Accession-specific parent-of-origin dependent and independent genome dosage effects on salt tolerance in Arabidopsis thaliana.

Improving the salt stress tolerance of crops is an important goal in plant breeding. Changes in the number of chromosome sets (i.e. ploidy level) cause genome dosage effects which can result in enhanced or novel traits. Maternal inheritance versus paternal inheritance of the same chromosome sets can have differential epigenetic effects on traits of F1 offspring. Hence, genome dosage effects can be parent-of-origin independent or dependent. The model plant Arabidopsis thaliana displays both genome dosage and parent-of-origin effects on plant growth under non-stress conditions. Using an isogenic ploidy series of diploid, triploid and tetraploid lines, we investigate the extent of genome dosage effects and their parent-of-origin dependency on in vitro salt stress tolerance of seedlings across 10 different A. thaliana accessions (genetic backgrounds). We detected genome dosage effects on salt stress tolerance for tetraploid lines in five accessions. In addition, through the generation of isogenic reciprocal F1 triploid lines, both parent-of-origin dependent and independent genome dosage effects on salt stress tolerance were detected. Thus, our results indicate not only that genome dosage balance effects can have significant impacts on abiotic stress tolerance in A. thaliana but also that parent-of-origin specific genome dosage effects can affect salt stress tolerance in plants.

Haploid rhapsody: the molecular and cellular orchestra of in vivo haploid induction in plants.

In planta haploid induction (HI), which reduces the chromosome number in the progeny after fertilization, has garnered increasing attention for its significant potential in crop breeding and genetic research. Despite the identification of several natural and synthetic HI systems in different plant species, the molecular and cellular mechanisms underlying these HI systems remain largely unknown. This review synthesizes the current understanding of HI systems in plants (with a focus on genes and molecular mechanisms involved), including the molecular and cellular interactions which orchestrate the HI process. As most HI systems can function across taxonomic boundaries, we particularly discuss the evidence for conserved mechanisms underlying the process. These include mechanisms involved in preserving chromosomal integrity, centromere function, gamete communication and/or fusion, and maintenance of karyogamy. While significant discoveries and advances on haploid inducer systems have arisen over the past decades, we underscore gaps in understanding and deliberate on directions for further research for a more comprehensive understanding of in vivo HI processes in plants.

Unravelling the Transcriptional Response of Agaricus bisporus under Lecanicillium fungicola Infection.

Mushrooms are a nutritionally rich and sustainably-produced food with a growing global market. Agaricus bisporus accounts for 11% of the total world mushroom production and it is the dominant species cultivated in Europe. It faces threats from pathogens that cause important production losses, including the mycoparasite Lecanicillium fungicola, the causative agent of dry bubble disease. Through quantitative real-time polymerase chain reaction (qRT-PCR), we determine the impact of L. fungicola infection on the transcription patterns of A. bisporus genes involved in key cellular processes. Notably, genes related to cell division, fruiting body development, and apoptosis exhibit dynamic transcriptional changes in response to infection. Furthermore, A. bisporus infected with L. fungicola were found to accumulate increased levels of reactive oxygen species (ROS). Interestingly, the transcription levels of genes involved in the production and scavenging mechanisms of ROS were also increased, suggesting the involvement of changes to ROS homeostasis in response to L. fungicola infection. These findings identify potential links between enhanced cell proliferation, impaired fruiting body development, and ROS-mediated defence strategies during the A. bisporus (host)-L. fungicola (pathogen) interaction, and offer avenues for innovative disease control strategies and improved understanding of fungal pathogenesis.

PICKLE RELATED 2 is a Neofunctionalized Gene Duplicate Under Positive Selection With Antagonistic Effects to the Ancestral PICKLE Gene on the Seed Transcriptome.

