Multilayered regulation of developmentally programmed pre-anthesis tip degeneration of the barley inflorescence.

Leaf and floral tissue degeneration is a common feature in plants. In cereal crops such as barley (Hordeum vulgare L.), pre-anthesis tip degeneration (PTD) starts with growth arrest of the inflorescence meristem dome, which is followed basipetally by the degeneration of floral primordia and the central axis. Due to its quantitative nature and environmental sensitivity, inflorescence PTD constitutes a complex, multilayered trait affecting final grain number. This trait appears to be highly predictable and heritable under standardized growth conditions, consistent with a developmentally programmed mechanism. To elucidate the molecular underpinnings of inflorescence PTD, we combined metabolomic, transcriptomic, and genetic approaches to show that barley inflorescence PTD is accompanied by sugar depletion, amino acid degradation, and abscisic acid responses involving transcriptional regulators of senescence, defense, and light signaling. Based on transcriptome analyses, we identified GRASSY TILLERS1 (HvGT1), encoding an HD-ZIP transcription factor, as an important modulator of inflorescence PTD. A gene-edited knockout mutant of HvGT1 delayed PTD and increased differentiated apical spikelets and final spikelet number, suggesting a possible strategy to increase grain number in cereals. We propose a molecular framework that leads to barley PTD, the manipulation of which may increase yield potential in barley and other related cereals.

Metabolomic and transcriptomic analyses of yellow-flowered crocuses to infer alternative sources of saffron metabolites.

BACKGROUND: The increasing demand for saffron metabolites in various commercial industries, including medicine, food, cosmetics, and dyeing, is driven by the discovery of their diverse applications. Saffron, derived from Crocus sativus stigmas, is the most expensive spice, and there is a need to explore additional sources to meet global consumption demands. In this study, we focused on yellow-flowering crocuses and examined their tepals to identify saffron-like compounds. RESULTS: Through metabolomic and transcriptomic approaches, our investigation provides valuable insights into the biosynthesis of compounds in yellow-tepal crocuses that are similar to those found in saffron. The results of our study support the potential use of yellow-tepal crocuses as a source of various crocins (crocetin glycosylated derivatives) and flavonoids. CONCLUSIONS: Our findings suggest that yellow-tepal crocuses have the potential to serve as a viable excessive source of some saffron metabolites. The identification of crocins and flavonoids in these crocuses highlights their suitability for meeting the demands of various industries that utilize saffron compounds. Further exploration and utilization of yellow-tepal crocuses could contribute to addressing the growing global demand for saffron-related products.

Combined nitrogen and drought stress leads to overlapping and unique proteomic responses in potato.

MAIN CONCLUSION: Nitrogen deficient and drought-tolerant or sensitive potatoes differ in proteomic responses under combined (NWD) and individual stresses. The sensitive genotype 'Kiebitz' exhibits a higher abundance of proteases under NWD. Abiotic stresses such as N deficiency and drought affect the yield of Solanum tuberosum L. tremendously. Therefore, it is of importance to improve potato genotypes in terms of stress tolerance. In this study, we identified differentially abundant proteins (DAPs) in four starch potato genotypes under N deficiency (ND), drought stress (WD), or combined stress (NWD) in two rain-out shelter experiments. The gel-free LC-MS analysis generated a set of 1177 identified and quantified proteins. The incidence of common DAPs in tolerant and sensitive genotypes under NWD indicates general responses to this stress combination. Most of these proteins were part of the amino acid metabolism (13.9%). Three isoforms of S-adenosyl methionine synthase (SAMS) were found to be lower abundant in all genotypes. As SAMS were found upon application of single stresses as well, these proteins appear to be part of the general stress response in potato. Interestingly, the sensitive genotype 'Kiebitz' showed a higher abundance of three proteases (subtilase, carboxypeptidase, subtilase family protein) and a lower abundance of a protease inhibitor (stigma expressed protein) under NWD stress compared to control plants. The comparably tolerant genotype 'Tomba', however, displayed lower abundances of proteases. This indicates a better coping strategy for the tolerant genotype and a quicker reaction to WD when previously stressed with ND.

