Disentangling hydroxynitrile glucoside biosynthesis in a barley (Hordeum vulgare) metabolon provides access to elite malting barleys for ethyl carbamate-free whisky production.

Barley produces several specialized metabolites, including five α-, ß-, and γ-hydroxynitrile glucosides (HNGs). In malting barley, presence of the α-HNG epiheterodendrin gives rise to undesired formation of ethyl carbamate in the beverage production, especially after distilling. Metabolite-GWAS identified QTLs and underlying gene candidates possibly involved in the control of the relative and absolute content of HNGs, including an undescribed MATE transporter. By screening 325 genetically diverse barley accessions, we discovered three H. vulgare ssp. spontaneum (wild barley) lines with drastic changes in the relative ratios of the five HNGs. Knock-out (KO)-lines, isolated from the barley FIND-IT resource and each lacking one of the functional HNG biosynthetic genes (CYP79A12, CYP71C103, CYP71C113, CYP71U5, UGT85F22 and UGT85F23) showed unprecedented changes in HNG ratios enabling assignment of specific and mutually dependent catalytic functions to the biosynthetic enzymes involved. The highly similar relative ratios between the five HNGs found across wild and domesticated barley accessions indicate assembly of the HNG biosynthetic enzymes in a metabolon, the functional output of which was reconfigured in the absence of a single protein component. The absence or altered ratios of the five HNGs in the KO-lines did not change susceptibility to the fungal phytopathogen Pyrenophora teres causing net blotch. The study provides a deeper understanding of the organization of HNG biosynthesis in barley and identifies a novel, single gene HNG-0 line in an elite spring barley background for direct use in breeding of malting barley, eliminating HNGs as a source of ethyl carbamate formation in whisky production.

Detecting variation in starch granule size and morphology by high-throughput microscopy and flow cytometry.

Starch forms semi-crystalline, water-insoluble granules, the size and morphology of which vary according to biological origin. These traits, together with polymer composition and structure, determine the physicochemical properties of starch. However, screening methods to identify differences in starch granule size and shape are lacking. Here, we present two approaches for high-throughput starch granule extraction and size determination using flow cytometry and automated, high-throughput light microscopy. We evaluated the practicality of both methods using starch from different species and tissues and demonstrated their effectiveness by screening for induced variation in starch extracted from over 10,000 barley lines, yielding four with heritable changes in the ratio of large A-granules to small B-granules. Analysis of Arabidopsis lines altered in starch biosynthesis further demonstrates the applicability of these approaches. Identifying variation in starch granule size and shape will enable identification of trait-controlling genes for developing crops with desired properties, and could help optimise starch processing.

Perennials as Future Grain Crops: Opportunities and Challenges.

Perennial grain crops could make a valuable addition to sustainable agriculture, potentially even as an alternative to their annual counterparts. The ability of perennials to grow year after year significantly reduces the number of agricultural inputs required, in terms of both planting and weed control, while reduced tillage improves soil health and on-farm biodiversity. Presently, perennial grain crops are not grown at large scale, mainly due to their early stages of domestication and current low yields. Narrowing the yield gap between perennial and annual grain crops will depend on characterizing differences in their life cycles, resource allocation, and reproductive strategies and understanding the trade-offs between annualism, perennialism, and yield. The genetic and biochemical pathways controlling plant growth, physiology, and senescence should be analyzed in perennial crop plants. This information could then be used to facilitate tailored genetic improvement of selected perennial grain crops to improve agronomic traits and enhance yield, while maintaining the benefits associated with perennialism.

FIND-IT: Accelerated trait development for a green evolution.

Improved agricultural and industrial production organisms are required to meet the future global food demands and minimize the effects of climate change. A new resource for crop and microbe improvement, designated FIND-IT (Fast Identification of Nucleotide variants by droplet DigITal PCR), provides ultrafast identification and isolation of predetermined, targeted genetic variants in a screening cycle of less than 10 days. Using large-scale sample pooling in combination with droplet digital PCR (ddPCR) greatly increases the size of low-mutation density and screenable variant libraries and the probability of identifying the variant of interest. The method is validated by screening variant libraries totaling 500,000 barley (Hordeum vulgare) individuals and isolating more than 125 targeted barley gene knockout lines and miRNA or promoter variants enabling functional gene analysis. FIND-IT variants are directly applicable to elite breeding pipelines and minimize time-consuming technical steps to accelerate the evolution of germplasm.

