Systemic reprogramming of phytohormone profiles and metabolic traits by virulent Diplodia infection in its pine (Pinus sylvestris L.) host.

The widespread ascomycetous fungus Diplodia pinea is a latent, necrotrophic pathogen in Pinus species causing severe damages and world-wide economic losses. However, the interactions between pine hosts and virulent D. pinea are largely not understood. In the present study, systemic defence responses were investigated in non-inoculated, asymptomatic needles and roots of D. pinea infected saplings of two P. sylvestris provenances under controlled greenhouse conditions. Here, we show that D. pinea infection induced a multitude of systemic responses of the phytohormone profiles and metabolic traits. Shared systemic responses of both pine provenances in needles and roots included increased abscisic acid and jasmonic acid levels. Exclusively in the roots of both provenances, enhanced salicylic acid and reduced indole-3-acetic acid levels, structural biomass, and elevated activities of anti-oxidative enzymes were observed. Despite these similarities, the two pine provenances investigated different significantly in the systemic responses of both, phytohormone profiles and metabolic traits in needles and roots. However, the different systemic responses did not prevent subsequent destruction of non-inoculated needles, but rather prevented damage to the roots. Our results provide a detailed view on systemic defence mechanisms of pine hosts that are of particular significance for the selection of provenances with improved defence capacity.

Barley strigolactone signalling mutant hvd14.d reveals the role of strigolactones in abscisic acid-dependent response to drought.

Strigolactones (SLs) are a group of plant hormones involved in many aspects of plant development and stress adaptation. Here, we investigated the drought response of a barley (Hordeum vulgare L.) mutant carrying a missense mutation in the gene encoding the SL-specific receptor HvD14. Our results clearly showed that hvd14.d mutant is hyper-sensitive to drought stress. This was illustrated by a lower leaf relative water content (RWC), impaired photosynthesis, disorganization of chloroplast structure, altered stomatal density and slower closure of stomata in response to drought in the mutant compared to the wild type parent cultivar Sebastian. Although the content of abscisic acid (ABA) and its derivatives remained unchanged in the mutant, significant differences in expression of genes related to ABA biosynthesis were observed. Moreover, hvd14.d was insensitive to ABA during seed germination. Analysis of Arabidopsis thaliana mutant atd14-1 also demonstrated that mutation in the SL receptor resulted in increased sensitivity to drought. Our results indicate that the drought-sensitive phenotype of barley SL mutant might be caused by a disturbed ABA metabolism and/or signalling pathways. These results together uncovered a link between SL signalling and ABA-dependent drought stress response in barley.

Boron Deficiency Effects on Sugar, Ionome, and Phytohormone Profiles of Vascular and Non-Vascular Leaf Tissues of Common Plantain (Plantago major L.).

Vascular tissues essentially regulate water, nutrient, photo-assimilate, and phytohormone logistics throughout the plant body. Boron (B) is crucial for the development of the vascular tissue in many dicotyledonous plant taxa and B deficiency particularly affects the integrity of phloem and xylem vessels, and, therefore, functionality of long-distance transport. We hypothesize that changes in the plants' B nutritional status evoke differential responses of the vasculature and the mesophyll. However, direct analyses of the vasculature in response to B deficiency are lacking, due to the experimental inaccessibility of this tissue. Here, we generated biochemical and physiological understanding of B deficiency response reactions in common plantain (Plantago major L.), from which pure and intact vascular bundles can be extracted. Low soil B concentrations affected quantitative distribution patterns of various phytohormones, sugars and macro-, and micronutrients in a tissue-specific manner. Vascular sucrose levels dropped, and sucrose loading into the phloem was reduced under low B supply. Phytohormones responded selectively to B deprivation. While concentrations of abscisic acid and salicylic acid decreased at low B supply, cytokinins and brassinosteroids increased in the vasculature and the mesophyll, respectively. Our results highlight the biological necessity to analyze nutrient deficiency responses in a tissue- rather organ-specific manner.

Whole-Genome Association Mapping and Genomic Prediction for Iron Concentration in Wheat Grains.