The evolution and diversification of proteins capable of remodeling domains has been critical for transcriptional reprogramming during cell fate determination in multicellular eukaryotes. Chromatin remodeling proteins of the CHD3 family have been shown to have important and antagonistic impacts on seed development in the model plant, Arabidopsis thaliana, yet the basis of this functional divergence remains unknown. In this study, we demonstrate that genes encoding the CHD3 proteins PICKLE (PKL) and PICKLE-RELATED 2 (PKR2) originated from a duplication event during the diversification of crown Brassicaceae, and that these homologs have undergone distinct evolutionary trajectories since this duplication, with PKR2 fast evolving under positive selection, while PKL is subject to purifying selection. We find that the rapid evolution of PKR2 under positive selection reduces the encoded protein's intrinsic disorder, possibly suggesting a tertiary structure configuration which differs from that of PKL. Our whole genome transcriptome analysis in seeds of pkr2 and pkl mutants reveals that they act antagonistically on the expression of specific sets of genes, providing a basis for their differing roles in seed development. Our results provide insights into how gene duplication and neofunctionalization can lead to differing and antagonistic selective pressures on transcriptomes during plant reproduction, as well as on the evolutionary diversification of the CHD3 family within seed plants.

Regulation of Carotenoid Biosynthesis and Degradation in Lettuce (Lactuca sativa L.) from Seedlings to Harvest.

Lettuce (Lactuca sativa L.) is one of the commercially important leafy vegetables worldwide. However, lettuce cultivars vary widely in their carotenoid concentrations at the time of harvest. While the carotenoid content of lettuce can depend on transcript levels of key biosynthetic enzymes, genes that can act as biomarkers for carotenoid accumulation at early stages of plant growth have not been identified. Transcriptomic and metabolomic analysis was performed on the inner and outer leaves of the six cultivars at different developmental stages to identify gene-to-metabolite networks affecting the accumulation of two key carotenoids, ß-carotene and lutein. Statistical analysis, including principal component analysis, was used to better understand variations in carotenoid concentration between leaf age and cultivars. Our results demonstrate that key enzymes of carotenoid biosynthesis pathway can alter lutein and ß-carotene biosynthesis across commercial cultivars. To ensure high carotenoids content in leaves, the metabolites sink from ß-carotene and lutein to zeaxanthin, and subsequently, abscisic acid needs to be regulated. Based on 2-3-fold carotenoids increase at 40 days after sowing (DAS) as compared to the seedling stage, and 1.5-2-fold decline at commercial stage (60 DAS) compared to the 40 DAS stage, we conclude that the value of lettuce for human nutrition would be improved by use of less mature plants, as the widely-used commercial stage is already at plant senescence stage where carotenoids and other essential metabolites are undergoing degradation.

Climate change and land-use change impacts on future availability of forage grass species for Ethiopian dairy systems.

Forage grasses are central feed resources for livestock globally. In Ethiopian dairy systems, they serve as feed sources during both wet and dry seasons, yet escalating climate change could threaten forage supply. Here, we investigate projected climate change impacts on three forage grasses currently recommended for Ethiopian dairy systems. We determine areas of geographical suitability for each species using three climate projections generated by General Circulation Models (GCMs) and calculate their ability to meet predicted dry matter demand under four scenarios for livestock intensification and land availability. By 2050, Buffel grass (Cenchrus ciliaris) is likely to be negatively affected by climate change in regions such as Tigray, while Rhodes grass (Chloris gayana) and Napier grass (Cenchrus purpureus) may have improved suitability under future climates. Our findings suggest that feed demands could theoretically be met by production of these forage grasses under current and future climates. However, if land availability is reduced and herd composition shifts towards higher-productivity exotic breeds, forage resources will not meet cattle demand even with improved agronomic management.

Clinical Performance of Direct RT-PCR Testing of Raw Saliva for Detection of SARS-CoV-2 in Symptomatic and Asymptomatic Individuals.

RT-PCR tests based on RNA extraction from nasopharyngeal swabs (NPS) are promoted as the "gold standard" for SARS-CoV-2 detection. However, the use of saliva samples offers noninvasive self-collection more suitable for high-throughput testing. This study evaluated performance of the TaqPath COVID-19 Fast PCR Combo kit 2.0 assay for detection of SARS-CoV-2 in raw saliva relative to a lab-developed direct RT-PCR test (SalivaDirect-based PCR, SDB-PCR) and an RT-PCR test based on RNA extraction from NPS. Saliva and NPS samples were collected from symptomatic and asymptomatic individuals (N = 615). Saliva samples were tested for SARS-CoV-2 using the TaqPath COVID-19 Fast PCR Combo kit 2.0 and the SDB-PCR, while NPS samples were tested by RT-PCR in RNA extracts according to the Irish national testing system. TaqPath COVID-19 Fast PCR Combo kit 2.0 detected SARS-CoV-2 in 52 saliva samples, of which 51 were also positive with the SDB-PCR. Compared to the NPS "gold standard" biospecimen method, 49 samples displayed concordant results, while three samples (35

Plastid ribosome protein L5 is essential for post-globular embryo development in Arabidopsis thaliana.