How do barley plants with impaired photosynthetic light acclimation survive under high-light stress?

MAIN CONCLUSION: WHIRLY1 deficient barley plants surviving growth at high irradiance displayed increased non-radiative energy dissipation, enhanced contents of zeaxanthin and the flavonoid lutonarin, but no changes in α-tocopherol nor glutathione. Plants are able to acclimate to environmental conditions to optimize their functions. With the exception of obligate shade plants, they can adjust their photosynthetic apparatus and the morphology and anatomy of their leaves to irradiance. Barley (Hordeum vulgare L., cv. Golden Promise) plants with reduced abundance of the protein WHIRLY1 were recently shown to be unable to acclimatise important components of the photosynthetic apparatus to high light. Nevertheless, these plants did not show symptoms of photoinhibition. High-light (HL) grown WHIRLY1 knockdown plants showed clear signs of exposure to excessive irradiance such as a low epoxidation state of the violaxanthin cycle pigments and an early light saturation of electron transport. These responses were underlined by a very large xanthophyll cycle pool size and by an increased number of plastoglobules. Whereas zeaxanthin increased with HL stress, α-tocopherol, which is another lipophilic antioxidant, showed no response to excessive light. Also the content of the hydrophilic antioxidant glutathione showed no increase in W1 plants as compared to the wild type, whereas the flavone lutonarin was induced in W1 plants. HPLC analysis of removed epidermal tissue indicated that the largest part of lutonarin was presumably located in the mesophyll. Since lutonarin is a better antioxidant than saponarin, the major flavone present in barley leaves, it is concluded that lutonarin accumulated as a response to oxidative stress. It is also concluded that zeaxanthin and lutonarin may have served as antioxidants in the WHIRLY1 knockdown plants, contributing to their survival in HL despite their restricted HL acclimation.

Unraveling metabolic patterns and molecular mechanisms underlying storability in sugar beet.

BACKGROUND: Sugar beet is an important crop for sugar production. Sugar beet roots are stored up to several weeks post-harvest waiting for processing in the sugar factories. During this time, sucrose loss and invert sugar accumulation decreases the final yield and processing quality. To improve storability, more information about post-harvest metabolism is required. We investigated primary and secondary metabolites of six sugar beet varieties during storage. Based on their variety-specific sucrose loss, three storage classes representing well, moderate, and bad storability were compared. Furthermore, metabolic data were visualized together with transcriptome data to identify potential mechanisms involved in the storage process. RESULTS: We found that sugar beet varieties that performed well during storage have higher pools of 15 free amino acids which were already observable at harvest. This storage class-specific feature is visible at harvest as well as after 13 weeks of storage. The profile of most of the detected organic acids and semi-polar metabolites changed during storage. Only pyroglutamic acid and two semi-polar metabolites, including ferulic acid, show higher levels in well storable varieties before and/or after 13 weeks of storage. The combinatorial OMICs approach revealed that well storable varieties had increased downregulation of genes involved in amino acid degradation before and after 13 weeks of storage. Furthermore, we found that most of the differentially genes involved in protein degradation were downregulated in well storable varieties at both timepoints, before and after 13 weeks of storage. CONCLUSIONS: Our results indicate that increased levels of 15 free amino acids, pyroglutamic acid and two semi-polar compounds, including ferulic acid, were associated with a better storability of sugar beet taproots. Predictive metabolic patterns were already apparent at harvest. With respect to elongated storage, we highlighted the role of free amino acids in the taproot. Using complementary transcriptomic data, we could identify potential underlying mechanisms of sugar beet storability. These include the downregulation of genes for amino acid degradation and metabolism as well as a suppressed proteolysis in the well storable varieties.

Cellulose defects in the Arabidopsis secondary cell wall promote early chloroplast development.