Malnutrition of iron (Fe) affects two billion people worldwide. Therefore, enhancing grain Fe concentration (GFeC) in wheat (Triticum aestivum L.) is an important goal for breeding. Here we study the genetic factors underlying GFeC trait by genome-wide association studies (GWAS) and the prediction abilities using genomic prediction (GP) in a panel of 369 European elite wheat varieties which was genotyped with 15,523 mapped single-nucleotide polymorphism markers (SNP) and a subpanel of 183 genotypes with 44,233 SNP markers. The resulting means of GFeC from three field experiments ranged from 24.42 to 52.42 µg·g-1 with a broad-sense heritability (H²) equaling 0.59 over the years. GWAS revealed 41 and 137 significant SNPs in the whole and subpanel, respectively, including significant marker-trait associations (MTAs) for best linear unbiased estimates (BLUEs) of GFeC on chromosomes 2A, 3B and 5A. Putative candidate genes such as NAC transcription factors and transmembrane proteins were present on chromosome 2A (763,689,738⁻765,710,113 bp). The GP for a GFeC trait ranged from low to moderate values. The current study reported GWAS of GFeC for the first time in hexaploid wheat varieties. These findings confirm the utility of GWAS and GP to explore the genetic architecture of GFeC for breeding programs aiming at the improvement of wheat grain quality.

Identifying Candidate Genes for Enhancing Grain Zn Concentration in Wheat.

Wheat (Triticum aestivum L.) is one of the major staple food crops worldwide. Despite efforts in improving wheat quality, micronutrient levels are still below the optimal range for human nutrition. In particular, zinc (Zn) deficiency is a widespread problem in human nutrition in countries relying mainly on a cereal diet; hence improving Zn accumulation in grains is an imperative need. This study was designed to understand the genetic architecture of Zn grain concentrations in wheat grains. We performed a genome-wide association study (GWAS) for grain Zn concentrations in 369 European wheat genotypes, using field data from 3 years. The complete wheat panel was genotyped by high-density arrays of single nucleotide polymorphic (SNP) markers (90k iSELECT Infinium and 35k Affymetrix arrays) resulting in 15,523 polymorphic markers. Additionally, a subpanel of 183 genotypes was analyzed with a novel 135k Affymetrix marker array resulting in 28,710 polymorphic SNPs for high-resolution mapping of the potential genomic regions. The mean grain Zn concentration of the genotypes ranged from 25.05-52.67 µg g-1 dry weight across years with a moderate heritability value. Notably, 40 marker-trait associations (MTAs) were detected in the complete panel of varieties on chromosomes 2A, 3A, 3B, 4A, 4D, 5A, 5B, 5D, 6D, 7A, 7B, and 7D. The number of MTAs in the subpanel was increased to 161 MTAs whereas the most significant and consistent associations were located on chromosomes 3B (723,504,241-723,611,488 bp) and 5A (462,763,758-466,582,184 bp) having major effects. These genomic regions include newly identified putative candidate genes, which are related to Zn uptake and transport or represent bZIP and mitogen-activated protein kinase genes. These findings provide the basis for understanding the genetic background of Zn concentration in wheat grains that in turn may help breeders to select high Zn-containing genotypes to improve human health and grain quality.

Abscisic acid influences tillering by modulation of strigolactones in barley.

Strigolactones (SLs) represent a class of plant hormones that are involved in inhibiting shoot branching and in promoting abiotic stress responses. There is evidence that the biosynthetic pathways of SLs and abscisic acid (ABA) are functionally connected. However, little is known about the mechanisms underlying the interaction of SLs and ABA, and the relevance of this interaction for shoot architecture. Based on sequence homology, four genes (HvD27, HvMAX1, HvCCD7, and HvCCD8) involved in SL biosynthesis were identified in barley and functionally verified by complementation of Arabidopsis mutants or by virus-induced gene silencing. To investigate the influence of ABA on SLs, two transgenic lines accumulating ABA as a result of RNAi-mediated down-regulation of HvABA 8'-hydroxylase 1 and 3 were employed. LC-MS/MS analysis confirmed higher ABA levels in root and stem base tissues in these transgenic lines. Both lines showed enhanced tiller formation and lower concentrations of 5-deoxystrigol in root exudates, which was detected for the first time as a naturally occurring SL in barley. Lower expression levels of HvD27, HvMAX1, HvCCD7, and HvCCD8 indicated that ABA suppresses SL biosynthesis, leading to enhanced tiller formation in barley.

Root Engineering in Barley: Increasing Cytokinin Degradation Produces a Larger Root System, Mineral Enrichment in the Shoot and Improved Drought Tolerance.