Accelerated Domestication of New Crops: Yield is Key.

Sustainable agriculture in the future will depend on crops that are tolerant to biotic and abiotic stresses, require minimal input of water and nutrients and can be cultivated with a minimal carbon footprint. Wild plants that fulfill these requirements abound in nature but are typically low yielding. Thus, replacing current high-yielding crops with less productive but resilient species will require the intractable trade-off of increasing land area under cultivation to produce the same yield. Cultivating more land reduces natural resources, reduces biodiversity and increases our carbon footprint. Sustainable intensification can be achieved by increasing the yield of underutilized or wild plant species that are already resilient, but achieving this goal by conventional breeding programs may be a long-term prospect. De novo domestication of orphan or crop wild relatives using mutagenesis is an alternative and fast approach to achieve resilient crops with high yields. With new precise molecular techniques, it should be possible to reach economically sustainable yields in a much shorter period of time than ever before in the history of agriculture.

Site-specific, silicon-induced structural and molecular defence responses against powdery mildew infection in roses.

BACKGROUND: Silicon (Si) application to miniature potted roses can decrease severity of powdery mildew (Podosphaera pannosa) and this is associated with increased accumulation of callose and hydrogen peroxide (H2 O2 ) as well as hypersensitive (HR) cells. We used microscopy, gene expression and specific inhibitors of callose and H2 O2 to determine how effective these plant responses are in stopping infection. RESULTS: Pathogen arrest in Si-treated (Si+) plants was accompanied by increased accumulation of callose and H2 O2 in papillae and HR cells, respectively. These responses were reduced by application of specific inhibitors (2-deoxy-d-glucose for callose and catalase for H2 O2 ), which increased disease severity in Si+, but not in Si- plants. As markers for HR and callose, expression of the HR-specific gene hsr203J and the wound-related callose synthase GSL5, respectively, was studied. An up-regulation of expression was only seen after isolation of HR cells with laser capture microdissection. The up-regulation was higher in Si+ than in Si- plants and occurred concomitantly with more efficient photosynthesis in Si+ plants at high disease severity as compared to Si- plants. CONCLUSION: Silicon-mediated activation of callose and H2 O2 are decisive factors in the defence of rose against P. pannosa and these responses were accompanied with more efficient photosynthesis to strengthen the plant. Only by isolation of HR cells using laser capture microdissection as compared to analysis of whole leaf tissues allowed detection of elevated transcript levels of hsr203J and GSL5 at infection sites as markers for HR. © 2021 Society of Chemical Industry.

High light intensity aggravates latent manganese deficiency in maize.

Manganese (Mn) plays an important role in the oxygen-evolving complex, where energy from light absorption is used for water splitting. Although changes in light intensity and Mn status can interfere with the functionality of the photosynthetic apparatus, the interaction between these two factors and the underlying mechanisms remain largely unknown. Here, maize seedlings were grown hydroponically and exposed to two different light intensities under Mn-sufficient or -deficient conditions. No visual Mn deficiency symptoms appeared even though the foliar Mn concentration in the Mn-deficient treatments was reduced to 2 µg g-1. However, the maximum quantum yield efficiency of PSII and the net photosynthetic rate declined significantly, indicating latent Mn deficiency. The reduction in photosynthetic performance by Mn depletion was further aggravated when plants were exposed to high light intensity. Integrated transcriptomic and proteomic analyses showed that a considerable number of genes encoding proteins in the photosynthetic apparatus were only suppressed by a combination of Mn deficiency and high light, thus indicating interactions between changes in Mn nutritional status and light intensity. We conclude that high light intensity aggravates latent Mn deficiency in maize by interfering with the abundance of PSII proteins.

The iron-regulated transporter 1 plays an essential role in uptake, translocation and grain-loading of manganese, but not iron, in barley.