Lincomycin (LIN)-mediated inhibition of protein synthesis in chloroplasts prevents the greening of seedlings, represses the activity of photosynthesis-related genes in the nucleus, including LHCB1.2, and induces the phenylpropanoid pathway, resulting in the production of anthocyanins. In genomes uncoupled (gun) mutants, LHCB1.2 expression is maintained in the presence of LIN or other inhibitors of early chloroplast development. In a screen using concentrations of LIN lower than those employed to isolate gun mutants, we have identified happy on lincomycin (holi) mutants. Several holi mutants show an increased tolerance to LIN, exhibiting de-repressed LHCB1.2 expression and chlorophyll synthesis in seedlings. The mutations responsible were identified by whole-genome single-nucleotide polymorphism (SNP) mapping, and most were found to affect the phenylpropanoid pathway; however, LHCB1.2 expression does not appear to be directly regulated by phenylpropanoids, as indicated by the metabolic profiling of mutants. The most potent holi mutant is defective in a subunit of cellulose synthase encoded by IRREGULAR XYLEM 3, and comparative analysis of this and other cell-wall mutants establishes a link between secondary cell-wall integrity and early chloroplast development, possibly involving altered ABA metabolism or sensing.

Untargeted metabotyping to study phenylpropanoid diversity in crop plants.

Plant genebanks constitute a key resource for breeding to ensure crop yield under changing environmental conditions. Because of their roles in a range of stress responses, phenylpropanoids are promising targets. Phenylpropanoids comprise a wide array of metabolites; however, studies regarding their diversity and the underlying genes are still limited for cereals. The assessment of barley diversity via genotyping-by-sequencing is in rapid progress. Exploring these resources by integrating genetic association studies to in-depth metabolomic profiling provides a valuable opportunity to study barley phenylpropanoid metabolism; but poses a challenge by demanding large-scale approaches. Here, we report an LC-PDA-MS workflow for barley high-throughput metabotyping. Without prior construction of a species-specific library, this method produced phenylpropanoid-enriched metabotypes with which the abundance of putative metabolic features was assessed across hundreds of samples in a single-processed data matrix. The robustness of the analytical performance was tested using a standard mix and extracts from two selected cultivars: Scarlett and Barke. The large-scale analysis of barley extracts showed (1) that barley flag leaf profiles were dominated by glycosylation derivatives of isovitexin, isoorientin, and isoscoparin; (2) proved the workflow's capability to discriminate within genotypes; (3) highlighted the role of glycosylation in barley phenylpropanoid diversity. Using the barley S42IL mapping population, the workflow proved useful for metabolic quantitative trait loci purposes. The protocol can be readily applied not only to explore the barley phenylpropanoid diversity represented in genebanks but also to study species whose profiles differ from those of cereals: the crop Helianthus annuus (sunflower) and the model plant Arabidopsis thaliana.

Immunomodulatory Properties of Blackberry Anthocyanins in THP-1 Derived Macrophages.

An anthocyanin-rich diet is considered to protect against chronic inflammatory processes although the bioavailability of anthocyanins is regarded as rather low. Moreover, the immunomodulatory role of anthocyanins is not fully understood yet. In the present study, fractions of blackberry (Rubus fruticosus) juice were investigated in plasma-relevant concentrations with respect to their immunomodulatory properties in lipopolysaccharide (LPS)-challenged THP-1-derived macrophages. The complex blackberry extract acted ineffective as well as potential degradation products. Cyanidin-3O-glucoside (Cy3glc), the main constituent of blackberry anthocyanins, diminished TNF-α levels at a concentration of 0.02 µg/mL, indicating protective effects as measured with quantitative RT-PCR and multiplex cytokine assays. LPS-boosted activity of transcription factor nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) of differentiated THP-1 reporter gene cells was marginally inhibited by Cy3glc. LPS-induced microRNA-155 was further increased, supporting the evidence of protection. Of note, fractions obtained from blackberry juice, in particular cyanidin-3O-(6″-dioxalylglucoside), were displaying potential pro-inflammatory properties as these elevated IL-6 and TNF-α levels. In conclusion, highly purified anthocyanin fractions of blackberry juice display both anti- and pro-inflammatory properties at plasma-relevant concentrations depending on their structure and substitution pattern.

The Jacalin-Related Lectin HvHorcH Is Involved in the Physiological Response of Barley Roots to Salt Stress.

