Seedling root system adaptation to water availability during maize domestication and global expansion.

The maize root system has been reshaped by indirect selection during global adaptation to new agricultural environments. In this study, we characterized the root systems of more than 9,000 global maize accessions and its wild relatives, defining the geographical signature and genomic basis of variation in seminal root number. We demonstrate that seminal root number has increased during maize domestication followed by a decrease in response to limited water availability in locally adapted varieties. By combining environmental and phenotypic association analyses with linkage mapping, we identified genes linking environmental variation and seminal root number. Functional characterization of the transcription factor ZmHb77 and in silico root modeling provides evidence that reshaping root system architecture by reducing the number of seminal roots and promoting lateral root density is beneficial for the resilience of maize seedlings to drought.

Anatomical Limitations in Adventitious Root Formation Revealed by Magnetic Resonance Imaging, Infrared Spectroscopy, and Histology of Rose Genotypes with Contrasting Rooting Phenotypes.

Adventitious root (AR) formation is one of the most important developmental processes in vegetative propagation. Although genotypic differences in rooting ability of rose are well known, the causing factors are not well understood. The rooting of two contrasting genotypes, 'Herzogin Friederike' and 'Mariatheresia', was compared following a multiscale approach. Using magnetic resonance imaging, we noninvasively monitored the inner structure of stem cuttings during initiation and progression of AR formation for the first time. Spatially resolved Fourier-transform infrared spectroscopy characterised the chemical composition of the tissues involved in AR formation. The results were validated through light microscopy and complemented by immunolabeling. The outcome demonstrated similarity of both genotypes in root primordia formation, which however, did not result in root protrusion through the shoot cortex in the difficult-to-root genotype 'Mariatheresia'. The biochemical composition of the contrasting genotypes highlighted main differences in cell wall-associated components. Further spectroscopic analysis of 15 contrasting rose genotypes confirmed the biochemical differences between easy- and difficult-to-root groups. Collectively, data indicates, that not the lack of root primordia limits AR formation in these rose genotypes, but the firmness of the outer stem tissue and/or cell wall modifications that pose a mechanical barrier and prevent root extension and protrusion.

Mechanisms of metabolic adaptation in the duckweed Lemna gibba: an integrated metabolic, transcriptomic and flux analysis.

BACKGROUND: Duckweeds are small, rapidly growing aquatic flowering plants. Due to their ability for biomass production at high rates they represent promising candidates for biofuel feedstocks. Duckweeds are also excellent model organisms because they can be maintained in well-defined liquid media, usually reproduce asexually, and because genomic resources are becoming increasingly available. To demonstrate the utility of duckweed for integrated metabolic studies, we examined the metabolic adaptation of growing Lemna gibba cultures to different nutritional conditions. RESULTS: To establish a framework for quantitative metabolic research in duckweeds we derived a central carbon metabolism network model of Lemna gibba based on its draft genome. Lemna gibba fronds were grown with nitrate or glutamine as nitrogen source. The two conditions were compared by quantification of growth kinetics, metabolite levels, transcript abundance, as well as by 13C-metabolic flux analysis. While growing with glutamine, the fronds grew 1.4 times faster and accumulated more protein and less cell wall components compared to plants grown on nitrate. Characterization of photomixotrophic growth by 13C-metabolic flux analysis showed that, under both metabolic growth conditions, the Calvin-Benson-Bassham cycle and the oxidative pentose-phosphate pathway are highly active, creating a futile cycle with net ATP consumption. Depending on the nitrogen source, substantial reorganization of fluxes around the tricarboxylic acid cycle took place, leading to differential formation of the biosynthetic precursors of the Asp and Gln families of proteinogenic amino acids. Despite the substantial reorganization of fluxes around the tricarboxylic acid cycle, flux changes could largely not be associated with changes in transcripts. CONCLUSIONS: Through integrated analysis of growth rate, biomass composition, metabolite levels, and metabolic flux, we show that Lemna gibba is an excellent system for quantitative metabolic studies in plants. Our study showed that Lemna gibba adjusts to different nitrogen sources by reorganizing central metabolism. The observed disconnect between gene expression regulation and metabolism underscores the importance of metabolic flux analysis as a tool in such studies.

