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The regulation of lipid synthesis in oil seeds is still not fully understood. Oilseed rape (Brassica napus) is the third most productive vegetable oil crop on the global market; therefore, increasing our understanding of lipid accumulation in oilseed rape seeds is of great economic, as well as intellectual, importance. Matrix-assisted laser/desorption ionization-mass spectrometry imaging (MALDI-MSI) is a technique that allows the mapping of metabolites directly onto intact biological tissues, giving a spatial context to metabolism. We have used MALDI-MSI to study the spatial distribution of two major lipid species, triacylglycerols and phosphatidylcholines. A dramatic, heterogenous landscape of molecular species was revealed, demonstrating significantly different lipid compositions between the various tissue types within the seed. The embryonic axis was found to be particularly enriched in palmitic acid, while the seed coat/aleurone layer accumulated vaccenic, linoleic, and 𝛼-linoleic acids. Furthermore, the lipid composition of the inner and outer cotyledons differed from each other, a remarkable discovery given the supposed identical functionality of these two tissues. Triacylglycerol and phosphatidylcholine molecular species distribution was analyzed through a developmental time series covering early seed lipid accumulation to seed maturity. The spatial patterning of lipid molecular species did not vary significantly during seed development. Data gathered using MALDI-MSI was verified through gas chromatography analysis of dissected seeds. The distinct lipid distribution profiles observed imply differential regulation of lipid metabolism between the different tissue types of the seed. Further understanding of this differential regulation will enhance efforts to improve oilseed rape productivity and quality.

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.

The angiosperm embryo and endosperm are limited in space because they grow insidematernal seed tissues. The elimination of cell layers of the maternal seed coat by programmedcell death (PCD) could provide space and nutrition to the filial organs. Using the barley (Hordeumvulgare L.) seed as a model, we elucidated the role of vacuolar processing enzyme 4 (VPE4) in cereals by using an RNAi approach and targeting the enzymatic properties of the recombinant protein. A comparative characterization of transgenic versus wild-type plants included transcriptional and metabolic profiling, flow cytometry, histology and nuclear magnetic imaging of grains. The recombinant VPE4 protein exhibited legumain and caspase-1 properties in vitro. Pericarp disintegration was delayed in the transgenic grains. Although the VPE4 gene and enzymatic activity was decreased in the early developing pericarp, storage capacity and the size of the endosperm and embryo were reduced in the mature VPE4-repressed grains. The persistence of the pericarp in the VPE4-affected grains constrains endosperm and embryo growth and leads to transcriptional reprogramming, perturbations in signalling and adjustments in metabolism. We conclude that VPE4 expression executes PCD in the pericarp, which is required for later endosperm filling, and argue for a role of PCD in maternal control of seed size in cereals.

The Venus flytrap Dionaea muscipula captures insects and consumes their flesh. Prey contacting touch-sensitive hairs trigger traveling electrical waves. These action potentials (APs) cause rapid closure of the trap and activate secretory functions of glands, which cover its inner surface. Such prey-induced haptoelectric stimulation activates the touch hormone jasmonate (JA) signaling pathway, which initiates secretion of an acidic hydrolase mixture to decompose the victim and acquire the animal nutrients. Although postulated since Darwin’s pioneering studies, these secretory events have not been recorded so far. Using advanced analytical and imaging techniques, such as vibrating ion-selective electrodes, carbon fiber amperometry, and magnetic resonance imaging, we monitored stimulus-coupled glandular secretion into the flytrap. Trigger-hair bending or direct application of JA caused a quantal release of oxidizable material from gland cells monitored as distinct amperometric spikes. Spikes reminiscent of exocytotic events in secretory animal cells progressively increased in frequency, reaching steady state 1 d after stimulation. Our data indicate that trigger-hair mechanical stimulation evokes APs. Gland cells translate APs into touch-inducible JA signaling that promotes the formation of secretory vesicles. Early vesicles loaded with H⁺ and Cl⁻ fuse with the plasma membrane, hyperacidifying the “green stomach”-like digestive organ, whereas subsequent ones carry hydrolases and nutrient transporters, together with a glutathione redox moiety, which is likely to act as the major detected compound in amperometry. Hence, when glands perceive the haptoelectrical stimulation, secretory vesicles are tailored to be released in a sequence that optimizes digestion of the captured animal.

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.Radchuk et al. report on the role of vacuolar processing enzymes (VPEs) in mediating programmed cell death (PCD) in the maternal tissues of a barley grain and influencing the delivery of assimilate to the endosperm. This study presents a means of increasing the efficiency of the grain filling process in the major cereal crop species by manipulating the timing of PCD.

Homeodomain leucine zipper class I (HD-Zip I) transcription factors (TFs) play key roles in the regulation of plant growth and development under stresses. Functions of the TaHDZipI-2 gene isolated from the endosperm of developing wheat grain were revealed. Molecular characterization of TaHDZipI-2 protein included studies of its dimerisation, protein–DNA interactions and gene activation properties using pull-down assays, in-yeast methods and transient expression assays in wheat cells. The analysis of TaHDZipI-2 gene functions was performed using transgenic barley plants. It included comparison of developmental phenotypes, yield components, grain quality, frost tolerance and the levels of expression of potential target genes in transgenic and control plants. Transgenic TaHDZipI-2 lines showed characteristic phenotypic features that included reduced growth rates, reduced biomass, early flowering, light-coloured leaves and narrowly elongated spikes. Transgenic lines produced 25–40% more seeds per spike than control plants, but with 50–60% smaller grain size. In vivo lipid imaging exposed changes in the distribution of lipids between the embryo and endosperm in transgenic seeds. Transgenic lines were significantly more tolerant to frost than control plants. Our data suggest the role of TaHDZipI-2 in controlling several key processes underlying frost tolerance, transition to flowering and spike development.

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 T 1 -maps, a new approach visualizes an alteration of a tissue-specific MRI parameter T 1 (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 T 1 -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.

• 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.

Summary 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.

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.