Development of an efficient glucosinolate extraction method

Background: Glucosinolates, anionic sulfur rich secondary metabolites, have been extensively studied because of their occurrence in the agriculturally important brassicaceae and their impact on human and animal health. There is also increasing interest in the biofumigant properties of toxic glucosinolate hydrolysis products as a method to control agricultural pests. Evaluating biofumigation potential requires rapid and accurate quantification of glucosinolates, but current commonly used methods of extraction prior to analysis involve a number of time consuming and hazardous steps; this study aimed to develop an improved method for glucosinolate extraction. Results: Three methods previously used to extract glucosinolates from brassicaceae tissues, namely extraction in cold methanol, extraction in boiling methanol, and extraction in boiling water were compared across tissue type (root, stem leaf ) and four brassicaceae species (B. juncea, S. alba, R. sativus, and E. sativa). Cold methanol extraction was shown to perform as well or better than all other tested methods for extraction of glucosinolates with the exception of glucoraphasatin in R. sativus shoots. It was also demonstrated that lyophilisation methods, routinely used during extraction to allow tissue disruption, can reduce final glucosinolate concentrations and that extracting from frozen wet tissue samples in cold 80% methanol is more effective. Conclusions: We present a simplified method for extracting glucosinolates from plant tissues which does not require the use of a freeze drier or boiling methanol, and is therefore less hazardous, and more time and cost effective. The presented method has been shown to have comparable or improved glucosinolate extraction efficiency relative to the commonly used ISO method for major glucosinolates in the Brassicaceae species studied: sinigrin and gluconasturtiin in B. juncea; sinalbin, glucotropaeolin, and gluconasturtiin in S. alba; glucoraphenin and glucoraphasatin in R. sativus; and glucosatavin, glucoerucin and glucoraphanin in E. sativa

Data in support of genetic architecture of glucosinolate variations in Brassica napus

The transcriptome-based GWAS approach, Associative Transcriptomics (AT), which was employed to uncover the genetic basis controlling quantitative variation of glucosinolates in Brassica napus vegetative tissues is described. This article includes the phenotypic data of leaf and root glucosinolate (GSL) profiles across a diversity panel of 288 B. napus genotypes, as well as information on population structure and levels of GSLs grouped by crop types. Moreover, data on genetic associations of single nucleotide polymorphism (SNP) markers and gene expression markers (GEMs) for the major GSL types are presented in detail, while Manhattan plots and QQ plots for the associations of individual GSLs are also included. Root genetic association are supported by differential expression analysis generated from root RNA-seq. For further interpretation and details, please see the related research article entitled ‘Genetic architecture of glucosinolate variation in Brassica napus’ [1]

Increasing water-use efficiency directly through genetic manipulation of stomatal density

Improvement in crop water-use efficiency (WUE) is a critical priority for regions facing increased drought or diminished groundwater resources. Despite new tools for the manipulation of stomatal development, the engineering of plants with high WUE remains a challenge. We used Arabidopsis epidermal patterning factor (EPF) mutants exhibiting altered stomatal density to test whether WUE could be improved directly by manipulation of the genes controlling stomatal density. Specifically, we tested whether constitutive overexpression of EPF2 reduced stomatal density and maximum stomatal conductance (gw(max)) sufficiently to increase WUE. We found that a reduction in gw(max) via reduced stomatal density in EPF2-overexpressing plants (EPF2OE) increased both instantaneous and long-term WUE without altering significantly the photosynthetic capacity. Conversely, plants lacking both EPF1 and EPF2 expression (epf1epf2) exhibited higher stomatal density, higher gw(max) and lower instantaneous WUE, as well as lower (but not significantly so) long-term WUE. Targeted genetic modification of stomatal conductance, such as in EPF2OE, is a viable approach for the engineering of higher WUE in crops, particularly in future high-carbon-dioxide (CO2) atmospheres

Manipulating stomatal density enhances drought tolerance without deleterious effect on nutrient uptake

Manipulation of stomatal density was investigated as a potential tool for enhancing drought tolerance or nutrient uptake. Drought tolerance and soil water retention were assessed using Arabidopsis epidermal patterning factor mutants manipulated to have increased or decreased stomatal density. Root nutrient uptake via mass flow was monitored under differing plant watering regimes using nitrogen-15 (15N) isotope and mass spectrometry. Plants with less than half of their normal complement of stomata, and correspondingly reduced levels of transpiration, conserve soil moisture and are highly drought tolerant but show little or no reduction in shoot nitrogen concentrations especially when water availability is restricted. By contrast, plants with over twice the normal density of stomata have a greater capacity for nitrogen uptake, except when water availability is restricted. We demonstrate the possibility of producing plants with reduced transpiration which have increased drought tolerance, with little or no loss of nutrient uptake. We demonstrate that increasing transpiration can enhance nutrient uptake when water is plentiful