Wheat Grains as a Sustainable Source of Protein for Health.

Protein deficiency is recognized among the major global health issues with an underestimation of its importance. Genetic biofortification is a cost-effective and sustainable strategy to overcome global protein malnutrition. This study was designed to focus on protein-dense grains of wheat (Triticum aestivum L.) and identify the genes governing grain protein content (GPC) that improve end-use quality and in turn human health. Genome-wide association was applied using the 90k iSELECT Infinium and 35k Affymetrix arrays with GPC quantified by using a proteomic-based technique in 369 wheat genotypes over three field-year trials. The results showed significant natural variation among bread wheat genotypes that led to detecting 54 significant quantitative trait nucleotides (QTNs) surpassing the false discovery rate (FDR) threshold. These QTNs showed contrasting effects on GPC ranging from -0.50 to +0.54% that can be used for protein content improvement. Further bioinformatics analyses reported that these QTNs are genomically linked with 35 candidate genes showing high expression during grain development. The putative candidate genes have functions in the binding, remobilization, or transport of protein. For instance, the promising QTN AX-94727470 on chromosome 6B increases GPC by +0.47% and is physically located inside the gene TraesCS6B02G384500 annotated as Trehalose 6-phosphate phosphatase (T6P), which can be employed to improve grain protein quality. Our findings are valuable for the enhancement of protein content and end-use quality in one of the major daily food resources that ultimately improve human nutrition.

SWEET11b transports both sugar and cytokinin in developing barley grains.

Even though Sugars Will Eventually be Exported Transporters (SWEETs) have been found in every sequenced plant genome, a comprehensive understanding of their functionality is lacking. In this study, we focused on the SWEET family of barley (Hordeum vulgare). A radiotracer assay revealed that expressing HvSWEET11b in African clawed frog (Xenopus laevis) oocytes facilitated the bidirectional transfer of not only just sucrose and glucose, but also cytokinin. Barley plants harboring a loss-of-function mutation of HvSWEET11b could not set viable grains, while the distribution of sucrose and cytokinin was altered in developing grains of plants in which the gene was knocked down. Sucrose allocation within transgenic grains was disrupted, which is consistent with the changes to the cytokinin gradient across grains, as visualized by magnetic resonance imaging and Fourier transform infrared spectroscopy microimaging. Decreasing HvSWEET11b expression in developing grains reduced overall grain size, sink strength, the number of endopolyploid endosperm cells, and the contents of starch and protein. The control exerted by HvSWEET11b over sugars and cytokinins likely predetermines their synergy, resulting in adjustments to the grain's biochemistry and transcriptome.

Seeing plants as never before.

Imaging has long supported our ability to understand the inner life of plants, their development, and response to a dynamic environment. While optical microscopy remains the core tool for imaging, a suite of novel technologies is now beginning to make a significant contribution to visualize plant metabolism. The purpose of this review was to provide the scientific community with an overview of current imaging methods, which rely variously on either nuclear magnetic resonance (NMR), mass spectrometry (MS) or infrared (IR) spectroscopy, and to present some examples of their application in order to illustrate their utility. In addition to providing a description of the basic principles underlying these technologies, the review discusses their various advantages and limitations, reveals the current state of the art, and suggests their potential application to experimental practice. Finally, a view is presented as to how the technologies will likely develop, how these developments may encourage the formulation of novel experimental strategies, and how the enormous potential of these technologies can contribute to progress in plant science.

Causes and consequences of endogenous hypoxia on growth and metabolism of developing maize kernels.