Plastid ribosomal proteins (PRPs) can play essential roles in plastid ribosome functioning that affect plant function and development. However, the roles of many PRPs remain unknown, including elucidation of which PRPs are essential or display redundancy. Here, we report that the nuclear-encoded PLASTID RIBOSOMAL PROTEIN L5 (PRPL5) is essential for early embryo development in A. thaliana, as homozygous loss-of-function mutations in the PRPL5 gene impairs chloroplast development and leads to embryo failure to develop past the globular stage. We confirmed the prpl5 embryo-lethal phenotype by generating a mutant CRISPR/Cas9 line and by genetic complementation. As PRPL5 underwent transfer to the nuclear genome early in the evolution of Embryophyta, PRPL5 can be expected to have acquired a chloroplast transit peptide. We identify and validate the presence of an N-terminal chloroplast transit peptide, but unexpectedly also confirm the presence of a conserved and functional Nuclear Localization Signal on the protein C-terminal end. This study highlights the fundamental role of the plastid translation machinery during the early stages of embryo development in plants and raises the possibility of additional roles of plastid ribosomal proteins in the nucleus.

Parent-of-Origin Effects on Seed Size Modify Heterosis Responses in Arabidopsis thaliana.

Parent-of-origin effects arise when a phenotype depends on whether it is inherited maternally or paternally. Parent-of-origin effects can exert a strong influence on F1 seed size in flowering plants, an important agronomic and life-history trait that can contribute to biomass heterosis. Here we investigate the natural variation in the relative contributions of the maternal and paternal genomes to F1 seed size across 71 reciprocal pairs of F1 hybrid diploids and the parental effect on F1 seed size heterosis. We demonstrate that the paternally derived genome influences F1 seed size more significantly than previously appreciated. We further demonstrate (by disruption of parental genome dosage balance in F1 triploid seeds) that hybridity acts as an enhancer of genome dosage effects on F1 seed size, beyond that observed from hybridity or genome dosage effects on their own. Our findings indicate that interactions between genetic hybridity and parental genome dosage can enhance heterosis effects in plants, opening new avenues for boosting heterosis breeding in crop plants.

Corona citizens' science project-repeated surveys of the Irish response to COVID-19 and subsequent lockdown and restrictive measures.

BACKGROUND: Worldwide, many people have been affected by COVID-19, a novel respiratory illness, caused by a new type of coronavirus SARS-CoV2. The COVID-19 outbreak is considered a pandemic and has created a number of challenges for the general population, patients, and healthcare professionals. Lockdowns have been implemented to slow down the spread of the virus with the expectation that these restrictions will limit the number of cases, and hence the number of hospitalizations and ICU admissions. However, these restrictions, and in particular lockdowns, impact on the life of everyone living in Ireland. AIM: To record how the COVID-19 pandemic and subsequent restrictive measures impacted on people's activities, work, schooling, and childcare. METHODS: The Corona Citizens' Science Project was set up as a population-wide survey. A questionnaire was designed, and the survey was first launched on the 8th of April 2020. An overview of results was released in the press days later. Data was collected in four waves: April 8, April 22, May 6, and June 17, 2020. Each wave had core questions allowing to compare each wave, and wave-specific questions, to understand current impact of changing measures. RESULTS: Over four waves, 152,259 responses were collected. The mean age of respondents was 47 with about 10% over the age of 65. Around 75% were female and 85% had a higher degree. Nearly 70% of the respondents were in employment, and around 13% were retired. Up to 20% of the respondents were essential workers, and 10% of respondents indicated they were in receipt of the COVID-19 pandemic unemployment payment. Around 10% of the people who responded were living alone. The number of people talked to the previous day was on average 2.3 in the first survey; during the lockdown, this went up over time, and in the last survey, the mean was 3.9. The percentage of respondents who did not talk to anyone the previous day decreased from 40 to 22% over the waves. In the first wave, about 6% of respondents reported having had flu-like symptoms in the last 14 days, which declined to 3.3%, 2.5%, and 2.0% in waves 2, 3, and 4 respectively. Similarly, over the four waves, the respondents who indicated that someone they lived with had flu-like symptoms declined from 17 to 12%, 9%, and 11%. Throughout the four waves, nearly one third of people reported one or more underlying conditions. CONCLUSIONS: As a result of the COVID-19 pandemic, a number of restrictive measures, in particular lockdown, were implemented in Ireland to protect populations and healthcare systems. To record some of the major impacts on society, we launched a Corona Citizens Science Project, with the aim to support decision-making. This report provides detail of its findings.

