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.

Genome and time-of-day transcriptome of Wolffia australiana link morphological minimization with gene loss and less growth control.

Rootless plants in the genus Wolffia are some of the fastest growing known plants on Earth. Wolffia have a reduced body plan, primarily multiplying through a budding type of asexual reproduction. Here, we generated draft reference genomes for Wolffia australiana (Benth.) Hartog & Plas, which has the smallest genome size in the genus at 357 Mb and has a reduced set of predicted protein-coding genes at about 15,000. Comparison between multiple high-quality draft genome sequences from W. australiana clones confirmed loss of several hundred genes that are highly conserved among flowering plants, including genes involved in root developmental and light signaling pathways. Wolffia has also lost most of the conserved nucleotide-binding leucine-rich repeat (NLR) genes that are known to be involved in innate immunity, as well as those involved in terpene biosynthesis, while having a significant overrepresentation of genes in the sphingolipid pathways that may signify an alternative defense system. Diurnal expression analysis revealed that only 13% of Wolffia genes are expressed in a time-of-day (TOD) fashion, which is less than the typical ∼40% found in several model plants under the same condition. In contrast to the model plants Arabidopsis and rice, many of the pathways associated with multicellular and developmental processes are not under TOD control in W. australiana, where genes that cycle the conditions tested predominantly have carbon processing and chloroplast-related functions. The Wolffia genome and TOD expression data set thus provide insight into the interplay between a streamlined plant body plan and optimized growth.

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.

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.

Anatomical insights into the vascular layout of the barley rachis: implications for transport and spikelet connection.

BACKGROUND AND AIMS: Vascular patterning is intimately related to plant form and function. Here, using barley (Hordeum vulgare) as a model, we studied the vascular anatomy of the spike-type inflorescence. The main aim of the present work was to clarify the relationship between rachis (spike axis) vasculature and spike size, to define vascular dynamics and to discuss the implications for transport capacity and its interaction with the spikelets. METHODS: We used serial transverse internode sections to determine the internode area, vascular area and number of veins along the rachis of several barley lines. KEY RESULTS: Internode area and total vascular area show a clear positive correlation with spike size, whereas the number of veins is only weakly correlated. The lateral periphery of the rachis contains large mature veins of constant size, whereas the central part is occupied by small immature veins. Spikelet-derived veins entering the rachis often merge with the immature rachis veins but never merge with the mature veins. An increase in floret fertility through the conversion of a two-rowed barley into an isogenic six-rowed line, in addition to a decrease in floret fertility owing to enhanced pre-anthesis tip degeneration caused by the mutation tip sterile 2.b (tst2.b), significantly affected vein size but had limited to no effects on the number of veins or internode area. CONCLUSIONS: The rachis vasculature is the result of a two-step process involving an initial layout followed by size adjustment according to floret fertility/spike size. The restriction of large mature vessels to the periphery and that of small immature vessels to the centre of the rachis suggests that long-distance transport and local supply to spikelets are spatially separated processes. The identification of spikelet-derived veins entering the rachis without fusing with its vasculature indicates that a vascular continuity between rachis and spikelets might be non-essential.

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.

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.

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.

A functional imaging study of germinating oilseed rape seed.

Germination, the process whereby a dry, quiescent seed springs to life, has been a focus of plant biologist for many years, yet the early events following water uptake, during which metabolism of the embryo is restarted, remain enigmatic. Here, the nature of the cues required for this restarting in oilseed rape (Brassica napus) seed has been investigated. A holistic in vivo approach was designed to display the link between the entry and allocation of water, metabolic events and structural changes occurring during germination. For this, we combined functional magnetic resonance imaging with Fourier transform infrared microscopy, fluorescence-based respiration mapping, computer-aided seed modeling and biochemical tools. We uncovered an endospermal lipid gap, which channels water to the radicle tip, from whence it is distributed via embryonic vasculature toward cotyledon tissues. The resumption of respiration is initiated first in the endosperm, only later spreading to the embryo. Sugar metabolism and lipid utilization are linked to the spatiotemporal sequence of tissue rehydration. Together, this imaging study provides insights into the spatial aspects of key events in oilseed rape seeds leading to germination. It demonstrates how seed architecture predetermines the pattern of water intake, which sets the stage for the orchestrated restart of life.