Maize (Zea mays) kernels are the largest cereal grains, and their endosperm is severely oxygen deficient during grain fill. The causes, dynamics, and mechanisms of acclimation to hypoxia are minimally understood. Here, we demonstrate that hypoxia develops in the small, growing endosperm, but not the nucellus, and becomes the standard state, regardless of diverse structural and genetic perturbations in modern maize (B73, popcorn, sweet corn), mutants (sweet4c, glossy6, waxy), and non-domesticated wild relatives (teosintes and Tripsacum species). We also uncovered an interconnected void space at the chalazal pericarp, providing superior oxygen supply to the placental tissues and basal endosperm transfer layer. Modeling indicated a very high diffusion resistance inside the endosperm, which, together with internal oxygen consumption, could generate steep oxygen gradients at the endosperm surface. Manipulation of oxygen supply induced reciprocal shifts in gene expression implicated in controlling mitochondrial functions (23.6 kDa Heat-Shock Protein, Voltage-Dependent Anion Channel 2) and multiple signaling pathways (core hypoxia genes, cyclic nucleotide metabolism, ethylene synthesis). Metabolite profiling revealed oxygen-dependent shifts in mitochondrial pathways, ascorbate metabolism, starch synthesis, and auxin degradation. Long-term elevated oxygen supply enhanced the rate of kernel development. Altogether, evidence here supports a mechanistic framework for the establishment of and acclimation to hypoxia in the maize endosperm.

Quantitative monitoring of paramagnetic contrast agents and their allocation in plant tissues via DCE-MRI.

BACKGROUND: Studying dynamic processes in living organisms with MRI is one of the most promising research areas. The use of paramagnetic compounds as contrast agents (CA), has proven key to such studies, but so far, the lack of appropriate techniques limits the application of CA-technologies in experimental plant biology. The presented proof-of-principle aims to support method and knowledge transfer from medical research to plant science. RESULTS: In this study, we designed and tested a new approach for plant Dynamic Contrast Enhanced Magnetic Resonance Imaging (pDCE-MRI). The new approach has been applied in situ to a cereal crop (Hordeum vulgare). The pDCE-MRI allows non-invasive investigation of CA allocation within plant tissues. In our experiments, gadolinium-DTPA, the most commonly used contrast agent in medical MRI, was employed. By acquiring dynamic T1-maps, a new approach visualizes an alteration of a tissue-specific MRI parameter T1 (longitudinal relaxation time) in response to the CA. Both, the measurement of local CA concentration and the monitoring of translocation in low velocity ranges (cm/h) was possible using this CA-enhanced method. CONCLUSIONS: A novel pDCE-MRI method is presented for non-invasive investigation of paramagnetic CA allocation in living plants. The temporal resolution of the T1-mapping has been significantly improved to enable the dynamic in vivo analysis of transport processes at low-velocity ranges, which are common in plants. The newly developed procedure allows to identify vascular regions and to estimate their involvement in CA allocation. Therefore, the presented technique opens a perspective for further development of CA-aided MRI experiments in plant biology.

Probing the Metabolic Landscape of Plant Vascular Bundles by Infrared Fingerprint Analysis, Imaging and Mass Spectrometry.

Fingerprint analysis is a common technique in forensic and criminal investigations. Similar techniques exist in the field of infrared spectroscopy to identify biomolecules according to their characteristic spectral fingerprint features. These unique markers are located in a wavenumber range from 1800 to 600 cm-1 in the mid infrared region. Here, a novel bioanalytical concept of correlating these spectral features with corresponding mass spectrometry datasets to unravel metabolic clusters within complex plant tissues was applied. As proof of concept, vascular bundles of oilseed rape (Brassica napus) were investigated, one of the most important and widely cultivated temperate zone oilseed crops. The link between mass spectrometry data and spectral data identified features that co-aligned within both datasets. Regions of origin were then detected by searching for these features in hyperspectral images of plant tissues. This approach, based on co-alignment and co-localization, finally enabled the detection of eight distinct metabolic clusters, reflecting functional and structural arrangements within the vascular bundle. The proposed analytical concept may assist future synergistic research approaches and may lead to biotechnological innovations with regard to crop yield and sustainability.