Salt stress tolerance of crop plants is a trait with increasing value for future food production. In an attempt to identify proteins that participate in the salt stress response of barley, we have used a cDNA library from salt-stressed seedling roots of the relatively salt-stress-tolerant cv. Morex for the transfection of a salt-stress-sensitive yeast strain (Saccharomyces cerevisiae YSH818 Δhog1 mutant). From the retrieved cDNA sequences conferring salt tolerance to the yeast mutant, eleven contained the coding sequence of a jacalin-related lectin (JRL) that shows homology to the previously identified JRL horcolin from barley coleoptiles that we therefore named the gene HvHorcH. The detection of HvHorcH protein in root extracellular fluid suggests a secretion under stress conditions. Furthermore, HvHorcH exhibited specificity towards mannose. Protein abundance of HvHorcH in roots of salt-sensitive or salt-tolerant barley cultivars were not trait-specific to salinity treatment, but protein levels increased in response to the treatment, particularly in the root tip. Expression of HvHorcH in Arabidopsis thaliana root tips increased salt tolerance. Hence, we conclude that this protein is involved in the adaptation of plants to salinity.

The transcription factor WRKY22 is required during cryo-stress acclimation in Arabidopsis shoot tips.

Storage of meristematic tissue at ultra-low temperatures offers a mean to maintain valuable genetic resources from vegetatively reproduced plants. To reveal the biology underlying cryo-stress, shoot tips of the model plant Arabidopsis thaliana were subjected to a standard preservation procedure. A transcriptomic approach was taken to describe the subsequent cellular events which occurred. The cryoprotectant treatment induced the changes in the transcript levels of genes associated with RNA processing and primary metabolism. Explants of a mutant lacking a functional copy of the transcription factor WRKY22 were compromised for recovery. A number of putative downstream targets of WRKY22 were identified, some related to phytohormone-mediated defense, to the osmotic stress response, and to development. There were also alterations in the abundance of transcript produced by genes encoding photosynthesis-related proteins. The wrky22 mutant plants developed an open stomata phenotype in response to their exposure to the cryoprotectant solution. WRKY22 probably regulates a transcriptional network during cryo-stress, linking the explant's defense and osmotic stress responses to changes in its primary metabolism. A model is proposed linking WRKY53 and WRKY70 downstream of the action of WRKY22.

Effects of Arbuscular Mycorrhization on Fruit Quality in Industrialized Tomato Production.

Industrialized tomato production faces a decrease in flavors and nutritional value due to conventional breeding. Moreover, tomato production heavily relies on nitrogen and phosphate fertilization. Phosphate uptake and improvement of fruit quality by arbuscular mycorrhizal (AM) fungi are well-studied. We addressed the question of whether commercially used tomato cultivars grown in a hydroponic system can be mycorrhizal, leading to improved fruit quality. Tomato plants inoculated with Rhizophagus irregularis were grown under different phosphate concentrations and in substrates used in industrial tomato production. Changes in fruit gene expression and metabolite levels were checked by RNAseq and metabolite determination, respectively. The tests revealed that reduction of phosphate to 80% and use of mixed substrate allow AM establishment without affecting yield. By comparing green fruits from non-mycorrhizal and mycorrhizal plants, differentially expressed genes (DEGs) were found to possibly be involved in processes regulating fruit maturation and nutrition. Red fruits from mycorrhizal plants showed a trend of higher BRIX values and increased levels of carotenoids in comparison to those from non-mycorrhizal plants. Free amino acids exhibited up to four times higher levels in red fruits due to AM, showing the potential of mycorrhization to increase the nutritional value of tomatoes in industrialized production.

Accumulation of the coumarin scopolin under abiotic stress conditions is mediated by the Arabidopsis thaliana THO/TREX complex.

Secondary metabolites are involved in the plant stress response. Among these are scopolin and its active form scopoletin, which are coumarin derivatives associated with reactive oxygen species scavenging and pathogen defence. Here we show that scopolin accumulation can be induced in the root by osmotic stress and in the leaf by low-temperature stress in Arabidopsis thaliana. A genetic screen for altered scopolin levels in A. thaliana revealed a mutant compromised in scopolin accumulation in response to stress; the lesion was present in a homologue of THO1 coding for a subunit of the THO/TREX complex. The THO/TREX complex contributes to RNA silencing, supposedly by trafficking precursors of small RNAs. Mutants defective in THO, AGO1, SDS3 and RDR6 were impaired with respect to scopolin accumulation in response to stress, suggesting a mechanism based on RNA silencing such as the trans-acting small interfering RNA pathway, which requires THO/TREX function.