Plant Regeneration via Adventitious Shoot Formation from Immature Zygotic Embryo Explants of Camelina.

Camelina is an oil seed crop that is enjoying increasing interest because it has a particularly valuable fatty acid profile, is modest regarding its water and nutrient requirements, and is comparatively resilient to abiotic and biotic stress factors. The regeneration of plants from cells accessible to genetic manipulation is an essential prerequisite for the generation of genetically engineered plants, be it by transgenesis or genome editing. Here, immature embryos were used on the assumption that their incomplete differentiation was associated with totipotency. In culture, regenerative structures appeared adventitiously at the embryos' hypocotyls. For this, the application of auxin- or cytokinin-type growth regulators was essential. The formation of regenerative structures was most efficient when indole-3-acetic acid was added to the induction medium at 1 mg/L, zygotic embryos of the medium walking stick stage were used, and their hypocotyls were stimulated by pricking to a wound response. Histological examinations revealed that the formation of adventitious shoots was initiated by locally activated cell division and proliferation in the epidermis and the outer cortex of the hypocotyl. While the regeneration of plants was established in principle using the experimental line Cam139, the method proved to be similarly applicable to the current cultivar Ligena, and hence it constitutes a vital basis for future genetic engineering approaches.

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.

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.

DEFECTIVE ENDOSPERM-D1 (Dee-D1) is crucial for endosperm development in hexaploid wheat.

Hexaploid wheat (Triticum aestivum L.) is a natural allopolyploid and provides a usable model system to better understand the genetic mechanisms that underlie allopolyploid speciation through the hybrid genome doubling. Here we aimed to identify the contribution of chromosome 1D in the development and evolution of hexaploid wheat. We identified and mapped a novel DEFECTIVE ENDOSPERM-D1 (Dee-D1) locus on 1DL that is involved in the genetic control of endosperm development. The absence of Dee-D1 leads to non-viable grains in distant crosses and alters grain shape, which negatively affects grain number and thousand-grain weight. Dee-D1 can be classified as speciation locus with a positive effect on the function of genes which are involved in endosperm development in hybrid genomes. The presence of Dee-D1 is necessary for the normal development of endosperm, and thus play an important role in the evolution and improvement of grain yield in hexaploid wheat.

Immunoelectron microscopy for locating calvin cycle enzymes in the thylakoids of synechocystis 6803.

Unicellular cyanobacteria Synechocystis 6803 were fixed using high-pressure freezing (HPF) and freeze substitution without any chemical cross-linkers. Immunoelectron microscopy of these cells showed that five sequential enzymes of the Calvin cycle (phosphoriboisomerase, phosphoribulokinase, ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO), 3-phosphoglyceratekinase and glyceraldehyde-3-phosphate dehydrogenase) and the catalytic portion of the chloroplast H+-ATP synthase (CF1) are located adjacent to the thylakoid membranes. Cell-free extracts of Synechocystis were processed by ultracentrifugation to isolate thylakoid fractions sedimenting at 40,000, 90,000, and 150,000 g. Among these, the 150,000-g fraction showed the highest linked activity of the above five sequential Calvin cycle enzymes and also the highest coordinated activity of light and dark reactions as assessed by ribose-5-phosphate (R-5-P) +ADP dependent CO2 fixation. Immunogold labeling of this membrane fraction confirmed the presence of the above five enzymes as well as the catalytic portion of the CF1 ATP synthase. Notably, the protein A-gold labeling of the thylakoids was observed without use of chemical cross-linkers and in spite of the normal washing steps used during standard immunolabeling. The results showed that soluble Calvin cycle enzymes might be organized along the thylakoid membranes.