The metabolic environment of the developing embryo: A multidisciplinary approach on oilseed rapeseed.

Brassicaceae seeds consist of three genetically distinct structures: the embryo, endosperm and seed coat, all of which are involved in assimilate allocation during seed development. The complexity of their metabolic interrelations remains unresolved to date. In the present study, we apply state-of-the-art imaging and analytical approaches to assess the metabolic environment of the Brassica napus embryo. Nuclear magnetic resonance imaging (MRI) provided volumetric data on the living embryo and endosperm, revealing how the endosperm envelops the embryo, determining endosperm's priority in assimilate uptake from the seed coat during early development. MRI analysis showed higher levels of sugars in the peripheral endosperm facing the seed coat, but a lower sugar content within the central vacuole and the region surrounding the embryo. Feeding intact siliques with 13C-labeled sucrose allowed tracing of the post-phloem route of sucrose transfer within the seed at the heart stage of embryogenesis, by means of mass spectrometry imaging. Quantification of over 70 organic and inorganic compounds in the endosperm revealed shifts in their abundance over different stages of development, while sugars and potassium were the main determinants of osmolality throughout these stages. Our multidisciplinary approach allows access to the hidden aspects of endosperm metabolism, a task which remains unattainable for the small-seeded model plant Arabidopsis thaliana.

A mechanistic view on lodging resistance in rye and wheat: a multiscale comparative study.

The development of crop varieties that are resistant to lodging is a top priority for breeding programmes. Herein, we characterize the rye mutant ´Stabilstroh' ('stable straw') possessing an exceptional combination of high lodging resistance, tall posture and high biomass production. Nuclear magnetic resonance imaging displayed the 3-dimensional assembly of vascular bundles in stem. A higher number of vascular bundles and a higher degree of their incline were the features of lodging-resistant versus lodging-prone lines. Histology and electron microscopy revealed that stems are fortified by a higher proportion of sclerenchyma and thickened cell walls, as well as some epidermal invaginations. Biochemical analysis using Fourier-transform infrared spectroscopy and inductively coupled plasma-optical emission spectrometry further identified elevated levels of lignin, xylan, zinc and silicon as features associated with high lodging resistance. Combined effects of above features caused superior culm stability. A simplistic mathematical model showed how mechanical forces distribute within the stem under stress. Main traits of the lodging-resistant parental line were heritable and could be traced back to the genetic structure of the mutant. Evaluation of lodging-resistant wheat 'Babax' ('Baviacora') versus contrasting, lodging-prone, genotype ´Pastor´ agreed with above findings on rye. Our findings on mechanical stability and extraordinary culm properties may be important for breeders for the improvement of lodging resistance of tall posture cereal crops.

Return of the Lemnaceae: duckweed as a model plant system in the genomics and postgenomics era.

The aquatic Lemnaceae family, commonly called duckweed, comprises some of the smallest and fastest growing angiosperms known on Earth. Their tiny size, rapid growth by clonal propagation, and facile uptake of labeled compounds from the media were attractive features that made them a well-known model for plant biology from 1950 to 1990. Interest in duckweed has steadily regained momentum over the past decade, driven in part by the growing need to identify alternative plants from traditional agricultural crops that can help tackle urgent societal challenges, such as climate change and rapid population expansion. Propelled by rapid advances in genomic technologies, recent studies with duckweed again highlight the potential of these small plants to enable discoveries in diverse fields from ecology to chronobiology. Building on established community resources, duckweed is reemerging as a platform to study plant processes at the systems level and to translate knowledge gained for field deployment to address some of society's pressing needs. This review details the anatomy, development, physiology, and molecular characteristics of the Lemnaceae to introduce them to the broader plant research community. We highlight recent research enabled by Lemnaceae to demonstrate how these plants can be used for quantitative studies of complex processes and for revealing potentially novel strategies in plant defense and genome maintenance.