Gene dosage compensation of rRNA transcript levels in Arabidopsis thaliana lines with reduced ribosomal gene copy number.

The 45S rRNA genes (rDNA) are among the largest repetitive elements in eukaryotic genomes. rDNA consists of tandem arrays of rRNA genes, many of which are transcriptionally silenced. Silent rDNA repeats may act as 'back-up' copies for ribosome biogenesis and have nuclear organization roles. Through Cas9-mediated genome editing in the Arabidopsis thaliana female gametophyte, we reduced 45S rDNA copy number (CN) to a plateau of ∼10%. Two independent lines had rDNA CNs reduced by up to 90% at the T7 generation, named low copy number (LCN) lines. Despite drastic reduction of rDNA copies, rRNA transcriptional rates, and steady-state levels remained the same as wild-type plants. Gene dosage compensation of rRNA transcript levels was associated with reduction of silencing histone marks at rDNA loci and altered Nucleolar Organiser Region 2 organization. Although overall genome integrity of LCN lines appears unaffected, a chromosome segmental duplication occurred in one of the lines. Transcriptome analysis of LCN seedlings identified several shared dysregulated genes and pathways in both independent lines. Cas9 genome editing of rRNA repeats to generate LCN lines provides a powerful technique to elucidate rDNA dosage compensation mechanisms and impacts of low rDNA CN on genome stability, development, and cellular processes.

Iron, Zinc and Phytic Acid Retention of Biofortified, Low Phytic Acid, and Conventional Bean Varieties When Preparing Common Household Recipes.

Biofortification is an effective method to improve the nutritional content of crops and nutritional intake. Breeding for higher micronutrient mineral content in beans is correlated with an increase in phytic acid, a main inhibitor of mineral absorption in humans. Low phytic acid (lpa) beans have a 90% lower phytic acid content compared to conventional beans. This is the first study to investigate mineral and total phytic acid retention after preparing common household recipes from conventional, biofortified and lpa beans. Mineral retention was determined for two conventional, three biofortified and two lpa bean genotypes. Treatments included soaking, boiling (boiled beans) and refrying (bean paste). The average true retention of iron after boiling was 77.2-91.3%; for zinc 41.2-84.0%; and for phytic acid 49.9-85.9%. Soaking led to a significant decrease in zinc and total phytic acid after boiling and refrying, whereas for iron no significant differences were found. lpa beans did not exhibit a consistent pattern of difference in iron and phytic acid retention compared to the other groups of beans. However, lpa beans had a significantly lower retention of zinc compared to conventional and biofortified varieties (p < 0.05). More research is needed to understand the underlying factors responsible for the differences in retention between the groups of beans, especially the low retention of zinc. Combining the lpa and biofortification traits could further improve the nutritional benefits of biofortified beans, by decreasing the phytic acid:iron and zinc ratio in beans.

Thermal disruption of the food matrix of biofortified lettuce varieties modifies absorption of carotenoids by Caco-2 cells.

Amongst green leafy vegetables, new varieties of lettuce enriched in lutein and ß-carotene are being developed to provide increased supply of dietary carotenoids. We investigated the effect of lettuce genotypes (varieties) and thermal treatments on lutein and ß-carotene bioaccessibility to the micellar fraction (and also carotenoid bioavailability) using a human Caco-2 cell model system. Carotenoid absorption by mammalian cells is not correlated with initial carotenoid concentration in fresh lettuce leaves. While thermal treatment of lettuce leaves increases carotenoid availability, resulting in higher lutein and ß-carotene absorption, disruption of the food matrix by prior cooking results in reduced carotenoid levels and transfer to the micellar fraction. Unless the food matrix is disrupted through breeding or post-harvest treatments, absorption of carotenoids from biofortified lettuce remains similar to lettuce cultivars with low carotenoid levels. Genetic improvement programs for biofortified lettuce varieties need to focus on increasing the carotenoid bioavailability from the food matrix.