Purple corn extract induces long-lasting reprogramming and M2 phenotypic switch of adipose tissue macrophages in obese mice.

BACKGROUND: Obesity is a chronic and systemic inflammatory disorder and an important risk factor for the onset of several chronic syndromes. Adipose tissue (AT) plays a crucial role in the development of obesity, promoting the infiltration and accumulation of leukocytes in the tissue and sustaining adipocyte expansion. Anthocyanins exert a broad range of health benefits, but their effect in improving obesity-related inflammation in vivo has been poorly characterized. We examined the effects of a purple corn cob extract in the context of AT inflammation in a murine diet-induced obesity (DIO) model. METHODS: Male C57BL/6J mice were subjected to control diet (CTR + H2O), high fat diet (HF + H2O) or high fat diet plus purple corn extract (HF + RED) for 12 weeks. Blood glucose, AT, and liver gene expression, metabolism, biochemistry, and histology were analysed and flow cytometry was performed on AT leukocytes and Kupffer cells. RESULTS: RED extract intake resulted in lower MCP-1 mediated recruitment and proliferation of macrophages into crown-like structures in the AT. AT macrophages (ATM) of HF + RED group upregulated M2 markers (ArgI, Fizz1, TGFß), downregulating inflammatory mediators (TNF-α, IL-6, IL-1ß, COX-2) thanks to the suppression of NF-kB signalling. ATM also increased the expression of iron metabolism-related genes (FABP4, Hmox1, Ferroportin, CD163, TfR1, Ceruloplasmin, FtL1, FtH1) associated with a reduction in iron storage and increased turnover. ATM from HF + RED mice did not respond to LPS treatment ex vivo, confirming the long-lasting effects of the treatment on M2 polarization. Adipocytes of HF + RED group improved lipid metabolism and displayed a lower inflammation grade. Liver histology revealed a remarkable reduction of steatosis in the HF + RED group, and Kupffer cell profiling displayed a marked switch towards the M2 phenotype. CONCLUSIONS: RED extract attenuated AT inflammation in vivo, with a long-lasting reprogramming of ATM and adipocyte profiles towards the anti-inflammatory phenotype, therefore representing a valuable supplement in the context of obesity-associated disorders.

Barley cysteine protease PAP14 plays a role in degradation of chloroplast proteins.

Chloroplast protein degradation is known to occur both inside chloroplasts and in the vacuole. Genes encoding cysteine proteases have been found to be highly expressed during leaf senescence. However, it remains unclear where they participate in chloroplast protein degradation. In this study HvPAP14, which belongs to the C1A family of cysteine proteases, was identified in senescing barley (Hordeum vulgare L.) leaves by affinity enrichment using the mechanism-based probe DCG-04 targeting cysteine proteases and subsequent mass spectrometry. Biochemical analyses and expression of a HvPAP14:RFP fusion construct in barley protoplasts was used to identify the subcellular localization and putative substrates of HvPAP14. The HvPAP14:RFP fusion protein was detected in the endoplasmic reticulum and in vesicular bodies. Immunological studies showed that HvPAP14 was mainly located in chloroplasts, where it was found in tight association with thylakoid membranes. The recombinant enzyme was activated by low pH, in accordance with the detection of HvPAP14 in the thylakoid lumen. Overexpression of HvPAP14 in barley revealed that the protease can cleave LHCB proteins and PSBO as well as the large subunit of Rubisco. HvPAP14 is involved in the normal turnover of chloroplast proteins and may have a function in bulk protein degradation during leaf senescence.

Hydroxycinnamic acids in sunflower leaves serve as UV-A screening pigments.