Genetic architecture of glucosinolate variation in Brassica napus

The diverse biological activities of glucosinolate (GSL) hydrolysis products play significant biological and economical roles in the defense system and nutritional qualities of Brassica napus (oilseed rape). Yet, genomic-based study of the B. napus GSL regulatory mechanisms are scarce due to the complexity of working with polyploid species. To address these challenges, we used transcriptome-based GWAS approach, Associative Transcriptomics (AT), across a diversity panel of 288 B. napus genotypes to uncover the underlying genetic basis controlling quantitative variation of GSLs in B. napus vegetative tissues. Single nucleotide polymorphism (SNP) markers and gene expression markers (GEMs) associations identify orthologues of MYB28/HAG1 (AT5G61420), specifically the copies on chromosome A9 and C2, to be the key regulators of aliphatic GSL variation in leaves. We show that the positive correlation observed between aliphatic GSLs in seed and leaf is due to the amount synthesized, as controlled by Bna.HAG1.A9 and Bna.HAG1.C2, rather than by variation in the transport processes. In addition, AT and differential expression analysis in root tissues implicate an orthologue of MYB29/HAG3 (AT5G07690), Bna.HAG3.A3, as controlling root aromatic GSL variation. Based on the root expression data we also propose Bna.MAM3.A3 to have a role in controlling phenylalanine chain elongation for aromatic GSL biosynthesis. This work uncovers a regulator of homophenylalanine-derived aromatic GSLs and implicates the shared biosynthetic pathways between aliphatic and aromatic GSLs

Potential isothiocyanate release remains constant across biofumigant seeding rates

Biofumigation is an integrated pest management method involving the mulching of a glucosinolate containing cover crop into a field in order to generate toxic isothiocyanates, effective soil borne pest control compounds. Variation in biofumigation efficacy demonstrates a need to better understand the factors affecting pest control outcomes and develop best practice for biofumigant choice, growth conditions and mulching methods which allow the greatest potential isothiocyanate release. We measured the glucosinolate concentration of 6 different commercial varieties of three different biofumigant plant species: Brassica juncea (ISCI99, Vitasso, Scala) Raphanus sativus (Diablo, Bento) and Sinapis alba (Ida Gold). Plants were grown at a range of commercially appropriate seeding rates and sampled at three growth stages (early development, mature, and 50% flowering). Within biofumigant species, highest ITC release potential was achieved with B. juncea cv. ISCI99 and R. sativus cv. Bento. Highest ITC release potential occurred at 50% flowering growth stage across species. Seeding rate had minor impact on ITC release potential from R. sativus but had no significant effect on the ITC release potential of B. juncea or S. alba cultivars

Stomatal Function Requires Pectin De-methyl-esterification of the Guard Cell Wall

Stomatal opening and closure depends on changes in turgor pressure acting within guard cells to alter cell shape. The extent of these shape changes is limited by the mechanical properties of the cells, which will be largely dependent on the structure of the cell walls. Although it has long been observed that guard cells are anisotropic due to differential thickening and the orientation of cellulose microfibrils, our understanding of the composition of the cell wall that allows them to undergo repeated swelling and deflation remains surprisingly poor. Here, we show that the walls of guard cells are rich in unesterified pectins. We identify a pectin methylesterase gene, PME6, which is highly expressed in guard cells and required for stomatal function. pme6-1 mutant guard cells have walls enriched in methyl-esterified pectin and show a decreased dynamic range in response to triggers of stomatal opening/closure, including elevated osmoticum, suggesting that abrogation of stomatal function reflects a mechanical change in the guard cell wall. Altered stomatal function leads to increased conductance and evaporative cooling, as well as decreased plant growth. The growth defect of the pme6-1 mutant is rescued by maintaining the plants in elevated CO2, substantiating gas exchange analyses, indicating that the mutant stomata can bestow an improved assimilation rate. Restoration of PME6 rescues guard cell wall pectin methyl-esterification status, stomatal function, and plant growth. Our results establish a link between gene expression in guard cells and their cell wall properties, with a corresponding effect on stomatal function and plant physiology

Decreased photosynthesis in the erect panicle 3 (ep3) mutant of rice is associated with reduced stomatal conductance and attenuated guard cell development