Transporters involved in manganese (Mn) uptake and intracellular Mn homeostasis in Arabidopsis and rice are well characterized, while much less is known for barley, which is particularly prone to Mn deficiency. In this study we have investigated the role of the iron-regulated transporter 1 (IRT1) for Mn uptake and translocation in barley plants. We employed an RNAi approach to reduce HvIRT1 expression to 5% of the wild-type level. This enabled characterization of the functional role of HvIRT1 by use of advanced imaging and phenotyping techniques applied to plants growing in hydroponics or soils with different Mn availability. Our results highlight the importance of HvIRT1 for the transport of Mn across the root endodermis into the stele. In the hvirt1-RNAi lines, a chlorotic phenotype with reduced shoot Mn concentration and impaired photosynthetic functionality was observed, especially under conditions with low Mn availability. We also document that HvIRT1 controlled the Mn distribution within the barley grain. Surprisingly, unlike other IRT1 orthologues, HvIRT1 played no significant role in iron uptake. We conclude that the barley IRT1 orthologue has a novel function with respect to ensuring sufficient shoot Mn concentrations. The preference of IRT1 for Mn instead of Fe is discussed in an evolutionary context.

Photosystem II Functionality in Barley Responds Dynamically to Changes in Leaf Manganese Status.

A catalytic manganese (Mn) cluster is required for the oxidation of water in the oxygen-evolving complex (OEC) of photosystem II (PSII) in plants. Despite this essential role of Mn in generating the electrons driving photosynthesis, limited information is available on how Mn deficiency affects PSII functionality. We have here used parameters derived from measurements of fluorescence induction kinetics (OJIP transients), non-photochemical quenching (NPQ) and PSII subunit composition to investigate how latent Mn deficiency changes the photochemistry in two barley genotypes differing in Mn efficiency. Mn deficiency caused dramatic reductions in the quantum yield of PSII and led to the appearance of two new inflection points, the K step and the D dip, in the OJIP fluorescence transients, indicating severe damage to the OEC. In addition, Mn deficiency decreased the ability to induce NPQ in the light, rendering the plants incapable of dissipating excess energy in a controlled way. Thus, the Mn deficient plants became severely affected in their ability to recover from high light-induced photoinhibition, especially under strong Mn deficiency. Interestingly, the Mn-efficient genotype was able to maintain a higher NPQ than the Mn-inefficient genotype when exposed to mild Mn deficiency. However, during severe Mn deficiency, there were no differences between the two genotypes, suggesting a general loss of the ability to disassemble and repair PSII. The pronounced defects of PSII activity were supported by a dramatic decrease in the abundance of the OEC protein subunits, PsbP and PsbQ in response to Mn deficiency for both genotypes. We conclude that regulation of photosynthetic performance by means of maintaining and inducing NPQ mechanisms contribute to genotypic differences in the Mn efficiency of barley genotypes growing under conditions with mild Mn deficiency.

Identification of manganese efficiency candidate genes in winter barley (Hordeum vulgare) using genome wide association mapping.

BACKGROUND: Manganese (Mn) has several essential functions in plants, including a role as cofactor in the oxygen evolving complex (OEC) of photosystem II (PSII). Manganese deficiency is a major plant nutritional disorder in winter cereals resulting in significant yield reductions and winter kill in more severe cases. Among the winter cereals, genotypes of winter barley are known to differ considerably in tolerance to Mn deficiency, but the genes controlling the Mn deficiency trait remains elusive. RESULTS: Experiments were conducted using 248 barley varieties, cultivated in six distinct environments prone to induce Mn deficiency. High-throughput phenotyping for Mn deficiency was performed by chlorophyll a (Chl a) fluorescence analysis to quantify the quantum yield efficiency of PSII. High-throughput phenotyping in combination with ICP-OES based multi-element analyses allowed detection of marker-trait associations by genome wide association (GWA) mapping. Several key candidate genes were identified, including PSII subunit proteins, germin like proteins and Mn superoxide dismutase. The putative roles of the encoded proteins in Mn dependent metabolic processes are discussed. CONCLUSIONS: Fifty-four candidate genes were identified by Chl a fluorescence phenotyping and association genetics. Tolerance of plants to Mn deficiency, which is referred to as Mn efficiency, appeared to be a complex trait involving many genes. Moreover, the trait appeared to be highly dependent on the environmental conditions in field. This study provides the basis for an improved understanding of the parameters influencing Mn efficiency and is valuable in future plant breeding aiming at producing new varieties with improved tolerance to cultivation in soil prone to induce Mn deficiency.