Root size and architecture are important crop plant traits, as they determine access to water and soil nutrients. The plant hormone cytokinin is a negative regulator of root growth and branching. Here, we generated transgenic barley (Hordeum vulgare) plants with an enlarged root system by enhancing cytokinin degradation in roots to explore the potential of cytokinin modulations in improving root functions. This was achieved through root-specific expression of a CYTOKININ OXIDASE/DEHYDROGENASE gene. Enhanced biomass allocation to roots did not penalize shoot growth or seed yield, indicating that these plants were not source limited. In leaves of transgenic lines, the concentrations of several macroelements and microelements were increased, particularly those with low soil mobility (phosphorus, manganese, and zinc). Importantly, seeds contained up to 44% more zinc, which is beneficial for human nutrition. Transgenic lines also demonstrated dampened stress responses to long-term drought conditions, indicating lower drought sensitivity. Taken together, this work demonstrates that root engineering of cereals is a promising strategy to improve nutrient efficiency, biofortification, and drought tolerance.

Genome-Wide Association Study of Calcium Accumulation in Grains of European Wheat Cultivars.

Mineral concentrations in cereals are important for human health, especially for people who depend mainly on consuming cereal diet. In this study, we carried out a genome-wide association study (GWAS) of calcium concentrations in wheat (Triticum aestivum L.) grains using a European wheat diversity panel of 353 varieties [339 winter wheat (WW) plus 14 of spring wheat (SW)] and phenotypic data based on two field seasons. High genotyping densities of single-nucleotide polymorphism (SNP) markers were obtained from the application of the 90k iSELECT ILLUMINA chip and a 35k Affymetrix chip. Inductively coupled plasma optical emission spectrometry (ICP-OES) was used to measure the calcium concentrations of the wheat grains. Best linear unbiased estimates (BLUEs) for calcium were calculated across the seasons and ranged from 288.20 to 647.50 among the varieties (µg g-1 DW) with a mean equaling 438.102 (µg g-1 DW), and the heritability was 0.73. A total of 485 SNP marker-trait associations (MTAs) were detected in data obtained from grains cultivated in both of the two seasons and BLUE values by considering associations with a -log10 (P-value) ≥3.0. Among these SNP markers, we detected 276 markers with a positive allele effect and 209 markers with a negative allele effect. These MTAs were found on all chromosomes except chromosomes 3D, 4B, and 4D. The most significant association was located on chromosome 5A (114.5 cM) and was linked to a gene encoding cation/sugar symporter activity as a potential candidate gene. Additionally, a number of candidate genes for the uptake or transport of calcium were located near significantly associated SNPs. This analysis highlights a number of genomic regions and candidate genes for further analysis as well as the challenges faced when mapping environmentally variable traits in genetically highly diverse variety panels. The research demonstrates the feasibility of the GWAS approach for illuminating the genetic architecture of calcium-concentration in wheat grains and for identifying putative candidate genes underlying this trait.

Grain yield and quality responses of wheat expressing a barley sucrose transporter to combined climate change factors.

Crop yield stability must be ensured under future climate conditions such as elevated CO2 and high temperatures. We tested 'HOSUT', a winter wheat line expressing a grain-targeted sucrose transporter of barley in response to combinations of CO2 enrichment, a heat wave, and high nitrogen fertilization. Compared with wild-type Certo, HOSUT had a superior performance for grain yield, aboveground biomass, and ears per plant, obviously due to transgene activity in developing grains and young vegetative sinks. HOSUT grains were larger and contained more endosperm cells. HOSUT and high CO2 effects similarly improved phenological and yield-related traits. Significant HOSUT-CO2 interactions for biomass of stems, ears, grain yield, nitrogen yield, and grain number revealed that Certo was promoted by CO2 enrichment, whereas HOSUT responded weakly. CO2 enrichment strongly reduced and HOSUT effects weakly reduced grain nitrogen, storage proteins, and free amino acids. In contrast to CO2 enrichment, HOSUT effects did not impair grain micronutrient concentrations. Significant HOSUT-nitrogen fertilization interactions for ear biomass, grain yield, grain number per plant, and harvest index indicated that HOSUT benefited more from additional nitrogen. The heat wave decreased aboveground and ear biomass, grain yield, harvest index, grain size, and starch and water use, but increased grain sucrose concentration.