Transgenerational effects of inter-ploidy cross direction on reproduction and F2 seed development of Arabidopsis thaliana F1 hybrid triploids.

KEY MESSAGE: Reproduction in triploid plants is important for understanding polyploid population dynamics. We show that genetically identical reciprocal F1 hybrid triploids can display transgenerational epigenetic effects on viable F2 seed development. The success or failure of reproductive outcomes from intra-species crosses between plants of different ploidy levels is an important factor in flowering plant evolution and crop breeding. However, the effects of inter-ploidy cross directions on F1 hybrid offspring fitness are poorly understood. In Arabidopsis thaliana, hybridization between diploid and tetraploid plants can produce viable F1 triploid plants. When selfed, such F1 triploid plants act as aneuploid gamete production "machines" where the vast majority of gametes generated are aneuploid which, following sexual reproduction, can generate aneuploid swarms of F2 progeny (Henry et al. 2009). There is potential for some aneuploids to cause gametophyte abortion and/or F2 seed abortion (Henry et al. 2009). In this study, we analyse the reproductive success of 178 self-fertilized inter-accession F1 hybrid triploids and demonstrate that the proportions of aborted or normally developed F2 seeds from the selfed F1 triploids depend upon a combination of natural variation and cross direction, with strong interaction between these factors. Single-seed ploidy analysis indicates that the embryonic DNA content of phenotypically normal F2 seeds is highly variable and that these DNA content distributions are also affected by genotype and cross direction. Notably, genetically identical reciprocal F1 hybrid triploids display grandparent-of-origin effects on F2 seed set, and hence on the ability to tolerate aneuploidy in F2 seed. There are differences between reciprocal F1 hybrid triploids regarding the proportions of normal and aborted F2 seeds generated, and also for the DNA content averages and distributions of the F2 seeds. To identify genetic variation for tolerance of aneuploidy in F2 seeds, we carried out a GWAS which identified two SNPs, termed MOT and POT, which represent candidate loci for genetic control of the proportion of normal F2 seeds obtained from selfed F1 triploids. Parental and grandparental effects on F2 seeds obtained from selfed F1 triploids can have transgenerational consequences for asymmetric gene flow, emergence of novel genotypes in polyploid populations, and for control of F2 seed set in triploid crops.

Reduction in nutritional quality and growing area suitability of common bean under climate change induced drought stress in Africa.

Climate change impacts on food security will involve negative impacts on crop yields, and potentially on the nutritional quality of staple crops. Common bean is the most important grain legume staple crop for human diets and nutrition worldwide. We demonstrate by crop modeling that the majority of current common bean growing areas in southeastern Africa will become unsuitable for bean cultivation by the year 2050. We further demonstrate reductions in yields of available common bean varieties in a field trial that is a climate analogue site for future predicted drought conditions. Little is known regarding the impact of climate change induced abiotic stresses on the nutritional quality of common beans. Our analysis of nutritional and antinutritional compounds reveals that iron levels in common bean grains are reduced under future climate-scenario relevant drought stress conditions. In contrast, the levels of protein, zinc, lead and phytic acid increase in the beans under such drought stress conditions. This indicates that under climate-change induced drought scenarios, future bean servings by 2050 will likely have lower nutritional quality, posing challenges for ongoing climate-proofing of bean production for yields, nutritional quality, human health, and food security.

Hybridity has a greater effect than paternal genome dosage on heterosis in sugar beet (Beta vulgaris).