Molecular speciation and tissue compartmentation of zinc in durum wheat grains with contrasting nutritional status.

Low concentration of zinc (Zn) in the endosperm of cereals is a major factor contributing to Zn deficiency in human populations. We have investigated how combined Zn and nitrogen (N) fertilization affects the speciation and localization of Zn in durum wheat (Triticum durum). Zn-binding proteins were analysed with liquid chromatography ICP-MS and Orbitrap MS(2) , respectively. Laser ablation ICP-MS with simultaneous Zn, sulphur (S) and phosphorus (P) detection was used for bioimaging of Zn and its potential ligands. Increasing the Zn and N supply had a major impact on the Zn concentration in the endosperm, reaching concentrations higher than current breeding targets. The S concentration also increased, but S was only partly co-localized with Zn. The mutual Zn and S enrichment was reflected in substantially more Zn bound to small cysteine-rich proteins (apparent size 10-30 kDa), whereas the response of larger proteins (apparent size > 50 kDa) was only modest. Most of the Zn-responsive proteins were associated with redox- and stress-related processes. This study offers a methodological platform to deepen the understanding of processes behind endosperm Zn enrichment. Novel information is provided on how the localization and speciation of Zn is modified during Zn biofortification of grains.

Acetylation of cell wall is required for structural integrity of the leaf surface and exerts a global impact on plant stress responses.

The epidermis on leaves protects plants from pathogen invasion and provides a waterproof barrier. It consists of a layer of cells that is surrounded by thick cell walls, which are partially impregnated by highly hydrophobic cuticular components. We show that the Arabidopsis T-DNA insertion mutants of REDUCED WALL ACETYLATION 2 (rwa2), previously identified as having reduced O-acetylation of both pectins and hemicelluloses, exhibit pleiotrophic phenotype on the leaf surface. The cuticle layer appeared diffused and was significantly thicker and underneath cell wall layer was interspersed with electron-dense deposits. A large number of trichomes were collapsed and surface permeability of the leaves was enhanced in rwa2 as compared to the wild type. A massive reprogramming of the transcriptome was observed in rwa2 as compared to the wild type, including a coordinated up-regulation of genes involved in responses to abiotic stress, particularly detoxification of reactive oxygen species and defense against microbial pathogens (e.g., lipid transfer proteins, peroxidases). In accordance, peroxidase activities were found to be elevated in rwa2 as compared to the wild type. These results indicate that cell wall acetylation is essential for maintaining the structural integrity of leaf epidermis, and that reduction of cell wall acetylation leads to global stress responses in Arabidopsis.

A phospholipid uptake system in the model plant Arabidopsis thaliana.

Plants use solar energy to produce lipids directly from inorganic elements and are not thought to require molecular systems for lipid uptake from the environment. Here we show that Arabidopsis thaliana Aminophospholipid ATPase10 (ALA10) is a P4-type ATPase flippase that internalizes exogenous phospholipids across the plasma membrane, after which they are rapidly metabolized. ALA10 expression and phospholipid uptake are high in the epidermal cells of the root tip and in guard cells, the latter of which regulate the size of stomatal apertures to modulate gas exchange. ALA10-knockout mutants exhibit reduced phospholipid uptake at the root tips and guard cells and are affected in growth and transpiration. The presence of a phospholipid uptake system in plants is surprising. Our results suggest that one possible physiological role of this system is to internalize lysophosphatidylcholine, a signalling lipid involved in root development and stomatal control.

Sensitive Detection of Phosphorus Deficiency in Plants Using Chlorophyll a Fluorescence.