Response of the plant hormone network to boron deficiency.

Plant hormones (PH) adjust plant growth to environmental conditions such as nutrient availability. Although responses of individual PHs to growth-determining nutrient supplies have been reported, little is known about simultaneous dynamics in the metabolism of different PH species. Brassica napus seedlings were grown under increasing supply of B, and LC-MS/MS was used to characterize bioactive forms of different PH species together with several of their precursors, storage and inactivated forms. Increasing shoot B concentrations in response to B supply were accompanied by decreasing concentrations of abscisic acid (ABA) and indole-3-acetic acid (IAA), which appeared to be synthesized under B deficiency mainly via indole-3-acetonitrile (IAN). By contrast, shoot B concentrations correlated closely with cytokinins, and the B-dependent growth response appeared to be triggered primarily by de-novo synthesis of cytokinins and by re-routing less active towards highly active forms of cytokinin. Also gibberellin biosynthesis strongly increased with B supply, in particular gibberellin species from the non-13-hydroxylation pathway. The brassinosteroid castasterone appeared to support shoot growth primarily at suboptimal B nutrition. These results indicate that a variable B nutritional status causes coordinated changes in PH metabolism as prerequisite for an adjusted growth response.

VRS2 regulates hormone-mediated inflorescence patterning in barley.

Plant architecture has clear agronomic and economic implications for crops such as wheat and barley, as it is a critical factor for determining grain yield. Despite this, only limited molecular information is available about how grain-bearing inflorescences, called spikes, are formed and maintain their regular, distichous pattern. Here we elucidate the molecular and hormonal role of Six-rowed spike 2 (Vrs2), which encodes a SHORT INTERNODES (SHI) transcriptional regulator during barley inflorescence and shoot development. We show that Vrs2 is specifically involved in floral organ patterning and phase duration by maintaining hormonal homeostasis and gradients during normal spike development and similarly influences plant stature traits. Furthermore, we establish a link between the SHI protein family and sucrose metabolism during organ growth and development that may have implications for deeper molecular insights into inflorescence and plant architecture in crops.

Phloem-Specific Methionine Recycling Fuels Polyamine Biosynthesis in a Sulfur-Dependent Manner and Promotes Flower and Seed Development.

The Yang or Met Cycle is a series of reactions catalyzing the recycling of the sulfur (S) compound 5'-methylthioadenosine (MTA) to Met. MTA is produced as a by-product in ethylene, nicotianamine, and polyamine biosynthesis. Whether the Met Cycle preferentially fuels one of these pathways in a S-dependent manner remained unclear so far. We analyzed Arabidopsis (Arabidopsis thaliana) mutants with defects in the Met Cycle enzymes 5-METHYLTHIORIBOSE-1-PHOSPHATE-ISOMERASE1 (MTI1) and DEHYDRATASE-ENOLASE-PHOSPHATASE-COMPLEX1 (DEP1) under different S conditions and assayed the contribution of the Met Cycle to the regeneration of S for these pathways. Neither mti1 nor dep1 mutants could recycle MTA but showed S-dependent reproductive failure, which was accompanied by reduced levels of the polyamines putrescine, spermidine, and spermine in mutant inflorescences. Complementation experiments with external application of these three polyamines showed that only the triamine spermine could specifically rescue the S-dependent reproductive defects of the mutant plants. Furthermore, expressing gene-reporter fusions in Arabidopsis showed that MTI1 and DEP1 were mainly expressed in the vasculature of all plant parts. Phloem-specific reconstitution of Met Cycle activity in mti1 and dep1 mutant plants was sufficient to rescue their S-dependent mutant phenotypes. We conclude from these analyses that phloem-specific S recycling during periods of S starvation is essential for the biosynthesis of polyamines required for flowering and seed development.

Cell Type-Specific Gene Expression Analyses by RNA Sequencing Reveal Local High Nitrate-Triggered Lateral Root Initiation in Shoot-Borne Roots of Maize by Modulating Auxin-Related Cell Cycle Regulation.