BACKGROUND: The phenomenon of heterosis is critical to plant breeding and agricultural productivity. Heterosis occurs when F1 hybrid offspring display quantitative improvements in traits to levels that do not occur in the parents. Increasing the genome dosage (i.e. ploidy level) of F1 offspring can contribute to heterosis effects. Sugar beet (Beta vulgaris) provides a model for investigating the relative effects of genetic hybridity and genome dosage on heterosis. Sugar beet lines of different ploidy levels were crossed to generate diploid and triploid F1 offspring to investigate the effect of; (1) paternal genome dosage increase on F1 heterosis, and; (2) homozygous versus heterozygous tetraploid male parents on F1 triploid heterosis. A range of traits of agronomic and commercial importance were analyzed for the extent of heterosis effects observed in the F1 offspring. RESULTS: Comparisons of parental lines to diploid (EA, EB) and triploid (EAA, EBB) F1 hybrids for total yield, root yield, and sugar yield indicated that there was no effect of paternal genome dosage increases on heterosis levels, indicating that hybridity is the main contributor to the heterosis levels observed. For all traits measured (apart from seed viability), F1 triploid hybrids derived from heterozygous tetraploid male parents displayed equivalent levels of heterosis as F1 triploid hybrids generated with homozygous tetraploid male parents, suggesting that heterosis gains in F1 triploids do not arise by simply increasing the extent of multi-locus heterozygosity in sugar beet F1 offspring. CONCLUSIONS: Overall, our study indicates that; (1) increasing the paternal genome dosage does not enhance heterosis in F1 hybrids, and; (2) increasing multi-locus heterozygosity using highly heterozygous paternal genomes to generate F1 triploid hybrids does not enhance heterosis. Our findings have implications for the design of future F1 hybrid improvement programs for sugar beet.

Determination of Enzymes Associated with Sulfite Toxicity in Plants: Kinetic Assays for SO, APR, SiR, and In-Gel SiR Activity.

The amino acid cysteine plays a major role in plant response to abiotic stress by being the donor of elemental sulfur for the sulfuration of the molybdenum cofactor, otherwise the last step of ABA biosynthesis, the oxidation of abscisic aldehyde, is inactivated. Additionally, cysteine serves as a precursor for the biosynthesis of glutathione, the reactive oxygen species scavenger essential for redox status homeostasis during stress. Cysteine is generated by the sulfate reductive pathway where sulfite oxidase (SO; EC 1.8.3.1) is an important enzyme in the homeostasis of sulfite levels (present either as a toxic intermediate in the pathway or as a toxic air pollutant that has penetrated the plant tissue via the stomata). SO is localized to the peroxisomes and detoxifies excess sulfite by catalyzing its oxidation to sulfate. Here we show a kinetic assay that relies on fuchsin colorimetric detection of sulfite, a substrate of SO activity. This SO assay is highly specific, technically simple, and readily performed in any laboratory.5'-adenylylsulfate (APS) reductase (APR, E.C. 1.8.4.9) enzyme regulates a crucial step of sulfate assimilation in plants, algae and some human pathogens. The enzyme is upregulated in response to oxidative stress induced by abiotic stresses, such as salinity and hydrogen peroxide, to generate sulfite an intermediate for cysteine generation essential for the biosynthesis of glutathione, the hydrogen peroxide scavenger. Here we present two robust, sensitive, and simple colorimetric methods of APR activity based on sulfite determination by fuchsin.Sulfite reductase (SiR) is one of the key enzymes in the primary sulfur assimilation pathway. It has been shown that SiR is an important plant enzyme for protection plant against sulfite toxicity and premature senescence. Here we describe two methods for SiR activity determination: a kinetic assay using desalted extract and an in-gel assay using crude extract.Due to the energetically favorable equilibrium, sulfurtransferase (ST) activity measured as sulfite generation or consumption. Sulfite-generating ST activity is determined by colorimetric detection of SCN- formation at 460 nm as the red Fe(SCN)3 complex from cyanide and thiosulfate using acidic iron reagent. Sulfite-consuming (MST) activity is detected as sulfite disappearance in the presence of thiocyanate (SCN-) or as SCN- disappearance. To abrogate interfering SO activity, total ST activities is detected by inhibiting SO activity with tungstate.

Determination of Total Sulfur, Sulfate, Sulfite, Thiosulfate, and Sulfolipids in Plants.