Phosphorus (P) is a finite natural resource and an essential plant macronutrient with major impact on crop productivity and global food security. Here, we demonstrate that time-resolved chlorophyll a fluorescence is a unique tool to monitor bioactive P in plants and can be used to detect latent P deficiency. When plants suffer from P deficiency, the shape of the time-dependent fluorescence transients is altered distinctively, as the so-called I step gradually straightens and eventually disappears. This effect is shown to be fully reversible, as P resupply leads to a rapid restoration of the I step. The fading I step suggests that the electron transport at photosystem I (PSI) is affected in P-deficient plants. This is corroborated by the observation that differences at the I step in chlorophyll a fluorescence transients from healthy and P-deficient plants can be completely eliminated through prior reduction of PSI by far-red illumination. Moreover, it is observed that the barley (Hordeum vulgare) mutant Viridis-zb(63), which is devoid of PSI activity, similarly does not display the I step. Among the essential plant nutrients, the effect of P deficiency is shown to be specific and sufficiently sensitive to enable rapid in situ determination of latent P deficiency across different plant species, thereby providing a unique tool for timely remediation of P deficiency in agriculture.

A laser ablation ICP-MS based method for multiplexed immunoblot analysis: applications to manganese-dependent protein dynamics of photosystem II in barley (Hordeum vulgare L.).

Manganese (Mn) constitutes an essential co-factor in the oxygen-evolving complex of photosystem II (PSII). Consequently, Mn deficiency reduces photosynthetic efficiency and leads to changes in PSII composition. In order to study these changes, multiplexed protein assays are advantageous. Here, we developed a multiplexed antibody-based assay and analysed selected PSII subunits in barley (Hordeum vulgare L.). A selection of antibodies were labelled with specific lanthanides and immunoreacted with thylakoids exposed to Mn deficiency after western blotting. Subsequently, western blot membranes were analysed by laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS), which allowed selective and relative quantitative analysis via the different lanthanides. The method was evaluated against established liquid chromatography electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) methods, based on data-dependent acquisition (DDA) and selected reaction monitoring (SRM). Manganese deficiency resulted in a general decrease in PSII protein abundances, an effect that was shown to be reversible upon Mn re-supplementation. Specifically, the extrinsic proteins PsbP and PsbQ showed Mn-dependent changes in abundances. Similar trends in the response to Mn deficiency at the protein level were observed when comparing DDA, SRM and LA-ICP-MS results. A biologically important exception to this trend was the loss of PsbO in the SRM analysis, which highlights the necessity of validating protein changes by more than one technique. The developed method enables a higher number of proteins to be multiplexed in comparison to existing immunoassays. Furthermore, multiplexed protein analysis by LA-ICP-MS provides an analytical platform with high throughput appropriate for screening large collections of plants.

Golgi localized barley MTP8 proteins facilitate Mn transport.

Many metabolic processes in plants are regulated by manganese (Mn) but limited information is available on the molecular mechanisms controlling cellular Mn homeostasis. In this study, a yeast assay was used to isolate and characterize two genes, MTP8.1 and MTP8.2, which encode membrane-bound proteins belonging to the cation diffusion facilitator (CDF) family in the cereal species barley (Hordeum vulgare). Transient expression in onion epidermal cells showed that MTP8.1 and MTP8.2 proteins fused to the green fluorescent protein (GFP) are localized to Golgi. When heterologously expressed in yeast, MTP8.1 and MTP8.2 were found to be Mn transporters catalysing Mn efflux in a similar manner as the Golgi localized endogenous yeast protein Pmr1p. The level of MTP8.1 transcripts in barley roots increased with external Mn supply ranging from deficiency to toxicity, while MTP8.2 transcripts decreased under the same conditions, indicating non-overlapping functions for the two genes. In barley leaves, the expression of both MTP8 genes declined in response to toxic Mn additions to the roots suggesting a role in ensuring proper delivery of Mn to Golgi. Based on the above we suggest that barley MTP8 proteins are involved in Mn loading to the Golgi apparatus and play a role in Mn homeostasis by delivering Mn to Mn-dependent enzymes and/or by facilitating Mn efflux via secretory vesicles. This study highlights the importance of MTP transporters in Mn homeostasis and is the first report of Golgi localized Mn2+ transport proteins in a monocot plant species.

HvZIP7 mediates zinc accumulation in barley (Hordeum vulgare) at moderately high zinc supply.