Plants have evolved a unique plasticity of their root system architecture to flexibly exploit heterogeneously distributed mineral elements from soil. Local high concentrations of nitrate trigger lateral root initiation in adult shoot-borne roots of maize (Zea mays) by increasing the frequency of early divisions of phloem pole pericycle cells. Gene expression profiling revealed that, within 12 h of local high nitrate induction, cell cycle activators (cyclin-dependent kinases and cyclin B) were up-regulated, whereas repressors (Kip-related proteins) were down-regulated in the pericycle of shoot-borne roots. In parallel, a ubiquitin protein ligase S-Phase Kinase-Associated Protein1-cullin-F-box protein(S-Phase Kinase-Associated Protein 2B)-related proteasome pathway participated in cell cycle control. The division of pericycle cells was preceded by increased levels of free indole-3-acetic acid in the stele, resulting in DR5-red fluorescent protein-marked auxin response maxima at the phloem poles. Moreover, laser-capture microdissection-based gene expression analyses indicated that, at the same time, a significant local high nitrate induction of the monocot-specific PIN-FORMED9 gene in phloem pole cells modulated auxin efflux to pericycle cells. Time-dependent gene expression analysis further indicated that local high nitrate availability resulted in PIN-FORMED9-mediated auxin efflux and subsequent cell cycle activation, which culminated in the initiation of lateral root primordia. This study provides unique insights into how adult maize roots translate information on heterogeneous nutrient availability into targeted root developmental responses.

Dynamics and partitioning of the ionome in seeds and germinating seedlings of winter oilseed rape.

Germination and seedling establishment are among the most critical phases in the development of plants, and seed vigour has become an important trait for the selection of robust crop cultivars. Little is known about the potentially limiting role of mineral nutrients in early metabolic and developmental processes during germination. Therefore, we assessed the ionome and relative distribution of mineral elements in different seed and seedling tissues of oilseed rape (Brassica napus L.) and monitored the internal allocation of nutrients during germination. In seeds, cotyledons harboured the main pool of K, P, S, Mg, Fe, Mn and Zn, whereas the seed coat contained most of the Ca, Na, B, Cu and Mo. Although the early root and hypocotyl tissue expanded first, concentrations of most elements were initially low. Re-allocation of elements to the root/hypocotyl tissue from other pools set in two days after seed imbibition and was most rapid for K. Relative to the critical deficiency levels of vegetative tissues, seed tissues were particularly low in B, K and Fe. Further analyses of the ionome of seeds and seedlings, grouped according to their germination efficiency, indicated that in particular low S, Mg and Ca coincided with germination failure. This study documents highly dynamic changes in the ionome of seed and seedling tissues and provides evidence for potentially limiting elements during early germination and seedling establishment in rapeseed.

Proteome analysis of Fusarium head blight in grains of naked barley (Hordeum vulgare subsp. nudum).

The effect of artificial Fusarium graminearum and F. culmorum infection at the level of the proteome on grains of naked barley (Hordeum vulgare subsp. nudum) was investigated in comparison to naturally infected samples. Fusarium infection in barley led to numerous host-specific biochemical responses. NEPHGE 2-D PAGE and MS were used to identify proteins that were differentially expressed in response to fungal infection and growing location of the plants. Moreover, the mycotoxin concentration of the grains was evaluated to characterize the infection degree. Inoculation of naked barley with Fusarium led to grain deoxynivalenol concentrations of up to 1.2 mg/kg. The carbon and nitrogen grain concentrations were not significantly changed after fungal infection, but differed between growing locations. Eleven proteins related to fungal infection were detected as were three proteins with effects based on growing location. These proteins belong to different protein groups involved in various cell functions: transcription regulation, defence response, nutrient reservoirs and starch biosynthesis. The results gave indications on plant defence strategies and changes as response to Fusarium infection in mature grains after a long infection period as well as being influenced by the growing location.

Effects of fusarium infection on the phenolics in emmer and naked barley.

Inoculated or non-inoculated naked barley and emmer cultivars were investigated with regard to their influence on phenolic acid profiles and their arabinoxylan content. Two groups of phenolic compounds were differentiated-methanol-soluble and hydrolyzable covalent-bound phenolic compounds. Chromatographic methods were applied for their analysis. The results showed ferulic acid as the predominant phenol in both total and covalent-bound fractions. The inoculation significantly reduced the ferulic acid content within a range of 5.6-6.6% in the two cereals and all their cultivars. Naked barley cultivars additionally contained the flavonoid catechin in the soluble fraction. The innoculation led here to a significant increase in the catechin content of about 4.5%. These results document an induction of the synthesis of catechin in naked barley after artificial Fusarium infection, whereas the ferulic acid content declined.