The ERECT PANICLE 3 gene of rice encodes a peptide that exhibits more than 50% sequence identity with the Arabidopsis F-box protein HAWAIIAN SKIRT (HWS). Ectopic expression of the Os02g15950 coding sequence, driven by the HWS (At3g61950) promoter, rescued the hws-1 flower phenotype in Arabidopsis confirming that EP3 is a functional orthologue of HWS. In addition to displaying an erect inflorescence phenotype, loss-of-function mutants of Os02g15950 exhibited a decrease in leaf photosynthetic capacity and stomatal conductance. Analysis of a range of physiological and anatomical features related to leaf photosynthesis revealed no alteration in Rubisco content and no notable changes in mesophyll size or arrangement. However, both ep3 mutant plants and transgenic lines that have a T-DNA insertion within the Os02g15950 (EP3) gene exhibit smaller stomatal guard cells compared with their wild-type controls. This anatomical characteristic may account for the observed decrease in leaf photosynthesis and provides evidence that EP3 plays a role in regulating stomatal guard cell development

Effects of elevated carbon dioxide on stomatal characteristics and carbon isotope ratio of Arabidopsis thaliana ecotypes originating from an altitudinal gradient

Stomatal functioning regulates the fluxes of CO2 and water vapour between vegetation and atmosphere and thereby influences plant adaptation to their habitats. Stomatal traits are controlled by external environmental and internal cellular signalling. The objective of this work was to quantify the effects of CO2 enrichment (CE) on stomatal density-related properties, guard cell length (GCL) and carbon isotope ratio (δ13C) of a range of Arabidopsis thaliana ecotypes originating from a wide altitudinal range (50–1260 m above sea level (asl)), and grown at 400 and 800 ppm [CO2], and thereby elucidate the possible adaptation and acclimation responses controlling stomatal traits and water use efficiency (WUE). There was highly-significant variation among ecotypes in the magnitude and direction of response of stomatal traits namely, stomatal density (SD) and index (SI) and guard cell length (GCL), and δ13C to CE, which represented a short-term acclimation response. A majority of ecotypes showed increased SD and SI with CE with the response not depending on the altitude of origin. Significant ecotypic variation was shown in all stomatal traits and δ13C at each [CO2]. At 400 ppm, means of SD, SI and GCL for broad altitudinal ranges, i.e. low (400 m), increased with increasing altitude, which represented an adaptation response to decreased availability of CO2 with altitude. δ13C was negatively correlated to SD and SI at 800 ppm but not at 400 ppm. Our results highlight the diversity in the response of key stomatal characters to CE and altitude within the germplasm of A. thaliana and the need to consider this diversity when using A. thaliana as a model plant

A chloroplast retrograde signal, 3’phosphoadenosine 5’-phosphate, acts as a secondary messenger in abscisic acid signaling in stomatal closure and germination

Organelle-nuclear retrograde signaling regulates gene expression, but its roles in specialized cells and integration with hormonal signaling remain enigmatic. Here we show that the SAL1-PAP (3'-phosphoadenosine 5'- phosphate) retrograde pathway interacts with abscisic acid (ABA) signaling to regulate stomatal closure and seed germination in Arabidopsis. Genetically or exogenously manipulating PAP bypasses the canonical signaling components ABA Insensitive 1 (ABI1) and Open Stomata 1 (OST1); priming an alternative pathway that restores ABA-responsive gene expression, ROS bursts, ion channel function, stomatal closure and drought tolerance in ost1-2. PAP also inhibits wild type and abi1-1 seed germination by enhancing ABA sensitivity. PAP-XRN signaling interacts with ABA, ROS and Ca2+; up-regulating multiple ABA signaling components, including lowly-expressed Calcium Dependent Protein Kinases (CDPKs) capable of activating the anion channel SLAC1. Thus, PAP exhibits many secondary messenger attributes and exemplifies how retrograde signals can have broader roles in hormone signaling, allowing chloroplasts to fine-tune physiological responses.Wannarat Pornsiriwong, Gonzalo M Estavillo, Kai Xun Chan, Estee E Tee, Diep Ganguly, Peter A Crisp, Su Yin Phua, Chenchen Zhao, Jiaen Qiu, Jiyoung Park, Miing Tiem Yong, Nazia Nisar, Arun Kumar Yadav, Benjamin Schwessinger, John Rathjen, Christopher I Cazzonelli, Philippa B Wilson, Matthew Gilliham, Zhong-Hua Chen, Barry J Pogso