In response to oxidative stress the biosynthesis of the ROS scavenger, glutathione is induced. This requires the induction of the sulfate reduction pathway for an adequate supply of cysteine, the precursor for glutathione. Cysteine also acts as the sulfur donor for the sulfuration of the molybdenum cofactor, crucial for the last step of ABA biosynthesis. Sulfate and sulfite are, respectively, the precursor and intermediate for cysteine biosynthesis and there is evidence for stress-induced sulfate uptake and further downstream, enhanced sulfite generation by 5'-phosphosulfate (APS) reductase (APR, EC 1.8.99.2) activity. Sulfite reductase (SiR, E.C.1.8.7.1) protects the chloroplast against toxic levels of sulfite by reducing it to sulfide. In case of sulfite accumulation as a result of air pollution or stress-induced premature senescence, such as in extended darkness, sulfite can be oxidized to sulfate by sulfite oxidase. Additionally sulfite can be catalyzed to thiosulfate by sulfurtransferases or to UDP-sulfoquinovose by SQD1, being the first step toward sulfolipid biosynthesis.Determination of total sulfur in plants can be accomplished using many techniques such as ICP-AES, high-frequency induction furnace, high performance ion chromatography, sulfur combustion analysis, and colorimetric titration. Here we describe a total sulfur detection method in plants by elemental analyzer (EA). The used EA method is simple, sensitive, and accurate, and can be applied for the determination of total S content in plants.Sulfate anions in the soil are the main source of sulfur, required for normal growth and development, of plants. Plants take up sulfate ions from the soil, which are then reduced and incorporated into organic matter. Plant sulfate content can be determined by ion chromatography with carbonate eluents.Sulfite is an intermediate in the reductive assimilation of sulfate to the essential amino acids cysteine and methionine, and is cytotoxic above a certain threshold if not rapidly metabolized and can wreak havoc at the cellular and whole plant levels. Plant sulfite content affects carbon and nitrogen homeostasis Therefore, methods capable of determining sulfite levels in plants are of major importance. Here we present two robust laboratory protocols which can be used for sulfite detection in plants.Thiosulfate is an essential sulfur intermediate less toxic than sulfite which is accumulating in plants in response to sulfite accumulation. The complexity of thiosulfate detection is linked to its chemical properties. Here we present a rapid, sensitive, and accurate colorimetric method based on the enzymatic conversion of thiosulfate to thiocyanate.The plant sulfolipid sulfoquinovosyldiacylglycerol (SQDG) accounts for a large fraction of organic sulfur in the biosphere. Aside from sulfur amino acids, SQDG represents a considerable sink for sulfate in plants and is the only sulfur-containing anionic glycerolipid that is found in the photosynthetic membranes of plastids. We present the separation of sulfolipids from other fatty acids in two simple ways: by one- and two-dimensional thin-layer chromatography.

Aldehyde Oxidase 4 Plays a Critical Role in Delaying Silique Senescence by Catalyzing Aldehyde Detoxification.

The Arabidopsis (Arabidopsis thaliana) aldehyde oxidases are a multigene family of four oxidases (AAO1-AAO4) that oxidize a variety of aldehydes, among them abscisic aldehyde, which is oxidized to the phytohormone abscisic acid. Toxic aldehydes are generated in plants both under normal conditions and in response to stress. The detoxification of such aldehydes by oxidation is attributed to aldehyde dehydrogenases but never to aldehyde oxidases. The feasibility of the detoxification of aldehydes in siliques via oxidation by AAO4 was demonstrated, first, by its ability to efficiently oxidize an array of aromatic and aliphatic aldehydes, including the reactive carbonyl species (RCS) acrolein, hydroxyl-2-nonenal, and malondialdehyde. Next, exogenous application of several aldehydes to siliques in AAO4 knockout (KO) Arabidopsis plants induced severe tissue damage and enhanced malondialdehyde levels and senescence symptoms, but not in wild-type siliques. Furthermore, abiotic stresses such as dark and ultraviolet C irradiation caused an increase in endogenous RCS and higher expression levels of senescence marker genes, leading to premature senescence of KO siliques, whereas RCS and senescence marker levels in wild-type siliques were hardly affected. Finally, in naturally senesced KO siliques, higher endogenous RCS levels were associated with enhanced senescence molecular markers, chlorophyll degradation, and earlier seed shattering compared with the wild type. The aldehyde-dependent differential generation of superoxide and hydrogen peroxide by AAO4 and the induction of AAO4 expression by hydrogen peroxide shown here suggest a self-amplification mechanism for detoxifying additional reactive aldehydes produced during stress. Taken together, our results indicate that AAO4 plays a critical role in delaying senescence in siliques by catalyzing aldehyde detoxification.