Mechanisms underlying the delivery of assimilates to the developing seed are of highest relevance in crop research. The transport route of assimilates is hidden from human eyes and is challenging for investigation. We established non-invasive imaging technology based on nuclear magnetic resonance (NMR), allowing to visualize sucrose allocation along the path from source to sink and especially within the living seed itself.

Non-invasive imaging/monitoring provides information on tissues involved in transport processes, dynamic and spatiotemporal pattern of assimilate import, helping to link developmental and molecular events during seed growth. We apply an integrative approach which includes mass spectrometric biochemical analyses, metabolic modelling, chemical imaging based on infrared spectroscopy and optical microscopy. Such combination represents a powerful platform for the comprehensive study of seed filling in crops (Melkus et al., Plant Biotechnology Journal 2011; Rolletschek et al., Plant Cell 2011; Borisjuk et al., Plant Journal 2012; Rolletschek et al., Plant Physiology 2015, Munz et al., New Phytologist 2017; Radchuk et al., JExpBot 2017).

In current DFG projects, we investigate the functional role of sugar transporters (SUT-, SWEET-families) for seed development and filling.

In laufenden DFG-finanzierten Projekten untersuchen wir zum Beispiel die Rolle von spezifischen Zuckertransportern (SUT-, SWEET-Genfamilien) für die Samenentwicklung in Getreiden.

The hypoxic state comes about whenever the capacity for oxygen diffusion is restricted, so that the concentration of oxygen available falls below the level required for cellular metabolism. These conditions apply frequently to the developing seed (for review see Rolletschek H. (2012). “Hypoxia – a phenomenon which shapes seed metabolism”. Habilitation at the Naturwissenschaftliche Fakultät, Leibniz Universität Hannover/Germany). Seed hypoxia is not only because of high respiratory activities but mainly due to the low void space/porosity of seed tissues, and thus limited diffusive oxygen uptake (Verboeven et al., New Phytologist 2013).

In current work, funded by DFG, we are investigating the implications of internal hypoxia for assimilate uptake and partitioning in the developing maize kernel. In particular we work on the functional role of specific metabolic enzymes (PPDK; Lappe et al., PNAS 2018), the metabolic heterogeneity of endosperm (Rolletschek et al., 2017 In: Maize Kernel Development), phytoglobins and signalling components using a number of transgenic and mutant plants.

Our idea is that in vivo metabolic fluxes are locally regulated and connected to seed architecture. In cereal grains, the isolation of the filial from the maternal tissues creates a complex metabolic system, involving a tripartite interaction between the pericarp, endosperm, and embryo. These three grain components in effect form an interactive system of autonomous organs, each following their own genetic programs. To specify whole-grain metabolism at the level of its component tissue parts, we are applying spatially resolved molecular, biochemical and physiological analysis of metabolic activities as well as metabolic modelling approaches.

Recent findings quantified the in vivo metabolic contributions of distinct seed organs and suggest the presence of a mechanism(s) able to ensure metabolic homeostasis in the face of short-term environmental fluctuation (Rolletschek et al., Plant Physiology 2015).

In oilseed rapeseed (Brassica napus), noninvasive NMR-based imaging has previously demonstrated the establishment of steep gradients in lipid accumulation (Borisjuk et al., Progress Lipid Research 2013). We have been investigating how metabolism in the distinct seed organs of B. napus (seed coat, endosperm, inner and outer cotyledons, radicle) is adjusted to local conditions inside the seed (Borisjuk et al, Plant Cell 2013, Lorenz et al., J Proteomics 2014, Schwender et al., Plant Physiology 2015). In current research, we focus on the characterization of the transient endosperm compartment, and investigate how it contributes to growth control of embryo.

Seeds determine the reproductive capacity of plants and are vital to their existence. To ensure seed survival and germination, the embryo enters the maturation phase in late development, which encompasses the accumulation of reserve compounds and the acquisition of desiccation tolerance. Trehalose 6‐phosphate (T6P), which functions as a signal for sugar availability in plants, is believed to regulate storage processes in seeds, since disruption of T6P synthesis in Arabidopsis thaliana causes embryo abortion at the onset of seed filling phase. To investigate the role of T6P during seed development, we modulated the T6P content in pea embryos by ectopic expression of T6P synthase (OtsA) or T6P phosphatase (OtsB) genes from E. coli. We already showed that T6P promotes cotyledon growth and starch accumulation in maturing seeds, and that this requires transcriptional induction of auxin biosynthesis. Our data indicate that T6P integrates auxin signalling with sugar availability to facilitate seed filling (Dissertation T. Meitzel 2018; McAdam et al., New Phytologist 2017). Yet, many aspects of this process are still not understood. To fill these gaps, we plan to characterize the phenotypic and metabolic changes in our transgenic plant models via NMR imaging, metabolite profiling and metabolic flux analysis. Furthermore, special emphasis will be put on the role of T6P during seed germination and lipid reserve accumulation in the oil crop Brassica napus.