Grain filling in barley relies on developmentally controlled programmed cell death.

Cereal grains contribute substantially to the human diet. The maternal plant provides the carbohydrate and nitrogen sources deposited in the endosperm, but the basis for their spatial allocation during the grain filling process is obscure. Here, vacuolar processing enzymes have been shown to both mediate programmed cell death (PCD) in the maternal tissues of a barley grain and influence the delivery of assimilate to the endosperm. The proposed centrality of PCD has implications for cereal crop improvement.

Trehalose 6-phosphate promotes seed filling by activating auxin biosynthesis.

Plants undergo several developmental transitions during their life cycle. One of these, the differentiation of the young embryo from a meristem-like structure into a highly specialized storage organ, is believed to be controlled by local connections between sugars and hormonal response systems. However, we know little about the regulatory networks underpinning the sugar-hormone interactions in developing seeds. By modulating the trehalose 6-phosphate (T6P) content in growing embryos of garden pea (Pisum sativum), we investigate here the role of this signaling sugar during the seed-filling process. Seeds deficient in T6P are compromised in size and starch production, resembling the wrinkled seeds studied by Gregor Mendel. We show also that T6P exerts these effects by stimulating the biosynthesis of the pivotal plant hormone, auxin. We found that T6P promotes the expression of the auxin biosynthesis gene TRYPTOPHAN AMINOTRANSFERASE RELATED2 (TAR2), and the resulting effect on auxin concentrations is required to mediate the T6P-induced activation of storage processes. Our results suggest that auxin acts downstream of T6P to facilitate seed filling, thereby providing a salient example of how a metabolic signal governs the hormonal control of an integral phase transition in a crop plant.

Adaptation Strategies of Halophytic Barley Hordeum marinum ssp. marinum to High Salinity and Osmotic Stress.

The adaptation strategies of halophytic seaside barley Hordeum marinum to high salinity and osmotic stress were investigated by nuclear magnetic resonance imaging, as well as ionomic, metabolomic, and transcriptomic approaches. When compared with cultivated barley, seaside barley exhibited a better plant growth rate, higher relative plant water content, lower osmotic pressure, and sustained photosynthetic activity under high salinity, but not under osmotic stress. As seaside barley is capable of controlling Na+ and Cl- concentrations in leaves at high salinity, the roots appear to play the central role in salinity adaptation, ensured by the development of thinner and likely lignified roots, as well as fine-tuning of membrane transport for effective management of restriction of ion entry and sequestration, accumulation of osmolytes, and minimization of energy costs. By contrast, more resources and energy are required to overcome the consequences of osmotic stress, particularly the severity of reactive oxygen species production and nutritional disbalance which affect plant growth. Our results have identified specific mechanisms for adaptation to salinity in seaside barley which differ from those activated in response to osmotic stress. Increased knowledge around salt tolerance in halophytic wild relatives will provide a basis for improved breeding of salt-tolerant crops.

Cellular Plasticity in Response to Suppression of Storage Proteins in the Brassica napus Embryo.

The tradeoff between protein and oil storage in oilseed crops has been tested here in oilseed rape (Brassica napus) by analyzing the effect of suppressing key genes encoding protein storage products (napin and cruciferin). The phenotypic outcomes were assessed using NMR and mass spectrometry imaging, microscopy, transcriptomics, proteomics, metabolomics, lipidomics, immunological assays, and flux balance analysis. Surprisingly, the profile of storage products was only moderately changed in RNA interference transgenics. However, embryonic cells had undergone remarkable architectural rearrangements. The suppression of storage proteins led to the elaboration of membrane stacks enriched with oleosin (sixfold higher protein abundance) and novel endoplasmic reticulum morphology. Protein rebalancing and amino acid metabolism were focal points of the metabolic adjustments to maintain embryonic carbon/nitrogen homeostasis. Flux balance analysis indicated a rather minor additional demand for cofactors (ATP and NADPH). Thus, cellular plasticity in seeds protects against perturbations to its storage capabilities and, hence, contributes materially to homeostasis. This study provides mechanistic insights into the intriguing link between lipid and protein storage, which have implications for biotechnological strategies directed at improving oilseed crops.

