporting that EaF82a is an IAA responsive gene. EaF82a expression in etiolated transgenic Arabidopsis seedlings responded to IAA under the influence of light suggesting a microenvironment of uneven light condition affects the EaF82a transcript levels and protein accumulation in variegated leaves.

Elevated CO2 spurs reciprocal positive effects between a plant virus and an arbuscular mycorrhizal fungus.

Plants form ubiquitous associations with diverse microbes. These interactions range from parasitism to mutualism, depending partly on resource supplies that are being altered by global change. While many studies have considered the separate effects of pathogens and mutualists on their hosts, few studies have investigated interactions among microbial mutualists and pathogens in the context of global change. Using two wild grass species as model hosts, we grew individual plants under ambient or elevated CO(2), and ambient or increased soil phosphorus (P) supply. Additionally, individuals were grown with or without arbuscular mycorrhizal inoculum, and after 2 wk, plants were inoculated or mock-inoculated with a phloem-restricted virus. Under elevated CO(2), mycorrhizal association increased the titer of virus infections, and virus infection reciprocally increased the colonization of roots by mycorrhizal fungi. Additionally, virus infection decreased plant allocation to root biomass, increased leaf P, and modulated effects of CO(2) and P addition on mycorrhizal root colonization. These results indicate that plant mutualists and pathogens can alter each other's success, and predict that these interactions will respond to increased resource availability and elevated CO(2). Together, our findings highlight the importance of interactions among multiple microorganisms for plant performance under global change.

The interaction of O3 and CO2 concentration, exposure timing and duration on stem rust severity on winter wheat variety 'Coker 9553'.

Wheat rusts, elevated ozone (O3), and carbon dioxide (CO2) are simultaneously impacting wheat production worldwide, but their interactions are not well understood. This study investigated whether near-ambient O3 is suppressive or conducive to stem rust (Sr) of wheat, considering the interactions with ambient and elevated CO2. Winter wheat variety 'Coker 9553' (Sr-susceptible; O3 sensitive) was inoculated with Sr (race QFCSC) following pre-treatment with four different concentrations of O3 (CF, 50, 70, and 90 ppbv) at ambient CO2 levels. Gas treatments were continued during the development of disease symptoms. Disease severity, measured as percent sporulation area (PSA), significantly increased relative to the CF control only under near-ambient O3 conditions (50 ppbv) in the absence of O3-induced foliar injury. Disease symptoms at higher O3 exposures (70 and 90 ppbv) were similar to or less than the CF control. When Coker 9553 was inoculated with Sr while exposed to CO2 (400; 570 ppmv) and O3 (CF; 50 ppbv) in four different combinations, and seven combinations of exposure timing and duration, PSA significantly increased only under continuous treatment with O3 for six weeks or pre-inoculation treatment for three weeks, suggesting that O3-predisposes wheat to the disease rather than enhancing disease post-inoculation. O3 singly and in combination with CO2 increased PSA on flag leaves of adult Coker 9553 plants while elevated CO2 alone had little effect on PSA. These findings show that sub-symptomatic O3 conditions are conducive to stem rust, contradicting the current consensus that biotrophic pathogens are suppressed by elevated O3. This suggests that sub-symptomatic O3 stress may enhance rust diseases in wheat-growing regions.

Microbial community dynamics responding to nutrient allocation associated with soybean cultivar 'Jake' ozone adaptation.

Tropospheric ozone (O3), a major air pollutant, leads to significant global yield loss in soybean [Glycine max (L.) Merr.]. Soybean cultivar 'Jake' shows O3 resilient traits in above-ground organs, but the root system remains sensitive to elevated O3 (eO3). Changing carbon (C) and nitrogen (N) resource composition during eO3 stress suggests that eO3 presumably alters belowground soil microbial communities and their driven nutrient transformation. Yet, the responses of belowground microbes to eO3 and their feedback on nutrient cycling in 'Jake' are unknown. In this study, we holistically investigated soil microbial communities associated with C and N dynamics and bacterial-fungal inter-kingdom networks in the rhizosphere and bulk soil at different developmental stages of 'Jake' grown under sub-ambient O3 [charcoal-filtered (CF) air, 12 h mean: 20 ppb] or eO3 (12 h mean: 87 ppb). The results demonstrated eO3 significantly decreased fungal diversity and complexity of microbial networks at different 'Jake' developmental stages, whereas bacterial diversity was more tolerant to eO3 in both bulk soil and rhizosphere. In the bulk soil, no O3-responsive microbial biomarkers were found to be associated with C and N content, implying eO3 may stimulate niche-based processes during 'Jake' growth. In contrast, this study identified O3-responsive microbial biomarkers that may contribute to the N acquisition (Chloroflexales) and C dynamics (Caldilineales, Thermomicrobiales, and Hypocreales) in the rhizosphere, which may support the O3 resilience of the 'Jake' cultivar. However, further investigation is required to confirm their specific contributions by determining changes in microbial gene expression. Overall, these findings conduce to an expanding knowledge base that O3 induces temporal and spatial changes in the effects of microbial and nutrient networks in the O3-tolerant agriculture ecosystems.

A Rapid Alkalinization Factor-like Peptide EaF82 Impairs Tapetum Degeneration during Pollen Development through Induced ATP Deficiency.