We elucidated that programmed cell death (PCD) in maternal seed parts is required for endosperm development and seed filling, thus contributing to the control of seed size in cereal grains. The distributions of TUNEL-positive nuclei, expression of PCD-related genes and cascades of caspase-like activities have revealed that each seed tissue follows an individual PCD pattern. Earlier we found that Triticeae-specific Jekyll gene, exclusively expressed in the nucellar projection, is involved in terminal differentiation of the nucellar tissues switching their cell fate to death (Radchuk et al., Plant Cell 2006). Further, we have established that tissue-specific genes encoding vacuolar processing enzyme (VPE) are required for PCD in distinct grain tissues. Using transcriptional and metabolic profiling, flow cytometry, 13C-feeding experiments, histology and nuclear magnetic resonance imaging of grains we demonstrated that PCD in pericarp is required to provide space for the expanding endosperm and embryo (Radchuk et al., New Phytology 2018). PCD in the nucellar projection contributes to nutrient flow towards endosperm and is controlled by expanded VPE2 subfamily in barley (Mascher et al., Nature 2017) and other Triticeae, compared to other Poaceae species. In current research, we elucidate the functional role of specific gene family members of VPE.

Our studies promote NMR‐imaging as a versatile analytic tool for developmental biology, potent for in vivo study of the inner life of plants (Borisjuk Habilitation Thesis “The inner life of seed: from seeing to understanding”, Leibniz Universität Hannover/Germany 2017).

A major thrust of developmental biology is to understand how molecular and cellular processes produce 3D morphology. Nuclear Magnetic Resonance Imaging (MRI) has a great virtue in being non‐invasive and therefore has the potential to monitor physiological processes in vivo. In our hands, MRI is capable to capture the previously hidden growth/storage without seed destruction and thus allows us to monitor living seed (Borisjuk et al., Plant J. 2012). We prime MRI for non-invasive visualization and survey of flower/seed interior and apply high resolution chemical (FT IR) imaging to characterize structure and composition of tissues with close to cellular resolution (Gündel et al., Plant Physiology 2018).

The introduction of functional imaging on living seed enables us to display seed growth and to uncover intimate events of the awakening of life during germination (Munz et al., New Phytologist 2017). 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. 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, sugar metabolism and lipid utilization are linked to the spatiotemporal sequence of tissue rehydration.

Mechanosensing

It is virtually unknown whether and how morphological changes in the embryo during development influence embryonic metabolism. For example, the curvature of the embryonic axis or the developing cotyledons could change the local growth conditions. It is unclear how the process of embryonic shape formation is perceived and translated into corresponding metabolic signals. The processes involved - termed mechanosensing and proprioception - are being investigated in current DFG projects using the oilseed rape model. In particular, we are looking for mechanisms of how the growing embryo adapts its shape and size to the available space (limited by the seed coat) and how this is reflected in its metabolism.

Other models in which we describe developmental processes are barley, maize, pea, and Arabidopsis (Kovalchuk et al., New Phytologist 2016; Radchuk et al., 2018; Lappe et al., PNAS 2018). In addition, the above-mentioned research approaches and methods are used as part of collaborations with breeders (rapeseed, wheat).

The development of novel bioanalytical methods is an essential part of our work in the various projects funded by DFG and BMBF. In particular, we work on: (1) improvement of imaging tools for plants using NMR/MRI, (2) biochemical methods for micro sampling using novel Orbitrap technology (LC/MS), (3) FTIR-microscopy for the analysis of spatial gradients in sugars, hormones and more, (4) methods for the high throughput screening of seeds using NIRS, TD-NMR and robotized sample delivery systems.

Examples of important method developments are given by Melkus et al., (Plant Biotech J 2011), Fuchs et al. (Plant Physiology 2013), Borisjuk et al. (Plant Cell 2013), Rolletschek et al. (Plant Biotech J 2015), Munz et al. (New Phytologist 2017), and Gündel et al. (Plant Physiology 2018).