High expression of zinc (Zn)-regulated, iron-regulated transporter-like protein (ZIP) genes increases root Zn uptake in dicots, leading to high accumulation of Zn in shoots. However, none of the ZIP genes tested previously in monocots could enhance shoot Zn accumulation. In this report, barley (Hordeum vulgare) HvZIP7 was investigated for its functions in Zn transport. The functions of HvZIP7 in planta were studied using in situ hybridization and transient analysis of subcellular localization with a green fluorescent protein (GFP) reporter. Transgenic barley lines overexpressing HvZIP7 were also generated to further understand the functions of HvZIP7 in metal transport. HvZIP7 is strongly induced by Zn deficiency, primarily in vascular tissues of roots and leaves, and its protein was localized in the plasma membrane. These properties are similar to its closely related homologs in dicots. Overexpression of HvZIP7 in barley plants increased Zn uptake when moderately high concentrations of Zn were supplied. Significantly, there was a specific enhancement of shoot Zn accumulation, with no measurable increase in iron (Fe), manganese (Mn), copper (Cu) or cadmium (Cd). HvZIP7 displays characteristics of low-affinity Zn transport. The unique function of HvZIP7 provides new insights into the role of ZIP genes in Zn homeostasis in monocots, and offers opportunities to develop Zn biofortification strategies in cereals.

Barley metallothioneins differ in ontogenetic pattern and response to metals.

The barley genome encodes a family of 10 metallothioneins (MTs) that have not previously been subject to extensive gene expression profiling. We show here that expression of MT1a, MT2b1, MT2b2 and MT3 in barley leaves increased more than 50-fold during the first 10 d after germination. Concurrently, the root-specific gene MT1b1 was 1000-fold up-regulated. Immunolocalizations provided the first evidence for accumulation of MT1a and MT2a proteins in planta, with correlation to transcript levels. In developing grains, MT2a and MT4 expression increased 4- and 300-fold over a 28-day-period after pollination. However, among the MT grain transcripts MT2c was the most abundant, whereas MT4 was the least abundant. Excess Cu up-regulated three out of the six MTs expressed in leaves of young barley plants. In contrast, most MTs were down-regulated by excess Zn or Cd. Zn starvation led to up-regulation of MT1a, whereas Cu starvation up-regulated MT2a, which has two copper-responsive elements in the promoter. Arabidopsis lines constitutively overexpressing barley MT2a showed increased sensitivity to excess Cd and Zn but no Cu-induced response. We suggest that barley MTs are differentially involved in intracellular homeostasis of essential metal ions and that a subset of barley MTs is specifically involved in Cu detoxification.

Characterization of two genes encoding metal tolerance proteins from Beta vulgaris subspecies maritima that confers manganese tolerance in yeast.

Manganese (Mn(2+)) is an essential micronutrient in plants. However increased Mn(2+) levels are toxic to plant cells. Metal tolerance proteins (MTPs), member of cation diffusion facilitator protein (CDF) family, have important roles in metal homeostatis in different plant species and catalyse efflux of excess metal ions. In this study, we identified and characterized two MTP genes from Beta vulgaris spp. maritima (B. v. ssp. maritima). Overexpression of these two genes provided Mn tolerance in yeast cells. Sequence analyses displayed BmMTP10 and BmMTP11 as members of the Mn-CDF family. Functional analyses of these proteins indicated that they are specific to Mn(2+) with a role in reducing excess cellular Mn(2+) levels when expressed in yeast. GFP-fusion constructs of both proteins localized to the Golgi apparatus as a punctuated pattern. Finally, Q-RT-PCR results showed that BmMTP10 expression was induced threefold in response to the excess Mn(2+) treatment. On the other hand BmMTP11 expression was not affected in response to excess Mn(2+) levels. Thus, our results suggest that the BmMTP10 and BmMTP11 proteins from B. v. ssp. maritima have non-redundant functions in terms of Mn(2+) detoxification with a similar in planta localization and function as the Arabidopsis Mn-CDF homolog AtMTP11 and this conservation shows the evolutionary importance of these vesicular proteins in heavy metal homeostatis among plant species.