In plants, the timely degeneration of tapetal cells is essential for providing nutrients and other substances to support pollen development. Rapid alkalinization factors (RALFs) are small, cysteine-rich peptides known to be involved in various aspects of plant development and growth, as well as defense against biotic and abiotic stresses. However, the functions of most of them remain unknown, while no RALF has been reported to involve tapetum degeneration. In this study, we demonstrated that a novel cysteine-rich peptide, EaF82, isolated from shy-flowering 'Golden Pothos' (Epipremnum aureum) plants, is a RALF-like peptide and displays alkalinizing activity. Its heterologous expression in Arabidopsis delayed tapetum degeneration and reduced pollen production and seed yields. RNAseq, RT-qPCR, and biochemical analyses showed that overexpression of EaF82 downregulated a group of genes involved in pH changes, cell wall modifications, tapetum degeneration, and pollen maturation, as well as seven endogenous Arabidopsis RALF genes, and decreased proteasome activity and ATP levels. Yeast two-hybrid screening identified AKIN10, a subunit of energy-sensing SnRK1 kinase, as its interacting partner. Our study reveals a possible regulatory role for RALF peptide in tapetum degeneration and suggests that EaF82 action may be mediated through AKIN10 leading to the alteration of transcriptome and energy metabolism, thereby causing ATP deficiency and impairing pollen development.

Re-evaluating the role of ascorbic acid and phenolic glycosides in ozone scavenging in the leaf apoplast of Arabidopsis thaliana L.

Phenolic glycosides are effective reactive oxygen scavengers and peroxidase substrates, suggesting that compounds in addition to ascorbate may have functional importance in defence responses against ozone (O(3)), especially in the leaf apoplast. The apoplastic concentrations of ascorbic acid (AA) and phenolic glycosides in Arabidopsis thaliana L. Col-0 wild-type plants were determined following exposure to a range of O(3) concentrations (5, 125 or 175 nL L(-1)) in controlled environment chambers. AA in leaf apoplast extracts was almost entirely oxidized in all treatments, suggesting that O(3) scavenging by direct reactions with reduced AA was very limited. In regard to phenolics, O(3) stimulated transcription of numerous phenylpropanoid pathway genes and increased the apoplastic concentration of sinapoyl malate. However, modelling of O(3) scavenging in the apoplast indicated that sinapoyl malate concentrations were too low to be effective protectants. Furthermore, null mutants for sinapoyl esters (fah1-7), kaempferol glycosides (tt4-1) and the double mutant (tt4-1/fah1-7) were equally sensitive to chronic O(3) as Ler-0 wild-type plants. These results indicate that current understanding of O(3) defence schemes deserves reassessment as mechanisms other than direct scavenging of O(3) by extracellular AA and antioxidant activity of some phenolics may predominate in some plant species.

Minimal influence of G-protein null mutations on ozone-induced changes in gene expression, foliar injury, gas exchange and peroxidase activity in Arabidopsis thaliana L.

Ozone (O(3)) uptake by plants leads to an increase in reactive oxygen species (ROS) in the intercellular space of leaves and induces signalling processes reported to involve the membrane-bound heterotrimeric G-protein complex. Therefore, potential G-protein-mediated response mechanisms to O(3) were compared between Arabidopsis thaliana L. lines with null mutations in the α- and ß-subunits (gpa1-4, agb1-2 and gpa1-4/agb1-2) and Col-0 wild-type plants. Plants were treated with a range of O(3) concentrations (5, 125, 175 and 300 nL L(-1)) for 1 and 2 d in controlled environment chambers. Transcript levels of GPA1, AGB1 and RGS1 transiently increased in Col-0 exposed to 125 nL L(-1) O(3) compared with the 5 nL L(-1) control treatment. However, silencing of α and ß G-protein genes resulted in little alteration of many processes associated with O(3) injury, including the induction of ROS-signalling genes, increased leaf tissue ion leakage, decreased net photosynthesis and stomatal conductance, and increased peroxidase activity, especially in the leaf apoplast. These results indicated that many responses to O(3) stress at physiological levels were not detectably influenced by α and ß G-proteins.

Influence of atmospheric vapour pressure deficit on ozone responses of snap bean (Phaseolus vulgaris L.) genotypes.

Environmental conditions influence plant responses to ozone (O(3)), but few studies have evaluated individual factors directly. In this study, the effect of O(3) at high and low atmospheric vapour pressure deficit (VPD) was evaluated in two genotypes of snap bean (Phaseolus vulgaris L.) (R123 and S156) used as O(3) bioindicator plants. Plants were grown in outdoor controlled-environment chambers in charcoal-filtered air containing 0 or 60 nl l(-1) O(3) (12 h average) at two VPDs (1.26 and 1.96 kPa) and sampled for biomass, leaf area, daily water loss, and seed yield. VPD clearly influenced O(3) effects. At low VPD, O(3) reduced biomass, leaf area, and seed yield substantially in both genotypes, while at high VPD, O(3) had no significant effect on these components. In clean air, high VPD reduced biomass and yield by similar fractions in both genotypes compared with low VPD. Data suggest that a stomatal response to VPD per se may be lacking in both genotypes and it is hypothesized that the high VPD resulted in unsustainable transpiration and water deficits that resulted in reduced growth and yield. High VPD- and water-stress-induced stomatal responses may have reduced the O(3) flux into the leaves, which contributed to a higher yield compared to the low VPD treatment in both genotypes. At low VPD, transpiration increased in the O(3) treatment relative to the clean air treatment, suggesting that whole-plant conductance was increased by O(3) exposure. Ozone-related biomass reductions at low VPD were proportionally higher in S156 than in R123, indicating that differential O(3) sensitivity of these bioindicator plants remained evident when environmental conditions were conducive for O(3) effects. Assessments of potential O(3) impacts on vegetation should incorporate interacting factors such as VPD.