The genome of jojoba (Simmondsia chinensis): A taxonomically isolated species that directs wax ester accumulation in its seeds.

Seeds of the desert shrub, jojoba (Simmondsia chinensis), are an abundant, renewable source of liquid wax esters, which are valued additives in cosmetic products and industrial lubricants. Jojoba is relegated to its own taxonomic family, and there is little genetic information available to elucidate its phylogeny. Here, we report the high-quality, 887-Mb genome of jojoba assembled into 26 chromosomes with 23,490 protein-coding genes. The jojoba genome has only the whole-genome triplication (γ) shared among eudicots and no recent duplications. These genomic resources coupled with extensive transcriptome, proteome, and lipidome data helped to define heterogeneous pathways and machinery for lipid synthesis and storage, provided missing evolutionary history information for this taxonomically segregated dioecious plant species, and will support efforts to improve the agronomic properties of jojoba.

Discovery of key regulators of dark gland development and hypericin biosynthesis in St. John's Wort (Hypericum perforatum).

Hypericin is a molecule of high pharmaceutical importance that is synthesized and stored in dark glands (DGs) of St. John's Wort (Hypericum perforatum). Understanding which genes are involved in dark gland development and hypericin biosynthesis is important for the development of new Hypericum extracts that are highly demanded for medical applications. We identified two transcription factors whose expression is strictly synchronized with the differentiation of DGs. We correlated the content of hypericin, pseudohypericin, endocrocin, skyrin glycosides and several flavonoids with gene expression and DG development to obtain a revised model for hypericin biosynthesis. Here, we report for the first time genotypes which are polymorphic for the presence/total absence (G+/G-) of DGs in their placental tissues (PTs). DG development was characterized in PTs using several microscopy techniques. Fourier transform infrared microscopy was established as a novel method to precisely locate polyaromatic compounds, such as hypericin, in plant tissues. In addition, we obtained transcriptome and metabolome profiles of unprecedented resolution in Hypericum. This study addresses for the first time the development of dark glands and identifies genes that constitute strong building blocks for the further elucidation of hypericin synthesis, its manipulation in plants, its engineering in microbial systems and its applications in medical research.

The highly divergent Jekyll genes, required for sexual reproduction, are lineage specific for the related grass tribes Triticeae and Bromeae.

Phylogenetically related groups of species contain lineage-specific genes that exhibit no sequence similarity to any genes outside the lineage. We describe here that the Jekyll gene, required for sexual reproduction, exists in two much diverged allelic variants, Jek1 and Jek3. Despite low similarity, the Jek1 and Jek3 proteins share identical signal peptides, conserved cysteine positions and direct repeats. The Jek1/Jek3 sequences are located at the same chromosomal locus and inherited in a monogenic Mendelian fashion. Jek3 has a similar expression as Jek1 and complements the Jek1 function in Jek1-deficient plants. Jek1 and Jek3 allelic variants were almost equally distributed in a collection of 485 wild and domesticated barley accessions. All domesticated barleys harboring the Jek1 allele belong to single haplotype J1-H1 indicating a genetic bottleneck during domestication. Domesticated barleys harboring the Jek3 allele consisted of three haplotypes. Jekyll-like sequences were found only in species of the closely related tribes Bromeae and Triticeae but not in other Poaceae. Non-invasive magnetic resonance imaging revealed intrinsic grain structure in Triticeae and Bromeae, associated with the Jekyll function. The emergence of Jekyll suggests its role in the separation of the Bromeae and Triticeae lineages within the Poaceae and identifies the Jekyll genes as lineage-specific.