Flavonoids and hydroxycinnamic acid derivatives, which are located in the upper epidermis of plants, are well known to screen ultraviolet radiation, thus protecting the underlying tissue from these harmful wavelengths. Both classes of secondary products complement each other over the UV spectral region according to their absorption spectra: flavonoids are most efficient as UV-A attenuators while hydroxycinnamates (HCAs) screen well within the UV-B region. Analysis of epidermal transmittance revealed a substantial UV-A screen in Helianthus annuus L. cv. Peredovick. Identifying responsible pigments by HPLC-MS, we found surprisingly low amounts of flavonoids but dominant abundance of the HCA derivatives chlorogenic and di-caffeoyl quinic acid. Both display low UV-A absorbance and thus, should contribute only a little to UV-A protection. However, growth at high light led to a decrease of epidermal transmittance at 366 nm of up to 90%. Underpinning the screening role, HCA autofluorescence microscopy revealed storage to occur predominantly in vacuoles of the upper epidermis. UV-A treatment in the absence of D1-repair resulted in photosystem II inactivation proportional to epidermal UV-A transmittance. Our findings suggest that UV-A protection can be achieved solely with HCAs, apparently through accumulation of high amounts of these compounds.

Colour bio-factories: Towards scale-up production of anthocyanins in plant cell cultures.

Anthocyanins are widely distributed, glycosylated, water-soluble plant pigments, which give many fruits and flowers their red, purple or blue colouration. Their beneficial effects in a dietary context have encouraged increasing use of anthocyanins as natural colourants in the food and cosmetic industries. However, the limited availability and diversity of anthocyanins commercially have initiated searches for alternative sources of these natural colourants. In plants, high-level production of secondary metabolites, such as anthocyanins, can be achieved by engineering of regulatory genes as well as genes encoding biosynthetic enzymes. We have used tobacco lines which constitutively produce high levels of cyanidin 3-O-rutinoside, delphinidin 3-O-rutinoside or a novel anthocyanin, acylated cyanidin 3-O-(coumaroyl) rutinoside to generate cell suspension cultures. The cell lines are stable in their production rates and superior to conventional plant cell cultures. Scale-up of anthocyanin production in small scale fermenters has been demonstrated. The cell cultures have also proven to be a suitable system for production of 13C-labelled anthocyanins. Our method for anthocyanin production is transferable to other plant species, such as Arabidopsis thaliana, demonstrating the potential of this approach for making a wide range of highly-decorated anthocyanins. The tobacco cell cultures represent a customisable and sustainable alternative to conventional anthocyanin production platforms and have considerable potential for use in industrial and medical applications of anthocyanins.

Plasma membrane proteome analysis identifies a role of barley membrane steroid binding protein in root architecture response to salinity.

Although the physiological consequences of plant growth under saline conditions have been well described, understanding the core mechanisms conferring plant salt adaptation has only started. We target the root plasma membrane proteomes of two barley varieties, cvs. Steptoe and Morex, with contrasting salinity tolerance. In total, 588 plasma membrane proteins were identified by mass spectrometry, of which 182 were either cultivar or salinity stress responsive. Three candidate proteins with increased abundance in the tolerant cv. Morex were involved either in sterol binding (a GTPase-activating protein for the adenosine diphosphate ribosylation factor [ZIGA2], and a membrane steroid binding protein [MSBP]) or in phospholipid synthesis (phosphoethanolamine methyltransferase [PEAMT]). Overexpression of barley MSBP conferred salinity tolerance to yeast cells, whereas the knock-out of the heterologous AtMSBP1 increased salt sensitivity in Arabidopsis. Atmsbp1 plants showed a reduced number of lateral roots under salinity, and root-tip-specific expression of barley MSBP in Atmsbp1 complemented this phenotype. In barley, an increased abundance of MSBP correlates with reduced root length and lateral root formation as well as increased levels of auxin under salinity being stronger in the tolerant cv. Morex. Hence, we concluded the involvement of MSBP in phytohormone-directed adaptation of root architecture in response to salinity.

Phosphoproteomics Profiling of Tobacco Mature Pollen and Pollen Activated in vitro.

Tobacco mature pollen has extremely desiccated cytoplasm, and is metabolically quiescent. Upon re-hydration it becomes metabolically active and that results in later emergence of rapidly growing pollen tube. These changes in cytoplasm hydration and metabolic activity are accompanied by protein phosphorylation. In this study, we subjected mature pollen, 5-min-activated pollen, and 30-min-activated pollen to TCA/acetone protein extraction, trypsin digestion and phosphopeptide enrichment by titanium dioxide. The enriched fraction was subjected to nLC-MS/MS. We identified 471 phosphopeptides that carried 432 phosphorylation sites, position of which was exactly matched by mass spectrometry. These 471 phosphopeptides were assigned to 301 phosphoproteins, because some proteins carried more phosphorylation sites. Of the 13 functional groups, the majority of proteins were put into these categories: transcription, protein synthesis, protein destination and storage, and signal transduction. Many proteins were of unknown function, reflecting the fact that male gametophyte contains many specific proteins that have not been fully functionally annotated. The quantitative data highlighted the dynamics of protein phosphorylation during pollen activation; the identified phosphopeptides were divided into seven groups based on the regulatory trends. The major group comprised mature pollen-specific phosphopeptides that were dephosphorylated during pollen activation. Several phosphopeptides representing the same phosphoprotein had different regulation, which pinpointed the complexity of protein phosphorylation and its clear functional context. Collectively, we showed the first phosphoproteomics data on activated pollen where the position of phosphorylation sites was clearly demonstrated and regulatory kinetics was resolved.

Changes of soluble sugars and ATP content during DMSO droplet freezing and PVS3 droplet vitrification of potato shoot tips.

The potato's great genetic diversity needs to be maintained for future agricultural applications and can be preserved at ultra-low temperatures. To decipher detailed physiological processes, the aim of the study was to analyze the regrowth in 28 gene bank accessions and to reveal metabolite changes in a subset of four accessions that showed pronounced differences after shoot tip cryopreservation using DMSO droplet freezing and PVS3 droplet vitrification. Regrowth varied in all 28 genotypes ranging from 5% ('Kagiri') to 100% ('Karakter') and was higher after PVS3 droplet vitrification (71 ±â€¯19%) than after cryopreservation using DMSO (54 ±â€¯17%). Sucrose, glucose, and fructose were analyzed and showed significant increases after pre-culture in combination with PVS3 or DMSO and liquid nitrogen treatment and were reduced during regeneration. In contrast, adenosine triphosphate (ATP) reached its minimum concentration after cryoprotection and liquid nitrogen treatment and recovered most quickly after PVS3 droplet vitrification. A shortening of the explant pre-culture period reduced dramatically the regrowth after PVS3 vitrification. However, correlations between the shoot tip regrowth and sugar concentration were absent and significant at a low extent with ATP (r = 0.4, P < 0.01). Interestingly, several sub-cultivations of the donor plants from the previous stock affected negatively the regrowth. In conclusion, the cryopreservation protocol, genotypes, pre-culture period and number of sub-cultures affect the regrowth ability of explants, which was best estimated by the ATP concentration after low-temperature treatment. Due to the superior performance of PVS3, the routine potato cryopreservation at the Gatersleben gene bank was changed to PVS3 droplet vitrification.

Genome-based establishment of a high-yielding heterotic pattern for hybrid wheat breeding.

Hybrid breeding promises to boost yield and stability. The single most important element in implementing hybrid breeding is the recognition of a high-yielding heterotic pattern. We have developed a three-step strategy for identifying heterotic patterns for hybrid breeding comprising the following elements. First, the full hybrid performance matrix is compiled using genomic prediction. Second, a high-yielding heterotic pattern is searched based on a developed simulated annealing algorithm. Third, the long-term success of the identified heterotic pattern is assessed by estimating the usefulness, selection limit, and representativeness of the heterotic pattern with respect to a defined base population. This three-step approach was successfully implemented and evaluated using a phenotypic and genomic wheat dataset comprising 1,604 hybrids and their 135 parents. Integration of metabolomic-based prediction was not as powerful as genomic prediction. We show that hybrid wheat breeding based on the identified heterotic pattern can boost grain yield through the exploitation of heterosis and enhance recurrent selection gain. Our strategy represents a key step forward in hybrid breeding and is relevant for self-pollinating crops, which are currently shifting from pure-line to high-yielding and resilient hybrid varieties.